UNIVERSIDAD DE COSTA RICA

SISTEMA DE ESTUDIOS DE POSGRADO

Posfolipasas A2miotxicas de venenos de serpientes de la familia Viperidae:

caracterizacin estructural y funcional

Tesis sometida a la consideracin del Programa de Doctorado en Ciencias para optar por
el grado de Doctor en Ciencias (Ph.D.)

Yarnileth Angulo Ugalde

Instituto Clodomiro Picado, Facultad de Microbiologa, y Departamento de Bioquinica,

Escuela de Medicina, Universidad de Costa Rica

Ciiidad Ui~iversitariaRodrigo Facio

2005
 AGRADECIMIE-

Quiero expresar mi ms sincero agradecimiento a:

A mi tutor, el Dr. Bruno Lomonte por su gran dedicacin, su paciencia extrema,
enseanza y gua en la realizacin de esta tesis y por su sincera amistad. En realidad ha sido
una experiencia valiossima para mi haber trabajado con una persona tan dedicada al trabajo y
sobre todo con una gran capacidad y un enorme amor a la investigacin.
A mi asesor, el Dr. Jos Mara Gutirrez por el apoyo que siempre me ha brindado, por
sus enseanazas y porque su trabajo refleja un espritu emprendedor y siempre ser el gran
maestro y el buen consejero para todos los que hemos trabajado con l.
A mi asesor, el Dr. Alberto Alape por sus excelentes recomendaciones, porque ha sido
para m un ejemplo maravilloso de lo que es la dedicacin y el esfuerzo para alcanzar frutos en
la investigacin y en la vida acadmica.
A mi familia por su apoyo y comprensin por el tiempo que no he dedicado a ellos por
dedicarlo a mi trabajo.
A todos mis compaeros del Instituto Clodorniro Picado, quienes con la colaboracin,
apoyo y el trabajo en equipo hacen que la investigacin se convierta en una forma de vida y
estmulo.
A los evaluadores del anteproyecto de tesis, el Dr. Edgardo Moreno, el Dr. Esteban
Chaves y la Dra. Ximena Corts, por sus excelentes recomendaciones.
A mi amiga y compaera Alexandra Rucavado, quien siempre tiene para m una
excelente sugerencia, y un apoyo incondicional en el trabajo. Gracias amiga.
Al Dr. Luis Diego Calzada y a la Lic. Camen Castro por su apoyo para el ingreso al
Programa de Doctorado en Ciencias.
A los Drs. Lourival Possani, Fernando Zamudio, Timoteo Olamendi y Georgina
Gurrola, del Laboratorio de Protenas del Instituto de Biotecnologia, UNAM-Cuemavaca,
Mxico, por el apoyo en la secuenciacin de la miotoxina II de A. nummifer y en la
produccin de pptidos sintticos y por su cordialidad y amistad que me brindaran en su
laboratorio.
Al Dr. Raghuvir Ami, Leandra Watanabe, Lisandra M. Gava, del Departamento de
Fisica, IBILCE/UNESP R. Cristoviio Colombo, Siio Jos do Rio Preto, Brazil, por el trabajo
colaborativo en la cristalizacin de la miotoxina 11 de A. nuntinfler.
A los doctores Jonas Perales, Gilberto Domont y Ana Gisele Neves, as como a Surza
L. Rocha, del departamento de Fisiologa e Farmacodiniimica del Instituto Oswaldo Cruz,
Fiocruz, Rio de Janeiro, Brazil, por su apoyo en las investigaciones con el inhibidor de
Didelphis marsupialis, el DM64. Adems, quiero agradecerles el carifio, apoyo y excelente
trato que me dieran durante el tiempo que pas en su laboratorio.
A los coautores de los artculos incluidos en este trabajo, el Dr. Andreimar Soares de la
Universidad de SZo Paulo en Ribeirao Preto, Brazil, al Dr. Wonhwa Cho, Universidad de
Illinois-Chicago, EUA, y al M.Sc Carlos Santamana, Instituto Clodorniro Picado, Universidad
de Costa Rica.
Un agradecimiento a las entidades que financiaron este trabajo de investigacin:
Intemational Foundation for Science (IFS), Vicerrectora de Investigacin, Universidad de
Costa Rica, Consejo Nacional para Investigaciones Cientficas y Tecnolgicas (CONIC1T)-
FORJNVES, Secretara de Relaciones Exteriores de Mxico (Programa Costa Rica-Mxico),
Embajada de Japn, NeTropica Sweden-Central American network.
 Esta tesis esta basada en los siguientes artculos, los cuales son referidos en el texto con
numerales romanos:

(1) Angulo Y, Olamendi-Portugal T, Possani L, Lomonte B (2000). Isolation and

characterization of myotoxin 11 fiom Atropoides (Bothrops) nummfer snake venom, a
new Lys49 phospholipase A2 homologue. The hiternationol Journnl of Biochemistry &
Cell Biology 32, 63-7 1.

(11) Angulo Y, Olamendi-Portugal T, Alape-Girn A, Possani L, Lomonte B (2002) Structural

characterization and phylogenetic relationships of myotoxin 11 fiom Atropoides
(Bothrops) numlnifer snake venom, a Lys49 phospholipase Az homologue. The
Internntional Jozrrnal of Biochemistry & Cell Biology 34, 1268-1278.

(111) Watanabe L, Gava LM, Angulo Y, Lomonte B, Ami RK (2004) Crystallization of the
Lys49 PLAz homologue, rnyotoxin 11, fiom the venorn of Atropoides nummifer.
Biochimicn et Biophysica Acta 1703, 87-89.

(IV) Lomonte B, Angulo Y, Santamaria C (2003) Comparative study of synthetic peptides

corresponding to region 115- 129 in Lys49 myotoxic phospholipases A2 from snake
venoms. Toxicorz 42, 307-3 12.

(V) Angulo Y, Gutirrez JM, Soares AM, Cho W, Lomonte B (2005) The myotoxic and
cytolytic activities of dimerc Lys49 phospholipase A2 homologues are reduced, but not
abolished, by a pH-induced dissociation. Toxicon (en prensa).

(VI) Angulo Y, Lomonte B (2004) Differential susceptibility of C2C12 myoblasts and

myotubes to group II phospholipase Az rnyotoxins fiom crotalid snake venoms. Cell
Biochernistry and Function (en prensa)

(VII) Angulo Y, Lomonte B (2003) Inhibitory effect of fucoidan on fhe activities of crotaline
snake venom myotoxic phospholipases A2. Biochemical Pharmacology 66, 1993-2000.
 Esta tesis fue aceptada por la Comisin del Programa de Doctorado en Ciencias, como
requisito parcial para optar por el grado de Doctor en Ciencias (Ph.D.), con nfasis en Ciencias
Biomdicas.

/ "
el MacLya Trejos
e del Decano del Sistema de Estudios de Posgrado

r. Bruno Lomonte Vigliotti

Director de Tesis

Asesor de Tesis

Dr. Alberto Alape Girn

Asesor de Tesis

ama de Doctorado en Ciencias

(A,Cx,&/du
C+do C/
M.dT;lilethAngulo Ugalde
Candidata
 INDICE

Pgina

Agradecimientos
Lista de publicaciones
Hoja de aprobacin v
lndice vi
Resumen vii
Abstract X

Lista de Cuadros y Figuras xii

...
Lista de Abreviaturas Xlll

l . Introduccin
1.1. Serpientes venenosas y sus venenos
1.2. Importancia mdica del accidente ofdico
1.3. Envenenamientos por serpientes de la familia Viperidae
1.4. Toxinas que daan msculo aisladas de venenos de serpiente
1.5. Fosfolipasas A2 secretadas
1.6. Relacin estructura-funcin en fosfolipasas A2 del grupo 11
1.7. Inhibidores de fosfolipasas A2del grupo 11
2. Objetivos
3. Materiales y Mtodos
4. Resultados
5. Discusin General
6. Conclusiones
7. Perspectivas futuras
8. Referencias
9. Apndice: publicaciones
Angulo Ugalde, Yamileth
Fosfolipasas A2 miotxicas de venenos de serpiente de la familia Viperidae:caracterizacin
estructural y funcional
Tesis de doctorado en Ciencias, San Jos, C.R.
Y Angulo U, 2005
81h: 9 il- 209 ref

RESUMEN

Las miotoxinas tipo fosfolipasa A2 (PLA2) del grupo II se encuentran comunrnente en

venenos de las serpientes de la familia Viperidae. Con la finalidad de comprender las
relaciones entre la estructura y la funcin de estas protenas, se han caracterizado bioqumica y
famacolgicamente diversas variantes naturales. En la presente tesis se describe una nueva
miotoxina de este grupo, aislada del veneno de Atropoides nummifer de Costa Rica. La
rniotoxina 11 de A. nummifer es un homodimero con subunidades bsicas de 121 residuos, y
una masa molecular de 13,789.90 Da por subunidad. Se determino su secuencia completa de
aminocidos, la cual presenta una alta similitud con PLAzs Lys49 de los gneros
Cerrophidion, Trimeresurus, Bothrops y Agkistrodon, las cuales segn el anlisis filogentico
divergieron tempranamente de las PLAzs Asp49. Se model la estnictura terciaria de la
rniotoxina 11 de A. nummifer con base en las coordenadas de la miotoxina 11 de Cerrophidio~i
gorlmani, observndose solamente pequeas variaciones entre ambas estructuras. La nueva
miotoxina se cristaliz y se obtuvieron datos de difraccin de rayos X a una resolucin de 2.32
A. Actualmente se est determinando su estructura tridimensional.
La miotoxina Ii de A. nummifer induce mionecrosis rpidamente despus de la
inyeccin intramuscular en ratn, como se evidencia por un aumento temprano en la actividad
de creatina kinasa en plasma. Adems, esta toxina induce edema, y posee accin citotxica
sobre clulas de msculo esqueltico en cultivo.
Una serie de evidencias identifican el sitio txico de las PLA2s Lys49 en la regin C-
terminal, donde los arninocidos que comprenden la secuencia 115-129 parecen jugar un papel
central. En este trabajo se evalu la citotoxicidad y la miotoxicidad de una serie de pptidos
sintticos correspondientes a la secuencia 115-129 de diferentes miotoxinas Lys49. Los
pptidos variaron en sus actividades, desde los que mostraron una alta toxicidad, hasta los que
no presentaron ninguna. Esto indica que las acciones txicas de las miotoxinas Lys49 no
siempre son reproducidas por sus pptidos 115-129 libres. Tal fue el caso del pptido C-
terrninal de la miotoxina 11 de A. nummifer, el cual no indujo citotoxicidad en cultivo, ni
miotoxicidad in vivo. Interesantemente, se encontr que el pptido C-terminal de la miotoxina
Lys49 de Agkistrodon p. piscivorus sintetizado con D-aminocidos conserva ambas
actividades de su enantimero natural L, sugiriendo que el mecanismo de toxicidad no
involucra el reconocimiento de un receptorlaceptor proteico en las clulas musculares.
Recientemente se ha propuesto que el estado dimrico de una PLAi Lys49 es esencial
para su capacidad de romper liposomas, y que esta actividad desaparece cuando la toxina se
disocia en monmeros a pH 5.0. Por lo tanto, se evalu el efecto de la disociacin inducida por
pH sobre las actividades citotxica y rniotxica de cuatro PLA2s Lys49. Ambas actividades
fueron menores a pH 5.0 que a pH 7.2, en concordancia con la propuesta de que el estado
dimrico es relevante para la funcin. Sin embargo, el hallazgo de que a pH 5.0 estas toxinas
retienen una actividad significativa sugiere que el estado dimrico no es un requerimiento
absoluto para su mecanismo de accin.
Las miotoxinas PLAi del grupo II inducen un efecto citolitico sobre diversos tipos de
clulas in vitro.En el presente trabajo se demostr que la fusin y diferenciacin de mioblastos
a miotubos, induce cambios que hacen que estos sean ms susceptibles a los mecanismos
txicos de las PLA2s del grupo 11, pero no as a otros agentes citolticos. Estos cambios
podran involucrar sitios aceptores de rniotoxinas, que an no han sido identificados.
Adems, en el presente trabajo se investig el efecto del fucoidan, un polisacrido
natural sulfatado, en las actividades citotxica y miotxica de un grupo de miotoxinas PLA2
del gmpo 11. Ambas actividades fueron eficientemente inhibidas por el fucoidan, en todas las
toxinas. La base de esta inhibicin parece ser la formacin rpida de complejos entre el
fucoidan y las miotoxinas. El fucoidan tambin inhibi la actividad citoltica de los pptidos
correspondientes a los residuos de diferentes miotoxinas Lys49 (115-129), indicando su
capacidad de interactuar con la regin C-terminal de estas toxinas. El fucoidan redujo
significativamente el dao a msculo en ratn, cuando se administr localmente,
inmediatamente despus del envenenamiento experimental con veneno crudo de B. asper.
Estos resultados dan base para la realizacin de futuros estudios sobre el posible uso de
fucanos sulfatados en el tratamiento de envenenamientos por vipridos.
Finalmente, se caracteriz la actividad inhibitoria del DM64, una protena aislada del
suero de DideZphis marstrpialis, sobre las PLAzs rniotxicas. Se demostr que dicha protena
es capaz de formar complejos con las miotoxinas PLAi de grupo 11, inhibiendo de esta forma
sus actividades citolticas in vitro y rniotxicas in vivo.
Angulo Ugalde, Yamileth
Fosfolipasas A2 miotxicas de venenos de serpiente de la familia Viperidae:caractenzacin
estnictural y funcional
Tesis de doctorado en Ciencias, San Jos, C.R.
Y Angulo U, 2005
8 1h: 9 il- 209 ref
ABSTKACT

Myotoxic phospholipases Ai (PLA2s) of the group II are comrnonly found in the

venoms of crotalid snakes. As an approach to understand their stnicture-function
relationships, diverse natural variants, have been characterized, biochemically and
phamacologically. This thesis describes a new myotoxic PLAz homologue, isolated from the
venom of Atropoides numrnifer from Costa Rica. A. numrnifer rnyotoxin 11 occurs as a
homodimer of basic subunits of 121 residues, with a subunit molecular mass of 13,789.90 Da.
Its complete amino acid sequence was determined, showing high similarity to Lys49 PLA2s
from Cerrophidion, Trimeresurus, Bothrops and Agkistrodon species, which form a subfamily
of PLAzs that diverged early from Asp49 PLA2s present in the same species, as shown by
phylogenetic analysis. The tertiary structure of the toxin was modeled, based on the
coordinates of Cerrophidion g o h a n i myotoxin 11, showing only minor differences between
both structures. The new toxin was crystallized, and X-ray diffraction data were collected to a
resolution of 2.32 A. Its three-dimensional structure is currently being determined.
A. nummifer myotoxin II induces rapid myonecrosis iipon intramuscular injection in
rnice, evidenced by an early increase in plasma creatine kinase activity. It also induces
signifcant edema, and displays cytolytic activity towards cultured skeletal muscle cells.
Current evidence supports the mapping of the toxic site of the PLAzs to the C-terminal
region, where amino acids comprised within the sequence 115-129 appear to play a central
role in toxicity. This study evaluated the cytotoxic and myotoxic effects of severa1 synthetic
peptides corresponding to the sequence 115-129 of different Lys49 inyotoxins. Peptides varied
widely in their activities, raiiging from fully toxic to harmless. These results indicate that the
toxic actions of Lys49 myotoxins cannot always be reproduced by tlieir peptides 115-129.
This was the case of the peptide from A. num~nijermyotoxin 11, wliich was not toxic to
cultured or mature muscle in vivo. Interestingly, the C-terminal peptide of Agkistrodon p.
piscivorus synthesized with D-amino acids retained both activities of the natural L-
enantiomer, suggesting that its mechanism of action does not involve the recogiiition of a
proteic receptor/acceptor site on muscle cells.
Recently, the dimeric state has been proposed to be essential for the Iiposome-
dismpting activity of a Lys49 PLA2, which dissociates into monomers at pH 5.0. Therefore,
this study evaluated the effects of a pH-induced dissociation on the toxic activities of four
Lys49 PLA2s, using biological targets. Both their cytolytic and myotoxic activities were lower
at pH 5.0 than at pH 7.2, in agreement with the proposed functional relevance of their dimeric
state. However, the observation that these proteins still retain a significant toxicity at pH 5.0,
suggests that the dimeric state is not an absolute requirement in their mechanism of action.
Group II PLAz myotoxins exert a cytolytic effect on diverse cell types in vitro.
Previous studies indicate that differentiated myotubes are highly susceptible to this effect. In
this work, it was demonstrated that fusion and differentiation of myoblasts into myotubes
induces changes that render the latter cells more susceptible to the toxic mechanism of group
II PLAz myotoxins, but not to other cytolytic agents. Such changes could involve myotoxin
acceptor sites, which remain to be identified.
The protective effect of fucoidan, a natural sulfated polysaccharide, against the
cytotoxic and nlyotoxic activities of a group of group 11 PLA2 myotoxins was investigated. Al1
of the toxins were efficiently inliibited by fucoidan, in both their cytotoxic and rnyotoxic
effects. The basis for this iiiliibition appears to be the rapid formation of complexes between
fucoidaii and myotoxiiis. Fucoidaii also inhibited the cytolytic activity of peptides tliat
represent the membrane-damaging region (residues 115- 129), indicating its ability to interact
with the C-terminal niyotoxic region of these PLA2. Fucoidan significantly inhibited muscle
damage in mice, when administered locally, immediately after experimental envenornation
with cmde venom from B. asper. These results encourage fiirther studies of sulfated fucans as
cornpounds of potential use to improve the treatment of envenomations by crotaline snakes.
Finally, the inhibitory activity of DM64, a protein isolated from tlie seruni of the
mammalian DideZphis mnrstlpinlis, towards myotoxic PLA2s was cliaracterized. It was
deinonstrated that this protein foniis complexes with the group II myotoxins, inhibiting their i>l
vitro cytolytic aild i ~ rvivo myotoxic actions.
 LISTA DE CUADROS Y FIGURAS

Cuadro 1 Fosfolipasas Az tipo Lys49 aisladas de venenos de serpiente.

Figura 1 Mecanismo catalitico de las fosfolipasas A*.

Figura 2 Representacin esquemtica de la estructura tridimensional de las fosfolipasas

A2 del gmpo 11.

Figura 3: Representacin esquemtica de la estructura dimrica de la miotoxina 11 de B.

asper .

Figura 4 Estr~lcturapromedio del fucoidn.

Figura 5 Formacin de complejos entre el DM64 y las fosfolipasas A2 del gmpo 11

miotxicas del veneno de Bothrops nsper.

Figura 6 Formacin de complejos entre el DM64 y las fosfolipasas Ai del gmpo 11

miotxicas.

Figura 7 Ausencia de formacin de complejos entre el DM64 y dos hemorraginas de

venenos de serpiente.

Figura 8 Inhibicin de la actividad citotxica in vitro de las fosfolipasas A2 del gmpo II

miotxicas por el DM64.
 LISTA DE ABRIEVXATURAS

ADNc ADN copia

BS~ bis-sulfosuccinimidil suberato
CK creatina kinasa
CM-Sephadex carboximetil-Sephadex
cm dominios de reconocimiento de carbohidratos
DHL deshidrogenasa lctica
DMEM medio de Eagle modificado por Dulbecco
OMS Organizacin Mundial de la Salud
PBS amortiguador de salina fosfato, pH 7.2
PEG polietilenglicol
PLA2 fosfolipasa Ai
PLI inhibidor de fosfolipasa
RP-HPLC cromatografa lquida de alto desempeo en fase reversa
SDS-PAGE electroforesis en gel de poliacrilamida con duodecil sulfato de sodio
SFB suero fetal bovino
UREA-PAGE electroforesis en gel de poliacrilarnida con urea
 l . INTRODUCCION

1.1 Serpientes venenosas y sus venenos

Las serpientes venenosas se clasifican en tres familias: Viperidae, Elapidae y

Colubridae (Campbell y Lamar, 2004; Solrzano, 2004). En Centroamrica existe una gran
diversidad de serpientes, y particularmente en Costa Rica se han descrito 22 especies de
serpientes venenosas, las cuales estn distribuidas en una amplia variedad de hiibitats
(Solrzano, 2004).
Los venenos de serpiente se producen en glndulas exocrinas especializadas, y estn
compuestos por una mezcla compleja de molculas de diferente naturaleza qumica,
mayoritariamente protenas, las cuales constituyen ms del 70% del peso seco. En un
accidente ofidico estas proteinas pueden causar neurotoxicidad, cardiotoxicidad, alteraciones
en la coagulacin sanguinea, hemorragia y mionecrosis. Las toxinas asociadas a estas
diferentes actividades pueden actuar sinrgicamente para inducir un efecto determinado, o
bien algunas piieden inducir ms de un efecto (Kini e Iwanaga, 1986b).
Los estudios sobre el origen y evolucin de las toxinas de los venenos de elpidos y
vipridos indican que muchos de los genes de las familias de protenas encontradas
actualmente en los venenos, fueron reclutados para cumplir una funcin txica muy temprano ,

en la evolucin (Fry y Wster, 2004). Por lo tanto, la composicin de proteinas en los venenos
de diferentes gmpos de serpientes nos puede dar una visin de las relaciones evolutivas entre
estas. Por ejemplo, dichos anilisis muestran que las fosfolipasas A2 (PLA2s) de los veiienos de
vipridos y elpidos no tienen un origen monofiltico, ya que las toxinas de vipridos
presentan una mayor similitud con las PLA2s de tipo sinovial-inflamatorio, mientras que las
toxinas PLAzs de elpidos presentan mayor similitud con las de tipo pancretico (Fry y
Wster, 2004).

1.2 Importancia mdica del accidente ofdico

Los envenenamientos por mordedura de serpiente en humanos constituyen un

problema de salud pblica, especialmente en las regiones tropicales del mundo. Segn datos
de la Organizacin Mundial de la Salud (OMS), hace algunas dcadas se producan unos
500.000 accidentes por ao y unas 30.000 muertes (Swaroop y Grab, 1954). Estimaciones
ms recientes sugieren que la magnitud de este problema a nivel mundial podra alcanzar los
2.600.000 accidentes y 125.000 muertes anuales (Chippaux, 1998). En Costa Rica se estima
que unos 500-600 pacientes son atendidos en los centros hospitalarios cada ao como
consecuencia de envenenamientos por serpientes, implicando una tasa anual de morbilidad de
aproximadamente 15-20 por 100.000 habitantes (Gutirrez, 1995; Sasa y Vsquez, 2003). La
mortalidad por accidentes ofidicos en Costa Rica ha sido estimada en 0,2 por 100.000
habitantes (Rojas et al., 1997).
Las mordeduras de serpiente adems de ser una causa de mortalidad, en una cierta
proporcin de casos pueden dejar a sus vctimas con discapacidades permanentes, debido a la
severidad del dao tisular causado por el veneno de algunas especies (Jorge et al, 1999).

1.3 Envenenamientos por serpientes de la familia Viperidae

Las serpientes de la familia Viperidae (subfamilia Crotalinae) se encuentran

ampliamente distribudas en Amrica Latina (Campbell y Lamar, 1989), comprendiendo un
gran nmero de especies que son responsables de la mayora de envenenamientos en este
continente (Gutirrez, 1995). Dentro de estas, las especies del gnero Bothrops son las de
mayor importancia mdica (Rosenfeld, 1971; Bolaos, 1984; Otero et al., 1992). Atropoides
>zummifer,anteriormente llamada Bothrops nummfler, y conocida comunrnente como "mano
de piedra", es una serpiente terrestre distribuida a lo largo de Centroamrica (Solrzano, 1 989;
Campbell y Lamar, 2004). En Costa Rica se le encuentra en los bosques lluviosos tropicales
(Taylor et al., 1974).
En general, los envenenamientos por serpientes de la familia Viperidae se caracterizan
por causar un dao promineiite al tejido local, incluyendo mionecrosis, hemorragia y edema,
adems de poseer una serie de acciones con manifestaciones sistmicas (Rosenfeld, 1971;
G~itirrezy Lomonte, 1989; Otero et al. ,1992). La necrosis de msculo es uno de los efectos
ms importantes inducido por estos venenos, con el potencial de dejar secuelas graves, como
prdida de tejido y discapacidad (Nishioka y Silveira, 1992; Warrell, 1996). Adems, las
coagulopatas constituyen uno de los hallazgos ms comunes en los envenenamientos por
vipridos (Hutton y Warrell, 1993). Pruebas de laboratorio como el tiempo de protrombina y
el tiempo de coagulacin sangunea son usadas para evaluar el desarrollo de estos
envenenamientos. Otro efecto fisiopatolgico importante en los envenenamientos por
vipridos es el dao renal agudo, que puede manifestarse como oliguria o anuria, usualmente 6
hr o ms despus de la mordedura. Esta nefrotoxicidad, junto con las alteraciones
cardiovasculares sistmicas (hipotensin y choque) causadas por los venenos de vipridos,
constituyen las principales causas de muerte (Nishioka y Silveira, 1992; Otero et al, 2002)

1.4 Toxinas que daan msculo (miotoxinas) aisladas de venenos de serpiente

Muchas especies de serpientes venenosas secretan toxinas que inducen necrosis de

msculo esqueltico (miotoxinas), las cuales contribuyen a digerir la presa y causan un
significativo dao al tejido en los accidentes por envenenamiento en humanos (Harris y
Cullen, 1990). Estas toxinas son muy frecuentes y abundantes en los venenos de vipridos.
Las miotoxinas de serpientes se han clasificado de acuerdo con sus propiedades
bioquimicas y toxinologicas en: (a) cardiotoxinas de venenos de elpidos, (b) toxinas bsicas y
de bajo peso molecular (42-45 aminocidos) denominadas miotoxinas pequeas, (c) toxinas
hemorrgicas que inducen dao muscular de forma indirecta, posiblemente asociado a
fenmenos de isquemia, (d) PLAzs neurotxicas que son tambin miotxicas, y (e) miotoxinas
no neurotxicas con estructura de PLA2.

(a) Cardiotoxjnas
Son un grupo de proteinas bsicas de 60-62 residuos de aminocidos, sin actividad
enzimtica y estructuralmente similares a las a neurotoxinas (Dufton y Hider, 1991). Esas
proteinas de membrana activas se han encontrado en los venenos de elpidos incluyendo las
cobras. El nombre de cardiotoxina se origina de la capacidad de estos componentes de
producir dao cardaco in vitro e in vivo (Harris y Cullen, 1990). Sin embargo, presentan
actividad citolitica en muchos tipos de clulas.

(b) Miotoxinas pequeas

El grupo de miotoxinas pequeas est formado por protenas bsicas compuestas de 42
a 45 residuos de aminocidos. Ejemplos de estas protenas son la crotamina de Crotalus
durissus terrzficus y la miotoxina a de Crotalus v. viridis (Laure, 1975; Cameron y Tu, 1977).

(c) Metaloproteinasas hernorragicas con efecto miotxico

Las toxinas hemorrgicas presentes en los venenos de serpiente de la familia Viperidae
son metaloproteinasas que contienen zinc, con masas que varan entre 20 y 100 kDa, capaces
de inducir un rpido sangrado local (Bjarnason y Fox, 1994). Las enzimas de alto peso
molecular se clasifican como metaloproteinasas con dominios tipo disintegrina y ricas en
cistena. Estas enzimas tienen una potente actividad proteoltica sobre las protenas de la
matriz extracelular (Kamiguti et al., 1998). Algunas de estas toxinas ha sido aisladas de los
venenos de serpiente de la familia Vipendae de Costa Rica, como la BaPl y la BaH4 de B.
asper (Gutirrez y Ovadia, 1998; Franceschi et al., 2000).

(d) Miotoxinas PLAz neurotxicas

Los venenos de elpidos contienen mltiples toxinas que presentan la capacidad de
actuar sobre la uniOn neuromuscular. Algunas de estas son PLAzs que interfieren
presinpticamente con la liberacin de acetilcolina, constituyendo las toxinas ms letales de
estos venenos. Dichas PLA2s tambin ejercen una potente accin miotxica, por lo que estos
envenenamientos combinan la neurotoxicidad presinptica con la miotoxicidad (Hams y
Cullen, 1990). En los vipridos tambin pueden encontrarse algunas PLA2s miotxicas con
alta neurotoxicidad, tales como la crotoxina del veneno de Crotalus durissus terrificus
(Gutirrez y Lomonte, 2003).

(e) Miotoxinas PLA2no neurotxicas

Las PLA2s de grupo II miotxicas encontradas en el veneno de serpientes de la familia
Viperidae se pueden clasificar en dos subgrupos principales: unas con un residuo de aspartato
en la posicin 49 (Asp49) y otras en las que este residuo ha sido siistitudo por una lisina
(Lys49) (Maraganore et al., 1984; Yoshizumi et al., 1990; Liu et al., 1990; Francis et al.,
1991). Sin embargo, algunas toxinas no pueden ser clasificadas en ninguno de estos subgrupos
como la amrnodytina L y la ecarpholina S, dos PLA2s que presentan serina en la posicin 49
(Ser49), aisladas de los venenos de Vipera ammodytes (Krizaj et al., 1991) y de Echis
carinatus sochureki (Polgr et al., 1996), respectivamente; y una PLA2 cuya posicin 49 es
ocupada por glutamina (Gln49), del veneno de Agkzstrodon blomhoffii ussurensis (Bao et al.,
2005).
Las miotoxinas no neurotxicas con estructura de PLA2 son componentes muy
abundantes en los venenos de algunas especies de serpientes de la familia Viperidae, que
juegan un papel preponderante en el dao muscular inducido por sus venenos (Gutirrez y
Lomonte, 1997). La caracterizacin bioqumica de estas miotoxinas ha demostrado que todas
ellas presentan gran similitud en trminos de masa molecular, puntos isoelctricos y
composicin de aminocidos (Mebs y Samejima, 1986; Lomonte y Gutirrez, 1989). Sin
embargo, se han encontrado considerables variaciones inter- e intraespecficas de su estructura
primaria, que podrian dar claves relevantes para delinear los determinantes estructurales de su
toxicidad (Selistre et al., 1996a; Ward et al., 1998).

1.5 Fosfolipasas A2 secretadas

Las PLA2s (EC 3.1.1.4) catalizan la hidrlisis de los glicerofosfolpidos en la posicin

sn-2, liberando cidos grasos y lisofosfolpidos (Kudo y Murakami, 2002). Aunque todas las
PLAzs catalizan esencialmente la misma reaccin, sus actividades biolgicas pueden variar
notablemente. Por tal razn, el estudio de las relaciones estructura-funcin en las PLA2s
constituye actualmente un reto desde el punto de vista cientfico, abordado por numerosos
grupos de investigacin.
Las PLA2s extracelulares (o secretadas) son abundantes en las secreciones pancreticas
de mamferos y en los venenos de reptiles y artrpodos, donde han adquirido una variedad de
funciones txicas a lo largo de la evolucin. Adems de una accin digestiva, estas enzimas
muestran una serie de actividades biolgicas que incluyen neurotoxicidad (Chang et al., 1977;
Bon et al., 1979), miotoxicidad (Mebs y Samejima, 1986; Gutirrez y Lomonte, 19951,
estimulacin o inhibicin de la agregacin plaquetaria (Gerrad et al., 1993; Yuan et al., 1993),
acciones hernolitica, anticoagulante, hipotensora, cardiotxica (Fletcher et al., 198l),
edernatigena (Lloret y Moreno, 1993; Ogawa et al., 1995; Kini, 1997) y bactericida (Pramo et
al., 1998).
Las PLA2s se han clasificado en 11 grupos, con base en sus caractersticas estructurales
y su patrn de uniones disulfuro (Six y Dennis, 2000). Las PLAzs de venenos de serpientes de
la familia Viperidae corresponden al grupo 11 de esta clasificacin. Estas protenas poseen
masas moleculares de alrededor de 14 kDa, con 120-124 residuos de arninocidos, y un patrn
caracterstico de 7 enlaces disulfuro conservados (Ami y Ward, 1996). Algunas PLAzs de
venenos pueden presentarse como monmeros, aunque frecuentemente pueden encontrarse en
estados oligomricos, por lo general como dmeros (Magro et al., 2003).
La actividad cataltica de las PLA2s secretadas es dependiente de la unin al calcio, el
cual sirve como un cofactor. Los estudios espectroscpicos y cristalogrficos han contribudo
significativamente a elucidar el mecanismo cataltico de las PLA2s y se ha demostrado que el
cido asprtico en la posicin 49 (Asp49) es altamente conservado, jugando un papel cnicial
en la estabilizacin del estado intermedio de transicin tetrahdrico en las PLAzs
cataliticarnente activas (Scott et al., 1990a, 1990b, 1992; Scott y Sigler, 1994), en el cual el P
carboxilato del Asp49 interacciona con los oxgenos carbonilo de los residuos Tyr28, Gly30 y
Gly32, 2 molculas de agua y el calcio. Adems, en el mecanismo de accin la His48 y el
Asp99 participan en la catlisis ya que la His48 extrae un protn de la molcula de agua, por
lo que la enzima adquiere carga positiva, o sea que realiza el ataque nucleofilico por lo que
dicha carga se estabiliza con uniones de hidrgeno con el Asp99.

Figura 1: Mecanismo cataltico de las fosfolipasas A2 (Scott et al., 1990a)

 En un subgrupo de PLAzs originalmente descubierto por Maraganore et al. (1984), se
encontr que el Asp49 es sustitudo por lisina. Los anlisis de la estructura cristalina de estas
variantes de PLA2s tipo Lys49 han demostrado que el grupo &-amino de la lisina est
localizado en la posicin que normalmente ocupa el calcio en las PLA2s Asp49 (Holland et al.,
1990; Scott et al., 1992), eliminndose la accin cataltica de las mismas, sin que estas pierdan
sus actividades biolgicas (Rufini et al., 1992; Daz et al., 1991, 1992; Gutirrez y Lomonte,
1997; Lomonte et al., 2003a). El Cuadro 1 presenta una lista de las PLA2s tipo Lys49 descritas
en venenos de serpientes.
Las PLA2s de grupo II presentan una alta similitud estructiiral, tanto en sus secuencias
de aminocidos, como en su estructura tridimensional resuelta mediante difraccin de rayos X
(Wery et al., 1991), an cuando las fuentes de obtencin de estas protenas sean muy
diferentes. Ms recientemente, los avances en las tcnicas de resonancia magntica nuclear
(NMR) han abierto una alternativa como mtodo complementario para determinar la
estructura tridimensional de las PLA2s en solucin.
Muchas de estas miotoxinas estn presentes en solucin como dmeros, por ejemplo las
miotoxinas II y III de Bothrops asper (Gutirrez y Lomonte, 1997), las miotoxinas 1 y 11 de
Atropoides rtummifer (Gutikrrez et al., 1986b; Angulo et al., 2000), de B. insularis (Selistre et
al., 1990), aunque algunas otras se han encontrado en forma nionomrica, tanto en solucin
como en forma cristalina (Ami et al., 1999). Por otra parte estas protenas son muy bsicas,
por lo que se ha visto que ciertos polianiones como la heparina pueden inhibir su miotoxicidad
y su accin citoltica (Melo et al., 1993; Lomonte et al., 1994a, 1994b).
Las miotoxinas de tipo Lys49 carecen de actividad cataltica, o a lo sumo esta es
extremadamente baja. Al respecto, ha existido una controversia de si la baja actividad
cataltica es debida a una leve contaminacin con PLAzs Asp49 (Krizaj et al., 1991, Polgar et
al., 1996, van den Berg et al., 1988, Scott et al., 1992), o por el contrario, si es una actividad
intrnseca de las PLA2 Lys49 (Liu et al., 1990, Shimohigashi et al., 1995). Las evidencias ms
recientes, basadas en el anlisis de miotoxinas recombinantes, apoyan fuertemente la primera
posibilidad (Ward et al., 2002). El hallazgo de cidos grasos en algunas protenas Lys49 qiie
se han cristalizado, sugiere que estas podran tener actividad cataltica, pero que la misma se
ve interrumpida por la imposibilidad de liberar el producto, abortando el ciclo catalitico (Lee
et al., 2001). Por tanto, en la prctica, dichas protenas seran catalticamente inactivas.
 Cuadro 1 :Fosfolipasas A2tipo Lys49 aisladas de venenos de serpiente.

Serpiente * Protena Cdigo ' Referencias

Agkistrodon p piscivorus AppK49 PO436 1 Maraganore et al. (1984)
Agkistrodon bilin entus PLArII * ;k Nikai et al. (1994)
A. contortrix laticinctus rniotoxina ACL P49121 Johnson y Ownby (1993); Selistre et al. (1996b)
Atropoides irumrnifer miotoxina 1 ** Gutirrez et al. (1 986b)
A tropoides rr ummqer miotoxina Ih ** Rojas et al. (2001)
A tropoides numrnger rniotoxina II P82950 Angulo et al. (2000); Angulo et al. (2002)
Bothrops nsper rniotoxiiia II P24605 Lomonte y Gutirrez (1989); Fraiicis et al (1 99 1)
Bothrops crsper miotoxina IV ** Daz et al. (1995)
Bothrops asper rniotoxina IVa # Lizano et al. (2001)
Bothrops atrox Ba-K49 ** Maraganore et al (1984)
Bothrops atrox BaPLA2-1 ** Kanashiro et al. (2002)
Bothrops atrox rniotoxina 1 AY43 102 Nuez et al. (2004)
Bothrops jnrarncussu bothropstoxina 1 490249 Homsi-Brandebuxgo et al. (1 988)
Bothrops moojeni rniotoxina I P82114 Lomonte et al. (1990); Soares et al (2000b)
Bothrops moojeni miotoxina 11 491834 Lomonte et al. (1990b); Soa~eset al (3998)
Bothrops tzezrwiedi miotoxina E ** Geoghegan et al (1999)
B. neuwiedi pauloerlsis BnSP-7 Q9IAT9 Rodrigues et al. (1998); Soares et al. (2000a)
Bothrops pirajai piratoxina 1 P58399 Toyama et al. (1 995, 1998)
Bothrops pirajai piratoxina II P82287 Toyarna et al. (1995,2000)
Bothrops pradoi P M -1 ** Moura-da-Silva et al. (199 1a)
Bo thriechis schlegelii miotoxina 1 P80963 Angulo et al. (1997), Tsai et al (2001)
Calloselasma rhodostomn CRV-K49 Q9PVF3 Tsai et al. (2000)
Cerrophidion godnzani miotoxiria 11 P8 1165 Diaz et al. (1 992); de Sousa et al (1 998)
Cerrophidion godmnni PgoK49 Q8UV7 Tsai et al. (2001)
Crotalus ntl-ox Cax-K49 Q8UVZ7 Tsai et al (2001)
Ci-otalus m nzolossus C m - K 4 9 ** Tsai et al (2001)
Deinagkistroclon acutus Dac-K49 057385 Wang et al. (19961, Fan et al. (1 999)
Deinngkistrodon nctrtus Dac-K49b # Tsai et al. (2001)
T~.imerestnalsalbolabris Tal-K49 ** Tsai et al. (2001)
Trimeresurus jlavoviridis BP-1 P20381 Yoshizumietal.(l990)
Trimeresurus flavoviridis BP-II E48188 Liuetal.(1990)
Trirnereszlnu graminerns PLA2-V P70090 Nakai et al (1995)
Trimeresurtrsgrantinezls PLA2-VI1 P70089 Nakashima et al. (1995)
T. nzucrosquanzatus TMV-K49 P22640 Wang et al. (1996); Liu et al. (1991)
Trinzeresu~wsokinrrverlsis To3 492152 Nobuhisa et al. (1996)
Trimeresurus pun iceus Tpu-K49 ** Tsai et al. (2001)
Cdigos de identificacin en las base de datos SwissProt o GeneBank.
** Secuencias de aminocidos parciales.
# Secuencias no enviadas a bases de datos.
 La protenas Lys49 poseen una accin miotxica que se compara cuantitativamente y
cualitativamente con su contraparte, las PLAzs Asp49, que poseen dicha actividad cataltica
(Gutirrez y Lomonte, 1997). Adems, las miotoxinas Lys49 pueden lesionar rpidamente las
membranas biolgicas y artificiales por mecanismos independientes de calcio (Gutirrez et al.,
1986a; Daz et al., 1991; Rufini et al., 1992; Lomonte et al., 1999b; Ward et al., 1998, 2002;
Ambrosio et al., 2005). Es importante sealar que la regin hidrofbica cercana al C-terminal
de estas miotoxinas, rica en aminocidos hidrofbicos y bsicos, ha sido considerada como
relevante para su accin de dao a membranas (Lornonte et al., 1994a; Gutirrez y Lomonte,
1995; da Silva Giotto et al., 1998; Ward et al., 2002; Lomonte et al., 2003b). Sin embargo,
algunas observaciones sugieren adems una posible participacin de la regin N-terminal
(Dijkstra et al., 1981; Diaz et al., 1991).
Considerando que las miotoxinas Lys49 se han encontrado en los venenos de muchas
especies de vipridos (Homsi-Brandenburgo et al., 1988; Gutirrez y Lomonte 1995; Soares et
al., 1998; Ownby et al., 1999; Chijiwa et al., 2000; Tsai et al., 2001), las mismas constituyen
un modelo interesante para estudiar el mecanismo de dao muscular catalticamente
independiente, y establecer los determinantes moleculares de su toxicidad.
Se ha demostrado que la heparina se une al sitio C-terminal de la miotoxina 11 de
Bothrops asper, neutralizando de esta manera las acciones txicas de la protena (Lomonte et
al., 1994b). Posteriormente se demostr que el pptido sinttico correspondiente a la regin
115-129 de la PLA;! Lys49 de A. p. piscivorus, reproduce todas las actividades txicas de dicha
miotoxina, tanto in vitro como N1 vivo, induciendo mionecrosis (Nez et al., 2001).
Reforzando esos hallazgos, estudios recientes de mutagnesis dirigida de la PLAz Lys49
bothropstoxina 1de Bothrops jararacussu, demostraron la relevancia funcional de los residuos
comprendidos en la regin C-terminal (Ward et al., 2002; Chioato et al., 2002). Adems, los
trabajos usando pptidos sintticos han demostrado que la actividad citotxica de las PLA2
Lys49 depende de este sitio efector catinico/hidrofbico localizado en la regin C-terminal y
que comprende los residuos 115-129 (Lomonte et al., 1994b; Caldern y Lomonte, 1998,
1999; Lomonte et al., 2003b; Nez et al., 2001).
Debido a la observacin de que dos diferentes pptidos sintticos correspondientes a la
secuencia 115-129 de dos miotoxinas Lys49, son capaces de reproducir las acciones txicas
directas de las miotoxinas de la cual son obtenidos (Lomonte et al., 1994b; Nez et al., 2001)
se propuso como un objetivo de este estudio, investigar si este hallazgo representa una regla
general en las protenas de la familia PLA2. Para esto, se seleccion un grupo de secuencias
115-129 de varias rniotoxinas Lys49, y se evalu las posibles actividades txicas de sus
pptidos sintticos correspondientes, Nt vitro e Nt vivo.
La patognesis del dao muscular inducido por miotoxinas del gnero Bothrops ha sido
estudiada por Gutirrez y colaboradores (1 984a, 1984b, 1986a, 1989). Estas toxinas se unen
inicialmente a la membrana plasmtica muscular (sarcolema) y, por un mecanismo an no
conocido en detalle, inducen un dao rpido a dicha membrana, causando un influjo de calcio
y otros iones, seguido por una hipercontraccin de miofibrillas y liberacin de los
componentes celulares (Mebs y Ownby, 1990; Ownby, 1990). Las primeras alteraciones
histolgicas se evidencian como lesiones en la periferia de las clulas musculares descritas
como "lesiones delta", las cuales se han observado tambin en otras patologas musculares
(Mokri y Engel, 1975).
Muchos detalles del mecanismo de accin de las miotoxinas de veneno de serpiente
son an desconocidos. Existen estudios recientes que demuestran la existencia de receptores
de muy alta afinidad para PLA2s en una variedad de tejidos, que reconocen tanto a las PLAzs
de algunos venenos de serpientes, como a las PLA2s de mamferos (Lambeau et al., 1989,
1990, 1994, 1995; Krizaj et al., 1991). Sin embargo, an no se tiene evidencia de que dichos
receptores participen en el mecanismo de accin de las PLAz miotxicas de grupo 11.
Existe evidencia que indica que las PLAzs miotxicas actan primeramente en el
sarcolema, alterando rpidamente la permeabilidad de la membrana, ya sea por un mecanismo
cataliticamente dependiente o bien por uno independiente. Sin embargo, an se desconoce la
naturaleza del sitio aceptor de la membrana involucrado en este mecanismo, y los detalles de
los eventos moleculares que siguen a la unin de la toxina. Los estudios realizados con una
variedad de lneas celulares en cultivo han mostrado que las PLA2s del grupo I miotxicas no
son citolticas, mientras que las del gmpo 11 presentan una fuerte actividad citolitica, que
conelaciona con su actividad miotxica in vivo (Lomonte et al., 1999b). Algunas de las
miotoxinas del grupo 11 estudiadas en modelos de cultivo celular dentro del subgmpo de
PLAzs Lys49 producen una actividad citoltica similar a la producida por las Asp49,
demostrando que este efecto puede desarrollarse en la ausencia de una actividad enzimtica
intrinseca de las miotoxinas (Lomonte et al., 1999b).
 En estudios previos se ha observado que los miotubos diferenciados in vitro son
blancos ms susceptibles a la actividad citoltica de algunas miotoxinas PLA2 del grupo II
(Bmss et al., 1993; Bieber et al., 1994; Incerpi et al., 1995; Lomonte et al., 1999b). Esta
observacin, surnada a las variaciones de la acci6n citotxica observadas entre diferentes
lneas celulares, nos lleva a la pregunta: json los miotubos diferenciados ms susceptibles que
los mioblastos a la accin de estas miotoxinas, o son los rniotubos intrnsicamente ms
susceptibles a cualquier tipo de perturbacin de la membrana? Este interrogante se plante
tambin como un objetivo del presente estudio.

1.6 Relacin estructura-funcin en PLAzs de grupo 11 miotxicas

Durante los iiltimos aos, se han aislado un nmero importante de miotoxinas de la

familia de las PLAzs de grupo TI, y su estudio ha permitido progresar en el conocimiento de su
estructura y mecanismo de accin (Gutinez y Lomonte 1997; Fletcher et al., 1997). La
caracterizacin bioquirnica de las miotoxinas ha demostrado que estas poseen variaciones
inter- e intra-especficas en la estructura primaria, que podran sealar los determinantes
moleculares de su toxicidad, observables mediante alineamientos mltiples de secuencias de
aniinocidos (Selistre et al., 1996a; Ward et al., 1998). Ward y colaboradores (1998), usando
un anlisis de secuencia espacial, identificaron 9 residuos altamente conservados en las PLAzs
Lys49, que estn agrupados en el sitio activo, en el canal hidrofbico de unin al sustrato, y en
las regiones de la interfase dimrica, respectivamente.
La estructura de las PLAl de grupo II se caracteriza por poseer una pequea hlice alfa
N-terminal, y dos hlices alfa largas antiparalelas (hlices 2 y 3, residuos 37-54 y 90-109),
conectados por una regin donde se encuentra la denominada ala beta. Los aminocidos
His48, Asp49, Asp99 y Tyr52 constituyen el sitio catalitico y estn localizados en esas dos
hlices largas. Por otro lado, el sitio de unin al calcio est constituido por dos tomos de
oxigeno del carboxilo del Asp49 (residuos 25-33) y el asa de unin al calcio con los
aminocidos Tyr28, Gly30 y Gly32 y el enlace disulfuro Cys27-Cys44 que produce una
orientacin relativa correcta del sitio de unin al calcio en relacin con los aminocidos que
forman el sitio catalitico. Adems, estas PLA2s contienen una regin C-terminal (Fig. 2) que
comprende los alninocidos 1 15-129, con alta densidad de cargas positivas (Arni y Ward,
 Figura 2: Representacidn esquem4tica de la estructura tndimensional
de las fosfolipasas Ap de grupo II (Magro et al., 2003).

IA evolucin & las P h s ha sido estudiada por Davidson y Dennis (1990), quienes
elaboraron Arboles filogen6ticos derivados de sus secuencias de aminocidos. Este &ido ha
sido titi1 para clasificarlas en los grupos 1 y 11. Por otro lado, Ogawa y colaboradores (1995)
elaboraron rboles evolutivos construidos con secuencias de ADNc que codifican para el
grupo iI de PLA2. Tanto ias secuencias de aminocidos como & ADNc, e incluso de ADN
gen6mic0, de ms de 180 PLA2 se encuentran disponibles y esta informaci6n ha sido usada en
estudios de comparacidn de secuencias, para predecir los residuos de amindcidos
involucrados en funcioncilidades especficas de estas protenas (Kini e Iwmaga, 1986a, 1986b;
Kini y Evans, 1987; Tsai y Chang, 1987; Ward et d.,1998; Selistre de Araujo et al., 1996a,
1996b).
Por otro lado, el uso de pptidos sintticos ha contribudo a la comprensin de la
klaci6n estrucnira-funcin. Los estudios realiados con el pCptido sintetic0
KKYRYLKPLCKK,correspondiente a la regibn C-terminai de la rniotoxina II de B.usper,
mostraron que reproduce varias actividades de dicha toxina, como la citotoxicidad para Aulas
endotelides y musculares en cultivo (Lomonte et al,, 1 994% m 3 b ) , =tividd b a c h c i h
(Pramo et al., 1998), y la actividad edematgena (Nuez et al., 2001), y constituye adems un
epitopo neutralizante de la actividad miotxica (Caldern y Lornonte, 1998, 1999). La triple
sustitucin de Tyr-+Trp en este pptido aument su actividad de dao a membranas y
reprodujo los efectos miotxicos in vivo (Lomonte et al., 1999a). Otros estudios con pptidos
sintticos de esta misma regin de la PLAz Lys49 de Agkistradon piscivorus han mostrado
hallazgos similares. Sin embargo, los pptidos correspondientes de dos PLA2s Asp49, no
produjeron ningn efecto txico in vitro o in vivo, sugiriendo que las miotoxinas Lys49 y las
Asp49 del gnipo 11, podrian presentar diferentes mecanismos de accin miotxica (Nfiez et
al., 2001).
Otros estudios relevantes para la comprensin del mecanismo estructura-funcin han
sido realizados por Ward et al. (2002), utilizando tcnicas de mutagnesis dirigida en la
bothropstoxina I de Bothrops jararacussu. En estos se determin que el mecanismo miotxico
de la miotoxinas Lys49 es independiente del mecanismo cataltico. Se demostr que al
cambiar la Lys49 por Asp49 no se logr recuperar la actividad cataltica, por lo que no solo
ese aminocido es el responsable de la prdida de dicha actividad. Adems, la mutacin del
residuo Lys 122 por Ala1 22, result en una disminucin de la actividad miotxica, sealando
as la importancia de la Lys122 en las actividades txicas de la protena.
Las PLA2s Lys49, an careciendo de actividad catalitica, inducen una conspicua
mionecrosis i~zvivo, as como una serie de efectos in vitvo como la citolisis, la disrupcibn de
liposomas, el bloqueo neuromuscular y la accin bactericida (Lomonte et al, 2003a). Dado que
sus efectos no pueden ser atribuidos a la hidrlisis de fosfolpidos, numerosas investigaciones
se han enfocado en elucidar la base molecular de su toxicidad. Las evidencias basadas en los
estudios con pptidos sintticos, el mapeo de sitios de interaccin con molculas
neutralizantes, y la mutagnesis dirigida (Lomonte et al., 2003b; Chioato y Ward, 2003)
identifican la regin C-terminal de las PLA2s Lys49 como esencial para sus actividades
miotxica, citolitica, bactericida y de dismpcin de liposomas. La fuerte evidencia para un
papel efector de esta regin deriva de las siguientes observaciones: (a) algunos pptidos
sintticos de 13 residuos, que representan la secuencia 115-129 de miotoxinas Lys49, son
capaces de reproducir, aunque con menor potencia, las acciones txicas de las protenas de que
son derivados; (b) los mutantes especficos C-terminales de una PLA2 Lys49 tienen una
toxicidad reducida ; y (c) la regin C-terminal constituye un sitio de unin a heparina y esta
unin ulhibe la citotoxicidad y la miotoxicidad de alguna de estas miotoxinas (Lomonte et d.,
1994a, 1994b).
Los estudios estructu~escon la bothropstoxina 1, un hornodimero PLAzs Lys49 del
veneno de B. jaramcussu, han demostrado que esta protena presenta dos conformaciones, la
fonna "abierta"y la forma "cerrada".Estas difieren en el ngulo formado por los mon6mem.
Basndose en esto, se propuso que la interfase del dimero podra funcionar como una bisagra,
permitiendo un desplazamiento relativo & la regidn C-terminal que podria contribuir a ia
desorganizacin de la bicapa de fosfoipidos (da Silva Giotto et al., 1998). Aniisis
subsiguientes examinaron el efecto de pH en el equilibrio mon6mero-dmero de Ia
bothropstoxina 1, demostrando que el estado didrico de la protena es esencial para su
capacidad de romper Liposornas, y que la toxina p e d a completamente esta actividad a pH 5.0,
concomitantemente con su disociaci6n a mon6meros (de Oliveira et d.,200 1).

Figura 3 Representacidn esquemhtica de la estructura dimerica de la

miotoxina II de B. asper. La zona oscura representa la regi6n C-terminal
(175-129) en cada subunidad. La figura fue generada con el programa
RasMol, con las coordenadas del Protein Data Bank, nmem de acceso
1CLP.

Estudios mientes rertlizados por Ambrosio et d.(2005) con tres estructuras cristdinas
monomricas de la miotoxina PLA2 Lys49 & Agkistrodon confort& laticinctus, revelaron
que la presencia de un ligando (un posible cido graso) en el sitio activo nominal, induce un
cambio conformacional que expone la superficie hidrofdbica de la regi6n C-terminal. Este
"nudillohidmfbico",dado por las cadenas laterales de los residuos Phel2 1 y Phe124, forma
una estructura can6nica entre los residuos 118-125, que parece ser comdn a muchas
miotoxinas Lys49. Con base en esto se ha propuesto un mecanismo de accin que involucra la
retencin del cido graso liberado en el sitio activo, lo que provoca un cambio conformacional
en la regin C-terminal, lo cual establece una conexin estructural directa entre el sitio activo
de la fosfolipasa y el sitio miotxico C-terminal (Ambrosio et al., 2005).
En resumen, se han realizado algunos avances significativos en la identificacin de las
regiones moleculares de las PLAzs de grupo II que determinan sus actividades txicas. Sin
embargo, la comprensin detallada de las actividades requiere de mayores estudios
estructurales que permitan proponer un mecanismo de accin ms completo. Un mejor
conocimiento sobre la relacin estructura-funcin de estas toxinas es de suma importancia
para abordar la bsqueda de inhibidores eficientes, sobre una base mas racional.

1.7 Xlihibidores de PkAts de grupo I[I

La relevancia clnica de la mionecrosis en los envenenamientos por serpientes ha

motivado a buscar molculas neutralizantes (no inmunes) contra las PLA2s miotoxicas,
incluyendo inhibidores naturales de origen animal y vegetal (Nobuhisa et al., 1998; Lizano et
al., 1997, 2000, 2003; Rocha et al., 2002; Deepa y Gowda, 2002; Arruda et al., 2002), que
podran llegar a complementar la terapia convencional con antivenenos.

puma
1
a
1-3Fkaer
4
t
so,

Figura 4: Estructura promedio del fucoidn (izquierda), un polisacrido sulfatado extrado

del alga Fucus vesiculosus (derecha).

Algunos glicosarninoglicanos sulfatados como la heparina y el heparn sulfato, inhiben

la actividad citolitica y miotxica de las PLA2s de Bothrops asper y B. jararacussu (Melo et
al., 1993; Lomonte et al., 1994a, 1994b). Adems, se han descrito otros inhibidores de PLA2s
como la 4-alkoxibenzamidina (Aitdafoun et al., 1996). De acuerdo con esto, se decidi
investigar si el fucoidan, un polisacrido su1fatado complejo extrado del alga Fucus
vesicirlosus, podria inhibir las actividades txicas de un grupo de PLA2s rniotxicas de
venenos de vipridos. La estructura del fucoidan se ha caracterizado en detalle (Fig.4). La
regin central del fucan est compuesta primariamente de un polmero de fucosas unidas por
enlaces a 1-3, con grupos sulfato en la posicin 4 del residuo de fucosa. La fucosa tambin se
une a este polmero para formar puntos de ramificacin, uno de cada dos a tres residuos de
fucosas entre la cadena (Patankar et al., 1993; Nishino et al., 1994). Este polisacrido induce
una variedad de efectos biolgicos en mamferos incluyendo la inhibicin del reclutamiento de
leiicocitos (Preobrazhenskaya et al., 1997), revascularizacin del tejido isqumico (Liiyt et al,
2003), agregacin plaquetaria (Durig, 1997), contraccin del colgeno (Fujimura et al., 2000),
e inhibicin de la proliferacin de clulas de msculo liso (Religa et al., 2000). El fucoidan
interacta con algunos componentes de los sistemas de la coagulacin y fibrinolisis, como con
el cofactor 11 de heparina, la antitrombina 111, trombina, plasmingeno glutmico, activador de
plasmingeno de tejido y urokinasa de bajo peso inolecular (Church et al, 1989; Minix y
Doctor, 1997), y posee actividades antimicrobianas (Zapopozhets et al, 1995).
La resistencia de algunos animales a los venenos de serpiente, ya sean estos las
serpientes mismas o bien mamferos, se ha explicado por la presencia de factores proteicos
neutralizantes en su sangre, que inhiben importantes componentes txicos del veneno (Ohkura
et al., 1997; Prez y Snchez, 1999; Faure, 2000). Dentro de estos factores se encuentran los
inhibidores de rnetaloproteinasas (factores antihemorrgicos) y los inhibidores de fosfolipasas
(PLIs) (Lizano et al., 1997,2000,2003; Perales y Domont, 2002; Rocha et al., 2002).
Basndose en la comparacin de secuencias de aminocidos, los PLIs plasmticos se
han clasificado en tres grupos: a, P, y y. Los inhibidores a contienen dominios de
reconocimiento de carbohidratos (CRDs) similares a las lectinas dependientes de cat2 (tipo-
C), as como los receptores de PLA2tipo M (miisculo) que unen a las PLAzs de gmpo 1 como
las pancreticas, y de grupo 11. El gmpo de PLIs P contiene repeticiones ricas en leiicina y
poseen un 33% de identidad en la secuencia con las glicoprotenas a ricas en leucina del suero
humano. Finalmente, los inhibidores y poseen un patrn de residuos de cistena, formando las
agrupaciones de "tres dedos" caractersticas del receptor activador de plasmingeno urokinasa
(u-PAR) y de la superfamilia de proteinas Lys (Ohkiira et al., 1997). El primer PLI
caracterizado fue aislado del plasma sanguneo de Bothrops asper (Lizaiio et al., 1997) y
denominado BaMIP, una protena oligomrica acdica de 120 kDa compuesta por 5
subunidades de 23-25 kDa. La secuencia de esta protena es similar a la de algunos inhibidores
tipo a. El BaMIP inhibe las actividades miotxicas, edematognicas y citolticas de las cuatro
miotoxinas del veneno de B. asper. Adems, se han aislado, caracterizado y clonado dos
inhibidores del plasma de Cerrophidion godmani (Lizano et al., 2000), el CgMIP-I y el
CgMIP-11, ambos glicoprotenas con masas moleculares de 110 kDa y 180 kDa
respectivamente. El CgMIP-I especficamente neutraliza las actividades de la miotoxina I de
C. godmani (una PLAz cataliticamente activa), mientras que el CgMIP-11 selectivamente
inhibe las propiedades txicas de la rniotoxina 11 de C.godmnni (una PLA2 Lys49,
enzimticamente inactiva).
Por otro lado, el DM64 se 11a descrito como una protena antirniotxica acdica, aislada
del plasma de Didelphis marsupialis, que posee un 15% de glicosilacin y una masa molecular
de 63.659 Da. Al clonar dicha protena se encontr que la secuencia de aminocidos es
homloga a la proteina DM43, un inhibidor de metaloproteinasas encontrado en el mismo
animal (Rocha et al., 2002). Como parte del presente trabajo, se estudi la capacidad del
DM64 de interaccionar con distintas PLAzs miotxicas y neutralizarlas.
En forma general, esta tesis pretende profundizar sobre la estructura (secuencia,
modelaje y cristalizacin) de una miotoxina PLAz tipo Lys49 (art. 1, II y III), como
herramienta para la comprensin del mecanismo de accin de estas toxinas. Tambin persigue
evaluar y caracterizar a nivel inolecular la interaccin de estas toxinas con diferentes
inolculas que puedan inhibir sus actividades txicas (art. VI1 y datos no publicados), y
caracterizar las actividades de un conjunto de pptidos sintticos correspondientes a su regin
C-terminal (art. IV). Adems, se aborda la filogenia de estas protenas con el propsito de
relacionar su estructura y evolucin dentro de los distintos gneros y familias de serpientes
(art. 11). Tambin persigue evaluar el papel de la dimerizacin de estas protenas en sus
actividades txicas utilizando un modelo de clulas musculares en cultivo (art. V), y el efecto
que posee la diferenciacin de dichas clulas sobre la susceptibilidad al mecanismo de
toxicidad (art. VI).
 2. OBJETIVOS

Objetivo General

Analizar algunos aspectos de las relaciones estructura-funcin y filogenticas de las

miotoxinas tipo fosfolipasa A2 (PLA2) del gmpo 11.

Objetivos Especificas

2.1 Caracterizar la estructura primaria y tridimensional de la miotoxina II de Atropoirles

nummijer como un nuevo miembro de las miotoxinas tipo Lys49.

2.2 Caracterizar la actividad de pptidos sintticos correspondientes a la regin C-terminal

1 15-129 de distintas miotoxinas.

2.3 Analizar el papel del estado dimrico de las miotoxiiias tipo PLA2 en sus actividades
citotxica y rniotxica.

2.4 Caracterizar la actividad citotxica de las PLA2s de grupo II sobre clulas musculares
(C2C12) in vitro, como posible modelo de la funcin miotxica de estas protenas.

2.5 Caracterizar la capacidad de diferentes sustancias (polisacridos sulfatados e inhibidores

plasmticos) para inhibir las actividades biolgicas de las miotoxinas PLAi de gmpo 11.
 3. MATERIALES Y METODOS

3.1 Venenos de serpiente

Se obtuvo el veneno de cada especie de serpiente a partir de al ineiios 15 especinenes
colectados en Costa Rica y mantenidos en el Instituto Clodomiro Picado. Posteriormente a su
extraccin, el veneno fue centrifugado, liofilizado y almacenado A -20" C.

3.2 Aislamiento de miotoxinas tipo PLA2

Estas toxinas se purificaron por crornatografia de intercambio catininico en
carboximetil-Sephadex C-25. Se disolvieron aproximadamente 500 mg de veneno de la
especie respectiva en amortiguador Tris-HC1 0.05 M, KC1 0.1 M, pH 7.0 y se aplicaron a una
columna de CM-Sephadex (30 x 2.5 cm; Pharmacia), equilibrada previamente con la misma
sol~icin.Posteriormente, para la elucin de las protenas bsicas unidas a la columna, se
aplic un gradiente de KCI de 0.1 M a 0.75 M, a una tasa de flujo de 0.4 rnl/min (Lornonte y
Gutirrez, 1989). La absorbancia del eluente a 280 nm se midi en forma continua en un
cromatgrafo Bio-Rad.

3.3 Determinacin de la pureza (homogeneidad) de toxinas

La homogeneidad de las toxinas se determin mediante tres criterios: (a) electroforesis
en gel de poliacrilarnida en presencia de duodecilsulfato de sodio (SDS-PAGE; Laemmli,
1970); (b) electroforesis en gel de poliacrilarnida en presencia de urea, usando un sistema de
amortiguador catinico (UREA-PAGE; Traub et al., 1971); y (c) cromatografia liquida de alto
desempeo en fase reversa (RP-HPLC), usando una columna C4, a un flujo de 1.O ml/min con
un gradiente de O a 60% de acetonitrilo en 0.1% de cido trifluoroactico.

3.4 Determinacin de la masa molecular de la miotoxina 11 de Atropoides nurnrnifer

La determinacin de la masa rnolecular de la miotoxina 11 de A. nurnmifer se realiz
mediante espectrometra de masas (MALDI-TOF) en un instrumento Voyager System 6255,
en una matriz de cido sinapnico ( 3 3 dimetoxi-4 hidroxi-sinapinic acid).
3.5 Determinacin de la estructura primaria de la miotoxina II de A. nzilnmifer
En primera instancia se determin la secuencia N-terminal de la protena en forma
directa, mediante la degradacin de Edman. Posteriormente, la proteina se redujo y
carboximetil, tomando una alcuota de 2 mg de la proteina y disolvindola en una solucin
amortiguadora de Tris-HC1 0.2 M, pH 8.6 con cloruro de guanidina 6 M. Se le aadi una
preparacin de ditiotreitol fresca (200 mM) y se incub por 45 min a 55 "C. Adems, se
alquil la proteina con cido iodoactico para confirmar las posiciones de las cisteinas.
Para determinar la secuencia completa de aminocidos de la protena, se combin la
degradacin de Edman directa (N-terminal) con la de la proteina nativa, reducida y alquilada
en un secuenciador automtico. Adems, se determin la secuencia de los pptidos obtenidos
mediante la digestin de la protena por las siguientes endopeptidasas especficas: arginina-C
de Clostridium histolyticum (digestin en amortiguador Tris-HCl 100 mM, pH 7.6
conteniendo 10 mM de CaClz por 4 hr a 37 "C); proteinasa aspartato-N (digestin en
amortiguador de fosfatos 50 mM, pH 8.0 por 4 hr a 37 "C); lisina-C (digestin en
amortiguador Tris-HCl 50 mM, EDTA 1 M, pH 6.5 por 3 hr a 37 "C); y quimotripsina
(digestin en amortiguador Tris-HCI 100 mM, pH 8.0 por 4 hr a 37 "C). La purificacin de los
pptidos resultantes se realiz en una columna C18 mediante RP-HPLC.

3.6 Modelaje de la estructura tridirnensional de la miotoxina II de A. nuntmifer

Con base en la secuencia completa de aminocidos, se construy un modelo de la
estructura tridirnensional de la miotoxina 11 de A. numrnlfer usando como molde geonitrico
inicial las coordenadas del cristal de la miotoxina 11 de Cerrophidion godmani, obtenidas a
una resolucin de 2.8 A (Cdigo lGOD en el Protein Data Bank (Ami et al., 1999), con la
cual muestra un 90.9% de identidad de secuencia. La secuencia de la toxina fue enviada al
y la estructura resultante
servidor Swiss-Model (http:l/www.expas~.cW~p~lb~/~nai~~page.ltn)
fue visualizada y analizada con Swiss-PDB Viewer v.3.5 1 (Glaxo-Wellcome Experimental
Research) y W ebLabViewer v.4.0 (Molecular Simulations, Inc.).

3.7 Comparacin de secuencias y relaciones filogenticas de La miotoxina 11 de A.

nrrmnr ver
Para comparar la secuencia de aminocidos de la miotoxina 11 de A. numrnifer con
otras PLA2s de grupo 11, se realiz un alineamiento mltiple con los programas FASTA
(Pearson y Lipman, 1988) y CLUSTAL W (Higgins et al., 1996) con ajuste de algunas brechas
("gaps"). Los rboles filogenticos se calcularon usando PROTDIST (Felsenstein, 1995) y el
algoritmo "neighbor-joining" (Saitou y Nei, 1987). El diagrama del rbol fue generado con
DRAWTREE (Felsenstein, 1995).

3.8 Cristalizacin de la miotoxina II de A. nlrmrnifer

La cristalizacin de la miotoxina II de A. nummifer fue realizada por un mtodo de
difusin de vapor en gota suspendida, usando platos de cultivo de 24 hoyos. Las pruebas
iniciales de cristalizacin se realizaron usando la tcnica de tamizaje descrita por Jancarik y
I<in (1991). Posteriormente, las condiciones se refinaron y la forma de cristal iinico se obtuvo
cuando se mezclaron 2 pL de la protena con un volumen igual de una solucin de acetato de
sodio 0.1 M , pH 4.6, PEG 3350 20% y sulfato de amonio 0.2 M. Los datos de difraccin de
rayos X fueron colectados a partir de un nico cristal (dimensin mxima de 0.3 mm) en el
Departamento de Fsica de la USP/SZo Carlos. La radiacin fue generada por un instrumento
Rigaku RU300 de rotacin de nodo que opera a 50 k v y 100 rnA, equipado con espejos
smicos y un detector MAR435 con plato de imagen (MAR Research). Los datos se
colectaron en 78 imgenes usando el mtodo de oscilacin con un rango de 1" por imagen. El
cristal difiact a una resolucin nominal de 2.3 A. Los datos de difraccin de rayos X fueron
procesados e integrados usando Denzo/Scalepack (Otwinowski y Minor, 1997).

3.9 Actividades enzimticas y biolgicas de las PLA2s grupo II

A. Actividad fosfolipasa: muestras de 0.1 m1 con diferentes concentraciones de toxina se

incubaron con 1 m1 de suspensin de yema de huevo diluida 1:5 en una solucin de Tris-
HCI 0.1 M, CaClz 0.01 M, pH 8.5 con 1% de Tritn X-100, por 30 min a 37 "C.
Posteriormente, se extrajeron los cidos grasos liberados por accin de la enzima y se
titularon de acuerdo con e1 mtodo de Dole (1956), utilizando NaOH 0.018 N y azul de
bromotimol como indicador.
B. Citotoxicidad: la actividad citotxica se determin usando una lnea celular de
endotelio murino (tEnd) (Bussolino et al., 1991) o bien una lnea muscular de ratn
(C2C12) (ATCC CRL-1772). Ambos tipos de clulas se cultivaron en medio de Dulbecco
Modificado por Eagle (DMEM) suplementado con 15% de suero fetal bovino (SFB;
Sigma), glutamina 2 mM, acido pirvico 1 mM, penicilina (100 U/ml), estreptomicina (0.1
mglml), y anfotericina B (0.25 pglml), en una atmsfera hmeda con 7% de COI, a 37 "C.
Cuando las clulas estuvieron confluentes en botellas de 25 cmZ,se disgregaron con una
solucin de tripsina (1500 U /ml) con EDTA 5.3 mM, por 5 min a 37 "C. Las clulas
resuspendidas se sembraron en placas de 96 hoyos, a una densidad aproximada de 1-4 X
lo4 clulas/hoyo, en el mismo medio. Despus de 2 a 4 das, tanto las clulas tEnd como
los mioblastos C2C12 fueron utilizados directamente, mientras que los miotubos se
obtuvieron mediante la diferenciacin de mioblastos por 4 das ms, en medio con 1% de
SFB. EI efecto Itico en dichas clulas se estim despus de una inciibacin de 3 hr con las
toxinas, a diferentes concentraciones, en un volumen final de 150 pl/hoyo, y
posteriormente se detemin la actividad de la enzima deshidrogenasa lactica (DHL; EC
1.1.1.27) en el sobrenadante.

C. Mionecrosis: se disolvieron diferentes cantidades de toxinas en 50 pL de una solucin

amortiguadora de fosfatos (PBS; NaCl 0.12 M, fosfato de sodio 0.04 M, pH 7.2). Las
diferentes soluciones de toxinas se inyectaron en grupos de 4 ratones CD-1 (1 8-20 g), en
el msculo gastronemio derecho. Un grupo de ratones fue inyectado con 50 p1 de PBS,
como control negativo. Despus de 3 hr se tomaron muestras de sangre de la cola y se
determin la actividad de la enzima creatina kinasa (CK; E.C. 2.7.3.2). La actividad de
esta enzima se expres como U/ml, en donde una unidad corresponde a la fosforilacin de
1 nmol de creatina por min a 25C.

D. Edema: Para determinar el edema producido por las toxinas se inyect

subcutneamente diferentes concentraciones de estas disueltas en PBS, a grupos de 4
ratones, en la almohadilla plantar. Despus de 1 hr se estim el edema por el aumento en el
grosor de la pata, el cual fue medido con un caliper de baja presin (Lomoiite et al., 1993).
Un grupo de ratones control recibieron 50 p1 de PBS.
 E. Accin antieoagulante: para determinar la accin anticoagulante de la miotoxina II de
A. nur>rniifer,se prepar un plasma de oveja pobre en plaquetas mediante centrifugacin de
sangre citratada a 1000 xg, a 5 "C. Para la prueba se utiliz 0.5 m1 de plasma, el cual se
incub6 con diferentes concentraciones de la toxina en PBS, por 10 min a 37C.
Posteriormente, se aadi 0.1 ml de CaC120.25 M y se anot el tiempo que tarda el plasma
en coagular. Como control de la prueba se incubaron alicuotas de plasma con 0.1 m1 de
PBS y se determin el tiempo de coagulacin de forma idntica. Las pruebas se llevaron a
cabo por triplicado.

3.1 0 Estudios con pptidos sintticos

Para realizar el estudio comparativo de los pptidos correspondientes a la secuencia
115-129 de diferentes miotoxinas PLA2 Lys49, estos se sintetizaron mediante la estrategia F-
moc (Walker, 1994) ya sea manualmente en el Instituto Clodomiro Picado, o automticaniente
por proveedores comerciales (Chiron Inc., o SynPep Inc., EUA). La masa molecular de los
pptidos se estim por espectrometra de masas y su pureza (295%) se determin mediante
RP-HPLC analtica. Los pptidos secos se conservaron a -20" C y se disolvieron en PBS, pH
7.2, inmediatamente antes de ser probados para sus actividades biolgicas, tales coino
citotoxicidad en clulas en cultivo y rnionecrosis en ratones.
Para evaluar la actividad txica de los pptidos Ni vitro, se realizar011 pruebas de
citotoxicidad sobre mioblastos murinos C2C12, como se describi en el punto 3.8-B. Los
pptidos sintticos se disolvieron en PBS y se diluyeroii en medio de cultivo. Las clulas
fueron expuestas a diferentes concentraciones de los pptidos, en un volumen de 150 p1, desde
25 a 150 pghoyo (aproximadamente 580 pM).

3.1 1 Disociacin por pH de los dmeros de PLA2s

A. Entrecruzamiento quimico: se prepararon soluciones de 2 pg/pL de las PLA2s en

PBS, cuyo pH se ajust ya sea a 5.0 o a 7.2. Estas soluciones se incubaron solas, o en
presencia de 2 pg/pL bis-sulfosuccinimidi1 suberato ( B S ~ ;Sigma), por 30 iriin a 25 "C.
Posteriormente, las muestras se calentaron a 95" por 5 min en preseiicia de SDS y P-
 mercaptoetanol, y se analizaron mediante SDS-PAGE (12%), a lOOV por 2 hr. Las
protenas se tieron con azul de Coornassie R-250.

B. Efecto del pH sobre la actividad citotxica in vitro: la actividad citotxica de las

PLAzs se prob a pH 5.0 (DMEM 1% SFB, titulado con cido ctrico 2,O M) o pH 7.2
(DMEM 1% SFB), a diferentes concentraciones de toxina ( 5 , 10, 20 y 40 pg) sobre clulas
C2C12, como se describi en el punto 3.843. Ademas, se prob la accin de dos pptidos
correspondientes a la regin C-terminal (residuos 115-129) de la miotoxina 11 de Bothrops
asper (KKYRYYLKPFCKK) y la PLAz Lys49 de Agkistrohn p. piscivurus
(KKYKAYFKLCKK), a una concentracin de 150 pg/150 p1 en dichas clulas.

C. Efecto del pH sobre la actividad miotxica N1 vivo: la actividad miotxica de las

PLA2s se evalu por la inyeccin de 75 pg/75 pl de cada toxina en PBS a pH 7.2 o pH 5.0,
en el gastronernio, como se describi en el punto 3.9-C.

3.12 Susceptibilidad diferencial entre mioblastos y miotubos de la lnea celular C2C12

Se prob comparativamente la citolisis inducida por vanas miotoxinas tipo PLAzs
Lys49 en clulas C2C12, ya sea como mioblastos no diferenciados o como miotubos. La
diferenciacin se obtuvo con medio de cultivo con SFB al 1%. Las toxinas utilizadas en esta
comparacin fueron: miotoxinas 1, 11, 111 y IV de B. nsper, miotoxina 11 de C. godmani,
miotoxina 1 y 11 de A. nznninfler, miotoxina 1 de B. schlegelii, PLAz del grupo 111, y melitina
sinttica de abeja (Apis rnellifera) (Sima), as como dos pptidos sintticos (Ba-p115-129,
KKYRYYLKPFCKK y Acl-p115-129, KKYKAYFKFKCKK) derivados de la miotoxina 11 de
B. asper y de la miotoxina de Agkistrodon contortrix laticinctus, respectivamente.
Adems, se probaron diferentes agentes lticos inespecficos tales como los detergentes
neutro (Tritn X-1 OO), aninico (duodecil sulfato de sodio) y catinico (cetil-trimetil brornuro
de amonio). Las pruebas de citotoxicidad se realizaron como se describi en el punto 3.9-B,
exponiendo los mioblastos o los iniotubos a diferentes concentraciones de cada agente ltico.
3.13. Efecto inhibitorio del fucoidan

A. Formacin de complejos macrornoleculares: se determin la formacin de con~plejos

macron~olecularesentre el fucoidan y las miotoxinas 1, II, 111 y 1V de B. asper mediante
turbidimetria. Se disolvieron 2 pg de cada toxina en 2 m1 de una solucin de Tris-KCl 0.01
M, pH 7.0, adicionndole seguidamente alicuotas de 1 p1 de fucoidan (20 mg/ml), para
obtener diferentes proporciones molares de fucoidan/miotoxina. Despus de cada adicin,
la mezcla se incub por 1 min antes de leer la absorbancia a 340 nm.

B. Inhibicin de la actividad citotxica NI vitro: las toxinas, ya sea solas o mezcladas

con fucoidan (masa rnolecular 135 kDa), a diferentes razones molares, se incubaron por 30
min a temperatura ambiente. Las pruebas de citotoxicidad se realizaron como se describi
en el punto 3.9-B.

C. Inhibicin de la actividad citotxica de pptidos sintticos: se prob la capacidad

inhibitoria del fucoidan sobre tres pptidos sintticos liticos correspondientes a la regin
C-terminal (secuencia 1 15-129) de la miotoxina 11 de B. asper (KKYRYYLKPFCKK), la
PLAz Lys49 de Agkistrodon piscivorus piscivorus (KKYKAYFKLKCKK)y la miotoxina
de A. contortrix laticinctus (KKYKAYFKFKCKK), respectivamente. Estos pptidos solos
(1 00 pg/150 pl), o mezclados con fucoidan a una razn molar de 0.25 (fucoidanpptido),
se incubaron por 30 tnin a temperatura ambiente y posteriormente se aplicaron a los
cultivos de clulas C2C12 para probar citotoxicidad, como se describi en el punto 3.9-B.

D. Inhibicin de la actividad miot6xica in vivo: la actividad miotxica de las PLA2s

purificadas y su inhibicin fueron probadas en grupos de 4 ratones CD-1 (18-20 g de
peso), despus de la inyeccin intramuscular (en el gastronemio) de 75 pg de cada toxina,
ya sea sola, o preincubada con fucoidan a una razn molar de 1:1, por 30 min a
temperatura ambiente. Los grupos control recibieron una inyeccin idntica de 100 p1 de
PBS solo, o fucoidan solo. La miotoxicidad se cuantific como se describi en el punto
3.9-C.
 E. Inhibicin de la actividad fosfofipasa Af: se prob el efecto inhibitorio del fucoidan
sobre la actividad fosfolipasa de dos miotoxinas tipo Asp49 (miotoxinas I y 111 de B.
ospeu), preincubndolas con el fucoidan a diferentes razones molares, por 30 min a
temperatura ambiente y probando finalmente la actividad fosfolipasa como se describi en
el punto 3.8-A.

3.14 Efecto inhibitorio de1 DM64

A. Purificacin del DM64: el DM64 se purific como se describe en Rocha et al. (2002).
El suero de Dzdelphis marsupialis fue dializado y centrifugado. Posteriormente, el
sobrenadante se fraccion en una columna de DEAE-Sephacel (2.6 x 17 cm) equilibrada
con amortiguador de acetato de sodio 0.01 M, pH 3.7. La elucin se realiz utilizando un
gradiente lineal de NaCl de 0.15 a 0.5 M, a un flujo de 0.5 ml/min. La fraccin del DM64
se precipit con sulfato de amonio a 80% de saturacin, se disolvi en un amortiguador de
fosfato de sodio 0.02 M, pH 7.0 y se dializ. Finalmente, la preparacin se centrifug y el
sobrenadante se fraccion por cromatografia de afinidad en una columna HitrapB NHS-
activada conteniendo miotoxina 11 de B. asper inrnovilizada, de acuerdo con las
instrucciones del fabricante (Phamacia). La fraccin unida se eluy a un flujo de 1 rnl/min
con amortiguador glicina-HC1 0.1 M, pH 2.7, recolectndose sobre Tris 1.0 M para
neutralizar el pH.

B. Formacin de complejos entre el DM64 y las miotoxinas PLAzs grupo 11: la

formacin de complejos entre las miotoxinas y el DM64 se analiz por cromatografia de
afinidad, usando una columna Hitrapo NHS-activada conteniendo 1 mg de DM64
imnobilizado, de acuerdo con las instniccioiies del fabricante (Phamacia). Las miotoxinas
(50 pg1100 yl), disueltas en fosfato de sodio 0.02 M, pH 7.0 se aplicaron a la columna y
posteriormente la elucin se realiz con glicina/HCl 0.1 M pH 2.7, recolectando con
amortiguador Tris 1.0 M para neutralizar el pH, a una tasa de flujo de 1 ml/min en un
sistema FPLC (Phamacia).
C. Formacin de complejos entre el DM64 y hernorraginas: la formacin de complejos
entre la atrolisina C de Crotalt<satrox, o la jararagina de Bothrops jararnca (ambas a 50
pg/100 pl), y el DM64 se analiz por cromatografa de afinidad, usando una columna
HitrapB NHS-activada conteniendo 1 rng de DM64 inniobilizado, como se describi en el
punto anterior.

D. Inhibicin de la actividad fosfolipasa A2: se prob el efecto inhibitorio del DM64

sobre la actividad fosfolipasa de dos miotoxinas Asp49 (miotoxinas 1 y 111 de B. nsper),
como se describi en el punto 3.9-A usando el mtodo de Dole. Se incubaron por 30 min a
temperatura ambiente las toxinas (10 pg) solas o preincubadas con DM64 a diferentes
razones molares. Posteriormente, se determin la actividad enzimatica como se describi
en el punto 3.9-A.

E. Inhibicin de la actividad citotxica in vitro: la actividad citotxica de las PLA2s

purificadas y su inhibicin por DM64 fueron probadas en mioblastos C2C12. Las toxinas
solas, o mezcladas con DM64, se incubaron por 30 min a temperatura ambiente, a
diferentes razones molares. La prueba de citotoxicidad se realiz como se describi eii el
punto 3.9-B.
 4. RESULTADOS

4.1 Aislamiento y caracterizacin de la miotoxina II Atropoides ii~inzmifer

Al fraccionar el veneno crudo de A. nunin~ifev en CM-Sephadex a pH 7.0, se
obtuvieron varias fracciones de componentes bsicos, como se muestra en la Fig. la, art. 1. El
pico 5 coirespondi a la miotoxina I descrita previamente por Gutirrez et al. (1986b) y el pico
4 fue seleccionado para ser posteriormente recromatografiado en las mismas condiciones (Fig.
lb, art. I), y obtener una nueva miotoxina. La miotoxina 11 de A. nummlfer purificada apareci
como una unica banda en el sistema catdico urea-PAGE (Fig. 221, art. 1) y como una banda de
16 D a en SDS-PAGE despes de la reduccin con 2-mercaptoetanol (Fig. 2b, art. 1).
Adems, la toxina eluy como un solo pico en la corrida analtica en el sistema RP-HPLC
(Fig. 3, art. 1). El anlisis mediante espectrometra de masas indic que cada subunidad de la
protena posee una masa molecular de 13,789.90 Da.

4.2 Actividades biolgicas de la miotoxina II de A. rtz~mmifer

La miotoxina 11 de A. numnzfer no present actividad fosfolipasa A2 detectable, en
concentraciones hasta de 50 pglrnl, sobre fosfolpidos de yema de huevo y no mostr actividad
anticoagulante, en concentraciones hasta de 800 p g h l , sobre plasma pobre en plaquetas.
Sin embargo, la toxina indujo mionecrosis dosis-dependiente in vivo al ser inyectada
intramusculamente, como se evidenci por el aumento significativo de la actividad de la
enzima CK en plasma a las 3 hrs (Fig. 5a, art. 1). Adems, esta toxina present un efecto
citolitico importante, tanto en clulas endoteliales (Fig. 5b, art.1) como musculares C2C12
(Fig. 1, art. VII), en cultivo.
Otra actividad biolgica observada para esta toxina fue la capacidad para inducir un
edema significativo en el modelo de la almohadilla plantar del ratn (Fig. 6, art. 1).

4.3 Estructura primaria de La miotoxina 11 de A. nummifev y su comparacin con otras

toxinas
Se present un reporte inicial de la secuencia de aminocidos N-terminal de la
rniotoxina 11 de A. nummifer (Fig. 7, art. 1) con sus primeros 53 residuos. Sin embargo, al
completar y confirmar dicha secuencia, se encontr6 que la posicin 53 es ocupada por una
alanina, en lugar de la asparagina reportada inicialmente. Los primeros 53 residuos del N-
terminal se obtuvieron directamente, mientras que el resto de la secuencia proteica se obtuvo
por el traslape de secuencias de los pptidos generados por protelisis coi1 las enzimas Arg-C,
Asp-N, Lys-C y quimotripsina, como se muestra en la Fig. 1, art. 11. Esta secuencia se
introdujo en la base de datos de Swiss-Prot y se le asign el nmero de acceso P82950.
Al aiializar la secuencia de aminocidos de la miotoxina 11 de A. jztunrnfer, se observ
que esta toxina contiene todos los residuos conservados de las variantes Lys49, iiicluyendo
Leu5, Glnll, Asn28, Arg34, Lys49, Lys53, Thr56, Ser74, Trp77, Lysll5 y Lys128, adems
de los 14 residuos cistena caractersticos de las fosfolipasas secretadas del grupo 11. Se
encontr una sustitucin conservativa en la posicin 108, en donde la mayora de las variantes
Lys49 tienen Glu, mientras que la miotoxina II de A. nunzrnlfer tiene Asp. Otro hallazgo
interesante se presenta en la posicin 86, en donde la inayora de las pmteiias Lys49 tienen un
residuo acdico y esta toxina tiene Lys.
Al comparar la secuencia de aminocidos de esta protena con las variantes Lys49 y
Asp49, se encontr que su identidad vara entre 90.9 % y 58% con respecto a otras variantes
Lys49, mientras que varia eiilre 58% y 35% con respecto a las variantes Asp49 descritas en la
literatura.

4.4 Anlisis filogentico

Un anlisis filogentico inicial de 73 secueiicias de PLA2 de grupo II disponibles en las
bases de datos Swiss-Prot y TrEMBL, revel que las variantes Lys49 constituyen un grupo
monofiltico, teniendo una relacin estrecha con las PLA2s Asp49 grupo 11. La Fig. 2, art. 11,
presenta un alinearnieilto mltiple de secuencias del grupo de variantes Lys49, el cual fue
utilizado para construir uii rbol filogentico que delinea las relaciones de la miotoxina 11 de
Atropoides nummlfer (PA22.ATRNU) con otras proteinas de la familia de PLA2s (Fig. 3, al?.
11). El rbol filogentico mostr que todas las PLA2s Lys49 son divergentes del gmpo de
variantes Asp49, presentes en las serpientes tanto del viejo mundo (subfamilia Viperinae)
coiiio del nuevo mundo (subfamilia Crotalinae). Adems, se observ que las protenas Lys49
foriiian un grupo ms estrechameiite relacionado con las dos variantes Ser49 conocidas
(aminodytina L y ecarpholina S), compartiendo un ancestro comn con estas.
Otro aspecto interesante es que la miotoxina II de A. nur,zrnifer y la miotoxina 11 de C.
godmani estn muy estrechamente relacionadas, y divergen anteriormente a la rama que
contiene las secuencias de las variantes Lys49 presentes en algunas especies de Bothrops
(Fig. 3, art. 11).

4.5 Estructura tridimensional de la miotoxina 11 de A. nummifer: modelaje y

cristalizacin
Al comparar las secuencias de la miotoxina 11 de A. nzcmnzlfer con otras mioioxinas
Lys49, se encontr que el mayor porcentaje de identidad lo present con la miotoxina 11 de C.
godmani (90.9%). Por lo tanto, se utiliz la estructura tridimensional de esta ltima proteina
(acceso lGOD en el PDB) (Ami et al., 1999) para modelar la estructura terciaria del
rnonmero de la miotoxina 11 de A. numrnifer, como se muestra en la Fig. 4, art. 11. Se
observasoii solamente muy pequeas desviaciones en el esqueleto de los tomos de carbono a
en relacin con la toxina de C. godmani, con un valor de rmsd de 0.08 A.
El modelo obtenido predice que la region C-terminal de la protena, que combina
residuos catinicos e hidrofbicos, est altamente expuesta. La estructura tridimensional de la
regin C-terminal, a pesar de ser la regin ms variable en este grupo de protenas, se
superpone exactamente al molde de la miotoxina II de C. godmani, ya que son idnticas en
este segmento.
Posteriormente, la miotoxina 11 de A. nummfer pudo ser cristalizada en el laboratorio
del Dr. R. K. Ami, en Brasil. En la Fig. 1, art. 111, se muestra el patrn de difraccin de rayos
X de esta protena, y la fotomicrografla del cristal que difiacta a una resolucin de 2.3 A.La
estructura tridimensional final de la toxina est actualmente en proceso de resolverse, a partir
de estos datos de difraccin de rayos X.

4.6 Estudios con pptidos sintticos

El pptido sinttico 115-129 de la miotoxina 11 de A. nunzmifer no indujo lisis en las
clulas musculares C2C12 en cultivo (Fig. 1, art. IV), an a dosis tan altas como 150 pg/150
pl/lioyo (1 mg/ml). La observacin microscpica del cultivo confirm que se mantuvo la
morfologa celular normal. Concordantemeiite, la inyeccin intramuscular de 250 pg del
pptido 115-129 de A. nummifer en ratones no produjo aumento de la enzima CK en
comparacin con los animales control, indicando no tener actividad miotxica.
 Estudios previos han demostrado que la actividad citotxica en clulas C2C12 es una
propiedad comn a todas las miotoxinas PLAzs de grupo 11 que han sido analizadas en este
sistema y que la prueba in vitro para las iniotoxinas y sus pptidos es ms sensible que la
prueba de miotoxicidad in vivo en ratn (Lornonte et al., 1999b; Nez et al., 2001). La Fig. 1,
art. IV, muestra que los pptidos correspondientes a la regin 115-129 de las miotoxinas de
Agkistrorlon contortrix laticinctus (Acla), PLAz Lys49 de A. p. piscivorus (AppK), miotoxina
11 de Bothrops aspev (Basp-TI) y la forma alternativa de esta (Basp-IIF), producen una clara
actividad citotxica, demostrada por la liberacin de LDH dosis-dependiente, y por la
observacin directa del dario celular al microscopio. Los pptidos de las dos especies de
Agkistrorlon fueron mas activos que los derivados de B. asper en la prueba.
Por otro lado, los pptidos correspondieiites a la miotoxina II de R. moojelli, (Binoo-
II), bokl~ropstoxiiia 1 de B. jararacussu (Bjsu-1), piratoxina II de B. pir-ajai (Bpir-II),
iiliotoxina II de Atropoides nunzinger (Anu-11), miotoxina 11 de C. godnznni (Cgod-11) y
aminodytina L de Vipera amnznodytes amnzodytes (Vainm), no causaron dafo celular a
concentraciones tan altas como 150 pg/150 plihoyo (1 mg/ml), como lo muestra la Fig. 1, art.
IV. Esto indica que las actividades txicas de las proteinas Lys49 no siempre son reproducidas
por sus coirespondientes pptidos sintticos 115-129.
Adicionalmente a los pptidos correspondientes a las secuencias naturales halladas eii
las iniotoxi~ias,algunos de los pptidos sintticos evaluados fueron seleccionados para dos
propsitos:

(a) determinar si la actividad citotxica de los pptidos puede ser atribuda a efectos
inespecficos de sus numerosos residuos de aminocidos cargados positivainente. Para ello, se
recombin6 al azar la secuencia de uno de los pptidos liticos (AppK), encontrndose que este
pptido no posee capacidad de daar las clulas en cultivo (Fig. 1, art. IV). Lo anterior
demuestra que la secuencia especifica de residuos en el pptido natural AppK, ms que su
simple presencia o frecuencia, es el factor que le coiifiere la capacidad de inducir la rnoette de
clulas en cultivo.

(b) evaluar si los pptidos interactan con alguna estructura dependiente de su configuracin.
Para ello, se sintetiz el pptido AppK con D-amii~ocidosy se prob en clulas en cultivo.
Este enantimero "D" mostr claramente citotoxicidad, con una potencia comparable a la de
su contraparte natural "L". (Fig. 1, art. IV). Por lo tanto, esto sugiere fuertemente que el
mecanismo citotxico del pptido AppK no involucra el reconocimiento de un sitio aceptor
proteico en las clulas de msculo esqueltico C2C12, ya que de ser as su efecto dependera
de la configuracin. Entonces, podramos pensar que posiblemente el mecanismo de accin de
las miotoxinas PLAzs Lys49 tampoco involucra el reconocimiento de un receptor proteico.
La secuencia original de la miotoxina II de B. asper contiene una ~nicroheterogeneidad
en la posicin 124, ocupada por Leu o Phe (Francis et al., 1991), por lo que se probaron dos
pptidos de esta toxina, tanto con Leu124 como con Phe124. En ambos casos se observ
idntica actividad citotxica (Fig. 1, art. IV), lo cual indica que la presencia de cualquiera de
estos dos residuos no modifica la actividad de estos pptidos. Por otro lado, el pptido de
A-num-II/C.god-II se modific sustituyendo el Asp 11G de la secuencia original, por Lys 116.
Sin embargo, an con esta sustitucin, el pkptido mantuvo la ausencia de la actividad
citotxica, indicndo que la introduccin de una mayor carga positiva en el pptido C-tenninal
no modifica su actividad.
Por otro lado, al probar el pptido Basp-11-pEM-2 (basado en la secuencia de la
miotoxina 11 de B. asper) que contiene la triple sustitucin Tyr+Trp (Lornonte et al., 1999a),
adems de las sustituciones Pro-+Ala y Cys+Ala, se encontr que su citotoxicidad es mayor
en comparacin con el pptido Basp-11 original, indicando que la presencia de los Trp aumeiita
su capacidad de causar muerte celular. La sustitucin de Cys en este pptido se hizo para
evaluar si la posible dirnerizacin del pptido es necesaria para la actividad txica. Sin
embargo, este pptido provoc dao celular, demostrando que la dirnerizacin no se requiere
para esta actividad (Fig. 1, art IV).
Posteriormente, se evalu la actividad de los pptidos in vivo inyectando 250 pg/50 pl
intramuscularmente en ratn y estimando la accin rnionecrtica mediante cuantificacin de
los niveles de CK en plasma despus de 3 hr. Se obtuvo que tanto el pptido AppK, como el
Acla, fueron capaces de producir mionecrosis, con un aumento significativo de los niveles de
CK en plasma (Fig.2, art. IV). Este hallazgo demuestra que la regin 115-129 de la PLAz K49
de A. contortrix laticinctus constituye un sitio miotxico, en concordancia con las
observaciones previas con la PLAz Lys49 de A. p. piscivoms (Nez et al., 2001). Ambos
pptidos difieren solamente en la posicin 123 (Leu en AppK y Phe en Acla), mostrando que
esta diferencia en la secuencia no causa un cambio drstico en la actividad txica de los
pptidos. Apoyando los resultados obtenidos in vitro, el pptido con secuencia al azar de
AppK no produjo miotoxicidad en ratn, mientras el pptido enantiomrico AppK-D fue
claramente miotxico (Fig. 2, art. IV). Finalmente, el pptido correspondiente a la regin C-
teminal de la miotoxina II de B. asper, que fue citotxico iu vitro, no indujo miotoxicidad in
vivo (Fig. 2, art. IV).

4.7 Efecto del pH sobre las actividades txicas de PLAzs Lys49 dimricas
Estudios previos demostraron que la miotoxina Lys49 dimrica de B. jnrnracussz~
(bothropstoxina 1) se disocia en monomeros a pH 5.0, concomitantanente con una prdida
completa de sil capacidad para daar bicapas artificiales. La transicin en la estructura
cuatemaria de miotoxinas inducida por pH se apoya en los anlisis eletroforticos despus de
la incubacin con iin acoplante homobifuncional, el bis-sulfosuccinirnidil suberato (BS~).Este
reactivo une los grupos arnino separados hasta por 11.4 A a la forma estable de unin
covalente arnida, en reacciones intra - e intemoleculares. La Fig. 1, art. V, muestra la
estabilizacin covalente de la forma dimrica de la miotoxina II de B. asper causada por el
B S ~a pH 7.2, mientras que a pH 5.0 solamente se observa la forrna rnonornnca. Los
resultados indican que el estado dimrico se favoreci a pH 7.2, y que a pH 5.0 se obtiene una
disociacin a monmeros, como se describi6 originalmente por de Oliveira et al. (2001).
El efecto citolitico de las iniotoxinas PLA2s Lys49 sobre inioblastos C2C12 se redujo
cuando ambos fueron incubados a pH 5.0. (Fig. 2,art. V). La toxina que present la mayor
diferencia en su actividad a los diferentes pH fue la PLA2 Lys49 de A. p. piscivovirs. La
incubacin de cultivos control con medio solo a pH 5.0 no caus dao celular en el perodo de
tiempo utilizado, tanto en la evaluacin por liberacin de DHL, como en la observacin al
microscopio.
Interesantemente, los pptidos correspondientes a la regin C-terminal p-AppK49 de
Agkislvodon p piscivom y p-Ba-mt II de B.aspeu, que se probaron en cuanto a su actividad
citolitica en clulas C2C12, mostraron un efecto citoltico significativamente ms bajo a pH
5.0 que a pH 7.2 (Fig. 3, art. V).
Los experimentos in vivo en ratones mostraron que el dao muscular producido por
miotoxinas preincubadas a pH 5.0 es significativamente menor que el producido por las
mismas preincubadas a pH 7.2. (Fig. 4, art. V).

4.8 Susceptibilidad diferencial entre mioblastos y rniotubos de la luea celular C2C12

Dentro de las condiciones de cultivo descritas, una alta proporcin de mioblastos
C2C12 se fusionaron a miotubos en pocos dias, despus de bajar la concentracin de SFB en
el medio a 1%. La morfologa de las clulas utilizadas, mioblastos y miotubos, se muestra en
la Fig. 1, art. VI. Las curvas dosis-respuesta muestran que el efecto citoltico de las miotoxinas
PLA2s giupo 11 probadas, eii todos los casos, fue significativamente niayor en iniotubos
diferenciados que en mioblastos (Fig. 2, art. VI). Las diferencias en la susceptibilidad a la lisis
fueron ms evidentes a dosis submximas de la miotoxina (5-10 pg por hoyo),
correspondientes a una concentracin de protena de aproximadamente 2-4 pM.
En contraste con las PLA2s del grupo 11 n~iotxicasde serpientes, la PLA2 del veneno
de abeja (grupo III), y la melitina, mostraron actividad citolitica similar en mioblastos y en
iiiiotubos, resultando en curvas de dosis-respuesta superpuestas (Fig. 3, art. VI). Sirnilarmente,
los tres tipos de detergentes probados (neutro, aninico o catinico) causaron el mismo efecto
Mico en ambos tipos de cultivos celulares (Fig. 4, art. VI). Por otro lado, los dos pptidos
sintticos correspondientes a la regin C-terminal de las miotoxinas PLAzs Lys49 de B. asper
y A. c. laticinctus, respectivamente, fueron significativamente ms txicos en miotubos que en
mioblastos (Fig. 5, art. VI), reproduciendo en este sentido el coinportamiento de sus protenas
originales.

4.9 Efecto inhibitorio del fucoidan

Al probar el efecto del fucoidan sobre la actividad citotxica de miotoxiiias PLAL se
encontr inhibicin para todas las miotoxinas probadas, con algunas ligeras variaciones
cuantitativas entre ellas. Las miotoxinas 11 y IV de B. asper y iniotoxinas 1 y 11 de A.
rizlmmfer, miotoxinas I y II de C. godnzani y niiotoxina I de B. scizlegelii se ii~hibieron
coinpletamente por preincubacin con el fucoidan, mientras que la actividad de las miotoxinas
1 y 111 de B.asper se redujo en 50-65% en una relacin molar de 1: 1 (Fig. 1, art. VII). La
capacidad inliibitoria del fucoidan sobre las miotoxinas se evalu mediante la liberacin de
DHL, lo cual fue consistente con las observaciones al microscopio de la morfologa de las
clulas en cultivo. El fucoidan por s solo, a la coiicentracin miixima utilizada en
experimentos de inhibicibn, no alter la morfologa celular, ni caus liberacin de DHL.
La rniotoxina II de B. nsper fue seleccionada para estudiar el curso en el tiempo del
efecto inhibitorio del fucoidan sobre la actividad citotxica iut vitro. Como se muestra en la
Fig. 2, art. VII, la accin del fucoidan fue muy rpida, con una completa inhibicin de la
toxina con una preincubacin de 5 min a temperatura ambiente, antes de aplicar la riiezcla al
cultivo celular.
Los resultados anteriores coinciden con la formacin rpida de complejos
macromoleciilares entre el fucoidan y las miotoxinas 1, 11, III, y 1V de B. nsper observados por
pruebas de turbidimetra. La adicin de ft~coidana esas toxinas en soi~icinprovoc iin
aumento rpido de turbidez a 340 mn, iridicarido la fonnacin de complejos insolubles. Esos
complejos generaron una turbidez mxima a uiia razn molar de aproximadaniente 0.13
(fucoidan/iniotoxina), y posteriormente se redisolvieron por uiia adicin gradual de exceso de
polisacrido (Fig. 3, art. VII).
El posible sitio de interaccin del fucoidan eii las iniotoxinas PLA2 se investig con
una prueba de unin competitiva a una fase slida con iniotoxina 11 inniovilizada, en donde el
caus una reduccin significativa en la unin de antic~ierposde conejo hacia la
f~~coidan
regin C-terminal 115-129 de la miotoxina 11 de B. iisper (Fig. 4, arl. VII). Adems, usando
tres pptidos sintticos citoliticos correspondientes a la regin 115-129 de diferentes PLA2s
iiiiotxicas en pruebas de citotoxicidad, se mostr que el fucoidan iiihibi la accin litica eii
los tres casos (Fig. 5, art. VI1).
Por otro lado, usando dos PLA2s tipo Asp49 de R. nsper (miotoxiiias I y III), se probo
la posible inhibicin de su actividad catalitica por el fucoidaii. Cuando el fucoidan fue
incubado con esas toxinas a razones molares mayores de 2:l (fucoidaii/miotoxina), su
actividad PLA2 fue idntica a la de las toxinas control incubadas sin fucoidan, evidenciando
que su actividad enzirntica no fue iiiliibida.
En otro conjunto de experimentos, se examin la iiihibicin de las miotoxinas PLAz
por fucoidan iil vivo, por estimacin de los niveles de CK plasmtica en ratn. Los datos
resumidos en la Fig. 6, art. VI1 muestran una clara reduccin en la CK plasintica inducida
por todas las toxinas probadas, despus de su incubacin con fucoidan a una razn molar de
1:1. Aunque hay algunas leves diferencias cuantitativas entre las toxinas, las inhibiciones
variaron entre 70 y 95%. Ademis, el fucoidan inhibi la acciii miotxica del veneno crudo de
B. nsper, en los experimentos con preincubacin (Fig. 6, art. VII). La inyeccin del fucoidan
por s solo no produjo aumentos de los valores de CK en plasma, en comparacin con los
animales control que recibieron una inyeccin de PBS solo.
Finalmente, el posible efecto protector del fucoidan fue evaluado en pruebas de
administracin independiente, en las que el polisacrido fue inyectado localmente,
inmediatamente despus de una inyeccin intrarnuscular de veneno de B. asper. cii ratn. La
administracin del fucoidan redujo significativamente la mionecrosis, resiiltando eri valores de
CK que corresponden a aproximadamente 50% del grupo que recibi veneno solo (Fig. 7, art.
VI1). Sin embargo, un incremento en tres veces la dosis de fucoidan administrada i ~ rsitu (de
90 a 270 pg) no mejor el nivel de proteccin.

4.1 O Efecto inhibitorio del DM64

La unin entre el DM64, una protena plasmtica de Didelphis r~inrszipialis,y las
miotoxinas 1, 11, III y IV de B. asper, I y 11 de A. nummifer, 11 de C. godmnrii, piratoxina y
bothropstoxinas I y 11, evaluada mediante cromatografia de afinidad, se muestra en las Figs. 5
Y 6-

0 2 4 6 8 1 0 O 2 4 6 8 1 0 1 2

Fracciones

Figura 5 : Formacin de complejos entre el DM64 y las PLA2s del grupo It miotxicas del
veneno de Bofhrops asper. Los anlisis se realizaron en una columna de afinidad NHS
~ i t r a p "con DM64 inmobilizado. Las isoformas de 8. asper (50 pgJ100~il)miotoxinas I (A),
II (B), 111 (C), y IV (D) fueron aplicadas a la columna, y eludas con amortiguador de glicina-
HCI 0.1 M, pH 3.0.
 O-
0 2 4 6 8 1 0 O 2 4 6 8 1 0 1 2

Fracciones

Figura 6: Formacin de complejos entre el DM64 y las fosfolipasas A, del grupo II

miotxicas. Los anlisis se realizaron en una columna de afinidad NHS Hitrapm con
DM64 inmovilizado. Las miotoxinas I (A) y II (B) de Atropoides nummifer, miotuxina II de
Cerrophidion godmani (C), piratoxina i d e Bothrops pirajai (D), bothropstoxina I de B.
jararacussu (E) y bothropstoxina II d e 8. jararacussu (F) (50 pg/iOO id), fueron aplicadas
a la columna, y eludas con amortiguador de glicina-HCI O 1 M, pH 3.0.

Mediante el mismo mtodo de afinidad, el DM64 no form complejos con dos

hemorraginas, la atrolisina C de Crotalus atrox y la jararagina de Botltrops jarartrcn (Fig. 7),
indicando que esta proteina posee selectividad para las iniotoxinas de estos venenos, pero no
para Las metaloproteinasas hemorrgicas.
 O 2 4 6 8 1 0 O 2 4 6 8 1 0 1 2

Fracciones

Figura 7 : Ausencia de formacin de complejos entre el DM64 y dos hernorraginas de

veneno de serpiente. El anlisis con las hemorraginas (50 pgfI00yl) atrolisina de
Crotalus atrox (A) y jararagina de Bothrops jararaca ( B ) se realiz6 en una columna de
afinidad NHS ~ i t r a p "con DM64 inmovilizado.

El efecto inhibitorio del DM64 sobre la actividad citotxica de las PLAzs miotxicas
de serpieiites de la familia Viperidae, se prob a diferentes proporciones molares
DM64/iriiotoxina. En los mioblastos C2C12 en cultivo, se present variabilidad eii la
capacidad de inhibicin del DM64 con las diferentes miotoxinas probadas. Sin embargo, en
todos los casos se obtuvo una buena inhibicin a tina proporcin molar de 1:l (Fig. 8). Por
otra parte, el DM64 no fue capaz de inhibir la actividad fosfolipasa de dos miotoxinas Asp49
probadas (iniotoxinas 1y 111 de B. aspev).
 o
O 00 O25 O 50 O 75 1 O0 1 25

Razn molar DM64 1 rniotoxina

Figura 8: Inhibicin de la actividad citotxica in vitro de las fosfolipasas A2 del grupo ll

miotxicas por el DM64. La citotoxicidad fue determinada por la fiberacin de LDH en
mioblastos de msculo esqueltico C2C12, 3 hr despus de la exposicin a miotoxinas
(15 pg) solas, o preincubadas con DM64 a las proporciones molares indicadas.
Miotoxinas I (O), II (A), III (O) y IV (e) de B. asper, miotoxina I I de Atropoides nummifer
(A), y miotoxina 11 de Cerrophidion godmani (m). Cada punto representa el promedio I
SO de tres determinaciones.
 5. DISCUSIN GENERAL

En este estudio se purific y caracteriz una nueva miotoxina tipo PLA2 del veneno de
Atropoides numrnifer. La protena se obtuvo de manera homognea, de acuerdo con los
criterios utilizados: urea-PAGE catdico, SDS-PAGE y RP-HPLC. Las electroforesis en gel
de poliacrilamida nos indican que la rniotoxina II de A. iiirmnzifer es un homodinero de
subunidades bsicas. Mediante secuenciacin, se determiii que cada subunidad consta de 121
aminocidos, con una masa molecular de 13,789.90 Da por unidad, detenninada en un
espectrmetro de masas. Esta protena presenta actividad citolitica in vitro, as como como
actividad miatxica e inductora de edema in vivo. Estas actividades biolgicas soii
ciialitativamente y cuantitativamente similares a las descritas para otras PLA2s blsicas de los
venenos de serpiente de la familia Viperidae, que incluyen la capacidad de inducir: (a) una
rpida necrosis de msculo esquelttico en el sitio de la inyeccin, (b) un irunediato aumento
de la permeabilidad de la microvasculatura local y ( c ) un rpido efecto citoltico in vitro.
La secuencia de aminocidos de la miotoxina 11 de A. nirnzmlfer muestra que es una
PLA2 tipo Lys49, con la estructura tpica del grupo IIA de esta familia de protenas. Esta
toxina tiene una alta identidad de secuencia con otras PLA2 Lys49 aisladas de especies de
Cerrophidion, Trirneresurzs, Bothrops y Agkistrorlon . Iiit eresantemente, el rbol fi logentico
constnido con 24 PLAzs de grupo 11 en este estudio, muestra que las variantes Lys49 se
encuentran ms cercanamente relacionadas con las PLA2s Ser49 de vipridos del viejo mundo
de los gneros Vipera y Echis, sugiriendo un ancestro comn que se separ despus de su
divergencia de las PLAzs Asp49. El anlisis detallado de los genes que codifican las PLA2s
Asp49 y Lys49 de las especies asiticas de Trimeresurus demuestra que estas presentan u11
mecanismo de evolucin acelerada (Ogawa et al., 1992; Nakashima et al., 1995; Nobuhisa et
al., 1996). Se desconoce ain el valor adaptativo de una rpida evolucin de las miotoxinas del
grupo IIA Asp49, activas catalticamente, a las miotoxinas Lys49 o Ser49, inactivas
catalticamente. Los estudios con un nmero de miotoxinas bsicas Asp49 y Lys49 aisladas de
varias especies de Bothrops muestran una potencia y espectro de actividades txicas muy
similares (Gutirrez y Lomonte, 1997), indicando que este cambio no est relacionado con una
mayor toxicidad.
La miotoxina 11 de A. nummijer muestra una relacin filogentica cercana con la
miotoxina 11 de C. godmnni, tambin distribuida en Amrica Central, y con las isoforrnas
Lys49, BP-1 y BPII, aisladas de la serpiente asitica T. flnvovirirlis. Esta separacin relativa
del p i p o formado por las PLAz Lys49 de las especies de Bothvops de Arnrica Central y
Suramrica (Le. miotoxina II de B. nsper, miotoxiiia I de B. moojeni, bothropstoxiiia I de B.
jararacussu y piratoxina 11 de B. pirrjai), mostrada en el rbol filogentico, refleja la divisin
taxonmica que propuso el cambio del gnero Bothrops al nuevo gnero Atropoides (Wennan,
1992).
La alta identidad en la secuencia de la miotoxina 11 de A. nurnmfer con la miotoxina 11
de C. godmani permiti la constmccii>n de un modelo confiable de la estructiira tridirnensjonal
de la proteina. La diferencia entre ambas protenas es mnima, con un msd de 0.08 A en el
esqueleto de carbonos alfa. Ain en la regin C-terminal, que muestra la mayor variabilidad en
las diferentes PLAi Lys49 (Lomonte et al., 2003b), la estructura predicha de la rniotoxina II de
A. nummlfer es virtualmente superponible a la proteina de C. gorlrnnni.
La regin C-terminal es de inters particular en las variantes Lys49, ya que como lo
mostraron estudios previos con pbptidos sintticos de 13 aminocidos correspondientes a los
residuos 115-129, esta regin reproduce las actividades de lesin directa a inernbrana en el
caso de dos miotoxinas de este grupo: rniotoxiiia II de B. nsper (Lomonte et al., 1994a) y la
Lys49 de Agkistroclori p. piscivorus (Nuez et al., 2001). Interesanteinente, el pptido 115- 129
de la miotoxina 11 de A. nummEfer no muestra efectos txicos en las clulas de msculo en
cultivo o el tejido muscular N1 vivo. Por lo tanto, esta PLA2 Lys49 es un caso en donde las
acciones txicas de la molcula completa no son reproducidas por el pptido C-terminal libre.
Dado que la regin 1 15-129 de la miotoxina 11 de A. ntlmmfler es idntica a la de la miotoxina
II de C. godmani, la presente observacin puede ser extrapolada tambin a esta protena. Sin
embargo, tales hallazgos no necesariamente excluyen la participacin de la regin C-terminal
de la miotoxina II de A. nurnrnifev o de la miotoxina II de C. goclmarii, en el mecanismo txico
de esas protenas, pues podra ser que el pptido libre 115-129 no necesariamente est
adoptando una conformacin adecuada para la accin txica en estos casos.
Los aminocidos que difieren entre los pptidos que representan la regin 1 15-129 de
diversas PLAzs Lys49 se indican en el Cuadro 1, art. 11. Estos varan desde el no txico de la
miotoxina 11 de A. nummifer/miotoxina II de C. god~~zani,
el pptido moderadamente txico de
la miotoxina 11 de B. asper y el pptido ms potente de la PLAzs Lys49 de A. p. piscivorus.
Aunque hay una diferencia en la carga neta positiva en estos tres pptidos (+5, +6, +7
respectivamente) que podria correlacionar con su capacidad txica, ello no parece ser
suficiente para explicar las variaciones en la actividad txica, ya que aunque el pptido 115-
129 de la miotoxina II de B. nspev aument la potencia txica en un orden de magnitud
despus de sustituir tres residuos de Tyr por Trp, no se alter su carga neta positiva (Lomoiite
et al., 1999a).
Algunos de los pptidos sintticos estudiados (AppK y Acla) pueden expresar la alta
actividad citotxica y miotxica propia de sus protenas correspondientes, otros solamente
reproducen el efecto citotxico in vitro (Basp-11, Basp-II-F) y otros no tienen ningn efecto
txico detectable (Bmoo-11, Bjsu-UBpir-11, Anum-II/Cgod-11). La aparente discrepancia entre
la actividad citoltica in vitro del pptido Basp-11, y su ausencia de toxicidad in vivo, podria ser
explicada por una mayor sensibilidad de la prueba citotxica en comparacin con la prueba de
iniotoxicidad (Lomonte et al., 1999a, Nez et al., 2001). Podriamos asumir que el nmero de
sitios blanco para los pptidos sea significativamente ms bajo en el modelo de cultivo celular,
que en la masa de tejido muscular in vivo, por lo que el pptido podra concentrarse a altas
densidades en la superficie de las clulas en cultivo, en contraste con la dispersin que
probablemente ocurre a lo largo de las fibras musculares in vivo.
Por otro lado, la ausencia de las actividades txicas para algunos pptidos estudiados,
no excluye la posible participacin de la secuencia 115-129 como una regin txica de sus
correspondieiites PLAzs Lys49. Desde una perspectiva evolutiva, es claro que las miotoxinas
PLA2 Lys49 constituyen un grupo filogenticamente relacionado (Fig. 3, art. II), por lo que no
se esperara que hubiera un desarrollo marcadamente diferente de las regiones activas en
diferentes especies de serpientes. La evidencia directa de toxicidad por los pptidos 115-129
de algunas de las PLA2s Lys49, sugiere que esta regin puede tambin jugar un papel central
en el mecanisnio txico de otros miembros de esta familia de protenas. Sin embargo, las
razones por las cuales algunos pptidos no muestran actividad no estn claras an. Una posible
explicaci~npodria ser que algunos pptidos libres en solucin, adopten una conformacin
alterada, desfavorable para el mecanismo txico. El segmento 1 15-129 forma una asa en la
regin C-telminal de las rniotoxinas Lys49 (Ami y Ward, 1996), pero la conformacin de 10s
pptidos libres correspondientes no ha sido estudiada an. Adems, la participacin de otras
regiones (adyacentes o distantes) en el mecanismo txico no debe excluirse.
Independientemente de estas razones, se propone que la observacin positiva de toxicidad
directa por un pptido C-terminal particular demuestra que esta regin est involucrada en el
mecanismo de toxicidad, pero lo opuesto no necesariamente es verdadero, es decir, un
resultado negativo necesariamente no excluye la participaciiin de esta regin. En apoyo a esta
idea, en estudios con mutantes de la bothropstoxiria I recombinante de B. jnmraczrsszr se
demostr claramente una ftincin relevante de los residuos 1 15-125 en las acciones txicas de
esta protena (Chioato et al., 2002), a pesar de que los presentes resultados mostraron que el
pCptido 115-129 de esta toxina fue inactivo.
En el presente estudio se demostr que tanto la actividad citotxica de varias PLAzs
Lys49 iu vitvo, como su actividad miotbxica in vivo se reducen, aunque no se eliminan, a pH
5.0, en comparacin con su actividad a pH 7.2. Dado que se ha demostrado que a pH 5.0
ocurre una disociacin de las formas dimricas de estas toxinas (de Oliveira et al., 2001), los
resultados apoyan la idea de que la disociacin puede reducir la capacidad de alterar la
membrana plasmtica, originalmente propuesta por de Oliveira et al. (2001). Sin embargo, en
contraste con los hallazgos de dichos investigadores usando liposomas como blanco, los
resultados con mioblastos en cultivo demuestran que la accin txica de las PLA2s Lys49 se
reduce a pH 5.0, pero 1x0 se elimina. Los anklisis estnicturales realizados con la bothropstoxina
1 sugieren un cambio de la estructura cuateniaria entre las conformaciones "abierta" y
"cei~ada"(da Silva Giotto et al., 1998), lo cual podra jugar un papel en la capacidad de las
miotoxinas Lys49 para desorganizar la bicapa de fosfolpidos, usando la regin C-terminal
como un sitio efector. Una posibilidad adicional, pero no mutuamente excluyente, es que la
mayor actividad de los dmeros pueda estar relacionada a una unin ms eficiente a aceptores
de membrana, con una mayor avidez de una interaccin divalente, comparada con la afinidad
de unin inonovalente de los monmeros individuales.
En los experimentos zn vivo en ratones, en donde se demostr que el dao tisular por
miotoxinas preincubadas a pH 5.0 es significativamente menor que el producido por las
mismas toxinas a pH 7.2, se debe considerar que dentro de esas condiciones, podria ser que las
soluciones inyectadas sean rpidamente amortiguadas a pH fisiolgico. Es posible que las
iniotoxjnas Lys49 ingresen al tejido muscular a pH 5.0 en un estado monomrico, y puedan
unirse a su blanco sarcolemal antes de que ocurra una reasociacin completa del dimero,
explicando as la baja actividad txica observada in vitro. Sin embargo, estas observaciones Nz
vivo son ms dificiles de interpretar que los resultados obtenidos con cultivos celulares, en
donde el pH del medio puede ser controlado en los experimentos.
Interesantemente, los pptidos correspondientes a la regin C-terminal de Agkisrodo~i
p. piscivorus (p-AppK49) y de B. asper (p-Ba-mt II) tambin mostraron el mismo fenmeno
en las clulas C2C12, con un menor efecto a pH 5.0. Estos pptidos cortos no tienen una
estnictura cuaternaria. La reduccin en la actividad citolitica de los pptidos a pH 5.0 no
puede ser atribuida a variaciones en el grado de protonizacin de los grupos laterales de la
cadena (que afecten su conformacin o su interaccin con la membrana plasmtica del
inioblasto), ya que si nos basamos en el conocimiento de los valores de pK de los gnipos
amino y carboxilo presentes en su secuencia, no se cambiara la carga neta al disminuir el pH
de 7.2 a 5.0. Por lo tanto, es posible especular que la estructura blanco en la membrana celular
podn'a ser alterada por la disminucin de pH, o que el aumento extracelular en la
concentracin de protones pueda tener un efecto protector contra la permeabilizacin de
membrana. El fenmeno de proteccin de membranas mediado por protones ha sido
previamente demostrado para algunas molculas citolticas (Bashford et al., 1989; Rostovtseva
et al., 1994). Los resultados permiten concluir que el estado djmrico de las PLA2s Lys49
puede jugar un papel importante, aunque no esencial, en el mecanismo de rniotoxicidad
mediado por la regin C-terminal.
Se ha propuesto como un til modelo in vitro para estudiar el mecanismo iniotxico de
las PLAzs del grupo II, a los cultivos celulares de niioblastoshniotubos de msculo
esqueltico. En dichas clulas, este efecto correlaciona con la actividad de dao muscular
observada in vivo (Bniss et al., 1993; Incerpi et al., 1995; Lomonte et al., 1999b). En este
estudio se investig si la fusin y diferenciacin de rnioblastos C2C12 conduce a una mayor
susceptibilidad hacia la accin citotxica de las PLA2s del grupo 11miotxicas.
Se observ claramente una susceptibilidad aumentada de los miotubos usando un
grupo de rniotoxinas de serpiente, incluyendo variantes Asp49 y Lys49. Adems, otro tipo de
protenas cuya accin rniotxica ha sido documentada, como la PLAi del veneno de abeja
(grupo 111) y el pptido catinico melitina (Ownby et al., 1997), afectaron a ambos tipos de
clulas en cultivo de la misma forma. El uso de varios detergentes tambin mostr un efecto
citolitico similar en mioblastos y miotubos, por lo que la susceptibilidada aumentada de los
miotubos a las PLAzs del gmpo II no puede ser atribuida a la posibilidad de una capacidad
menor de esas clulas para hacer frente a perturbaciones generales de la homeostasia de la
membrana. Por tanto, la diferenciacin de mioblastos en miotubos debe conducir a cambios
que favorezcan la accin de las miotoxinas PLA2 del gmpo 11. Estos cambios podnan
involucrar la expresin de un mayor nmero, o de un tipo de alta afinidad, de sitios aceptores
para las PLAzs miotxicas en la membrana plasmhtica. Sin enibargo, la posible participacin
de procesos intracelulares que ocurren despus de la fusiOn de los mioblastos, podra tambin
jugar un papel y no puede exluirse por ahora.
La selectividad del efecto miotxico de las PLA2s de gmpo II in vivo se ha atribuido a
la existencia de sitios de unin especficos en la membrana plasmtica de las clulas de
msculo esqueltico, pero su naturaleza es completamente desconocida. Se han identificado
aceptores proteicos de alta afinidad para PLAzs neurotxicas activas presinpticamente, en
tejido neurona1 (Lambeau et al., 1989; Krizaj y Gubensek, 2000). Por otro lado, una protena
de membrana rnultidominio de 180 KDa, conocida como receptor tipo M, se ha caracterizado
como un sitio de unin para algunas PLA2s del gmpo 1 en msculo esqueltico (Lambeau et
al., 1990). Sin embargo, no hay ninguna evidencia reportada a la fecha que involucre a los
receptores tipo M en el efecto rniotxico de las PLA2s de gmpo 11.
Alternativamente, las miotoxinas PLA2s del gmpo II podran actuar reconociendo
lipidos de membrana, como glicerofosfolpidos o glicolipidos, que estn diferencialmente
expresados, o diferencialmente agrupados en dominios lipidicos o en balsas (rcfls), eii los
mioblastos y los miotubos. Algunas observaciones sugieren que los fosfolpidos cargados
negativamente podrian ser aceptores importantes para el mecanismo rniotxico de estas
proteinas (Diaz et al, 2001). En particular, la actividad citolitica demostrada por un pptido
sinttico derivado de la PLA2 Lys49 de Agkzstrodun p.pzscivoru, preparado con D-
aminoacidos (art. IV), sugiere fuertemente que el mecanismo de accin de esas miotoxinas no
involucra el reconocimiento de un sitio aceptor proteico en clulas musculares. Este fenmeno
de susceptibilidad diferencial indica que el sistema de cultivo celiilar mioblastos/miotubos
podria ser un excelente modelo para detectar los cambios fenotpicos que aparecen en esa
transicin pudiendo ser estos los "aceptores" de las PLA2s.
En las ltimas dcadas, se ha reportado un nmero creciente de iiihibidores de PLAzs
rniotxicas de venenos de serpiente, como protenas plasmiticas animales (Nobuhisa et al.,
1998; Lizano et al., 2000; Rocha et al., 2002), polisacridos (Melo et al., 1993; Lomoiite et al.,
1994b) y componentes de plantas (Mahanta y Mukherjee, 2001; Deepa y Gowda, 2002). Con
el avance gradual de la comprensin sobre los determinantes moleculares y los mecanismos de
accin de las miotoxinas, el hallazgo de inhibidores eficientes podra aprovecharse para el
desarrollo de tratamientos complementarios a la seroterapia durante los envenenamientos
ofidicos en el fiituro.
El descubrimiento de las PLA2s Lys49 enzimticamente inactivas en los venenos de
crotlidos (Maraganore et al., 1984, Maraganore y I-Ieinrikson, 1986), ha impulsado las
investigaciones para delinear el sitio txico en estas protenas. La evidencia existente indica
que la regin C-terminal de las variantes Lys49, que combina aminocidos catinicos e
hidrofbicos, constituye un sitio determinante para su actividad miotxica (Lomonte et al.,
1494a, 2003; Nez et al., 2001, Caldern y Lomonte, 1998; Chioato et al., 2002; Ambrosio et
al., 2005). Por tanto, las molciilas poliaiiinicas con la capacidad de unirse a este sitio txico
coiistituyen candidatos racionales para la evaluacin de su actividad inhibitoria de la
rniotoxicidad.
En el presente estudio se evalu la capacidad neutralizante del fucoidan, un
polisacrido sulfatado, extrado del alga FUCLIS
v e s i c ~ l o s ~ sAunque
. comparten algunas
propiedades con heparina, los fiicanos sulfatados constituyen molculas polianinicas
alternativas, con un amplio espectro de perfiles famacolgicos (Mourao y Pereira, 1999).
Usando un grupo de miotoxinas PLAi purificadas de diferentes venenos de vipridos, se
demostr que el fucoidan inhibe eficieiiteinente sus efectos citotxico (in vitro) y miotxico
(in vivo). Mas an, en el caso de dos variantes Asp49 activas cataliticamente, el fucoidan

inliibi sus accioiies txicas sin afectar su capacidad de hidrolizar fosfolipidos. Este resultado
ejernplifica la disociacin entre toxicidad y catlisis en la familia de las miotoxinas tipo Asp49
gmpo 11, reportadas en diversos estudios previos (Lomonte et al., 1992, 1994; Gutirrez y
Lomonte, 1997; Soares et al., 2001).
El mecanismo de inhibicin del fucoidan hacia las miotoxinas PLA2 se basa en la
formacin rpida de complejos, probablemente mediada por interacciones electrostticas
multivalentes entre los sulfatos aninicos del polisacrido y los numerosos residuos catinicos
de las toxinas, que tienen un punto isoelctrico bsico. La formaci0n de complejos unidos
reversiblemente se evidenci por anlisis de turbidimetria, en donde las propiedades de
dispersin de luz de las mezclas fucoidadmiotoxina varan de acuerdo con las proporciones
relativas de esos dos componentes, resultando en una tpica curva de absorbancia en forma de
campana.
El posible sitio de unin del fucoidkn en las miotoxinas se investig usando pptidos
sintticos que representan la regin de dao a membrana (residuos 115-129) para tres de las
toxinas (Lonioiite et al., 2003b). El fucoidn claramente inhibi la actividad citoltica de los
tres pptidos sintticos, demostrando su capacidad para interaccionar con la regin miotbxica
C-terminal de esas PLA2s. Esta conclusin tambin es apoyada por los datos de competencia
de unin entre los anticuerpos contra la regin 115-129 de la miotoxina II de B. nsper y el
fucoidan: la unin de dichos anticuerpos a la toxina irnovilizada se redujo significativamente
en presencia del fucoidan.
Por su naturaleza polianinica comn con la heparina, el fucoidn podria actuar de un
modo similar (Lomonte et al., 1994b). Sin embargo, la densidad de cargas negativas en un
esqueleto de polisacrido no es el nico factor que determina la capacidad de interaccionar con
las miotoxiiias. Por ejemplo, se ha observado que la unin del condroitn sulfato y el dermatn
sulfato a la miotoxina II de B. asper es mas dbil que la unin del heparn sulfato, aunque este
ltimo es menos sulfatado (Lomonte et al., 1994b). Esto indica que los distintos esqueletos de
los polisacridos poseen influencia para una interaccin ptima coii las protenas. Lin et al.
(1999), al caracterizar la interaccin entre la heparina y una PLA2 de grupo 1 del veneno de
Nnja >ligricollis, hallaron que participan tanto las fuerzas electrostAticas como las no
electrostticas.
Se evalu la capacidad del fucoidn para inhibir el dao a nisculo, administrndolo
localmente, inmediatamente despus del envenenamiento con veneno crudo de B. aspeu. Bajo
dichas condiciones, la inyeccin de fucoidn disminuye parcialmente la mionecrosis inducida
por el veneno, a un nivel de aproximadamente 50% de los animales no tratados. Sin embargo,
es importante sealar que no necesariamente es adecuado extrapolar los resultados obtenidos
en ratn con los de un accidente ofdico en humanos, ya que el envenenamiento en ratn se
desarrolla muy rpidamente.
Una limitacin importante del desarrollo de cualquier inhibidor efectivo clnicamente
para las miotoxinas es la rapidez dramtica con que estas protenas afectan el mUsculo
esqueltico, como se ha observado por tcnicas de microscopa intravital (Lomonte et al.,
1994~).El alto peso molecular del fucoidan (135kDa) podra tambin ser un factor que podra
limitar su distribucin y difusin en los tejidos. La caracterizacin de los fragmentos de
fucoidaii de baja masa, o de diferentes tipos de fucanes sulfatados obtenidos de una variedad
de fuentes naturales (Mourao y Pereira, 1999), podria ser de inters en este sentido.
El DM4, una proteina plasmtica acidica de Diclelphis rnarszlpinlis, tiene la capacidad
de forn~arcomplejos con las diferentes PLA2s iniotxicas probadas. Sin embargo, iio es capaz
de hacerlo con las metaloproteinasas hemorrgicas. Esto indica que el DM64 es uiia protena
con interaccin especifica hacia las rniotoxinas, que es capaz de inhibir su actividad, lo que
insta a realizar estudios para encontrar el sitio de interaccin, que contribuyan al
esclarecimiento del mecanismo de esta inhibicin.
 6. CONCLUSIONES

6.1 La miotoxina II de Atropoides ~iummiferes una PLAz de tipo Lys49, hornodirnrica, con
subunidades bsicas de 121 aminocidos y una masa molecular de 13,789.90 Da por unidad.
Su secuencia tiene un alto porcentaje de identidad con otras protenas de esta familia, halladas
en venenos de serpientes de los gneros Cerrophidion, Tri~neresurus,Bofhrops y Agkistrodon.
Sus actividades biolgicas incluyen citotoxicidad in vitro as como miotoxjcidad e induccin
de edema in vivo.

6.2 La miotoxina II de A. nummfer posee una relacin filogentica cercana con la miotoxina
11 de C. g o h a n i . El anlisis filogentico muestra que las PLA2s Lys49 divergeii de las Asp49,
y que las primeras se encuentran ms relacionadas con las PLA2s Ser49.

6.3 La regin C-terminal de las PLAzs Lys49 miotxicas juega un papel importante en su
mecanismo de accin. Algunos pptidos sintticos correspondientes a esta regin reproducen
los efectos txicos de las correspondientes miotoxinas, mientras otros no lo hacen, coino es el
caso del pptido 115-129 de la miotoxina 11 de A. nunimger.

6.4 El estado dimrico de las PLA2s Lys49 puede jugar un papel importante, aunque no
esencial, en el mecanismo de miotoxicidad mediado por la regin C-terniiiial.

6.5 Las clulas musculares en cultivo constituyen un modelo til para estudiar el mecaiiisnio
miotxico de las PLAzs del grupo 11. La diferenciacin de mioblastos C2C12 aumenta la
susceptibilidad a la accin citotxica de estas protenas, siendo esto una caracterstica
especifica para este grupo de miotoxinas, ya que otros agentes liticos se coiiiportan de la
misma manera en rnioblastos que en miotubos.

6.6 El fucoidan posee un efecto inhibitorio sobre las PLA2s miotxicas, forinaiido coinplejos
que involucran el reconocimiento de la regin C-terminal. Su capacidad neutralizante podra
ser de inters teraputico, aunque se requieren ms estudios para buscar formas de lograr una
mayor distribucin y una mayor difusin en tejidos.
6.7 La proteitia plasmtica DM64 de Didelphis marsupinlis interacta aparentemente en forma
especifica con las PLA2s miotxicas, inhibiendo su toxicidad. Por tanto, podra ser un buen
modelo para ser caracterizado a nivel molecular, con el fin de delinear los sitios de interaccin
entre anibos componentes. Esto podra ser til para comprender la relacin estructura-funcin
de estas toxiiias, as como para la bsqueda de molculas inhibidoras con fines teraputicos.
 7. PERSPECTIVAS FUTURAS

Este trabajo de investigacin plantea la necesidad de nuevas lineas de trabajo referente a :

1. Estudios para la bsqiieda de posibles aceptores en la membrana plasmtica de

miotoxinas PLAz del grupo 11, a traves de estudios con microscopa electrnica,
fluorescencia y con el uso del modelo de clulas in vitro de miotubos C2C12.

2. Estudios que permitan detallar el papel de la regin C-terminal de las Lys49 en la

disrupcin de membranas.

3. Estudios sobre los efectos intracelulares de las miotoxinas PLA2 del grupo II
(mecanismos de movilizacin con el calcio intracelular, o ingreso de calcio a travs de
la membrana plasmtica, uso de inhibidores de sntesis de ganglisidos u otros lipidos
de membrana y reguladores de la actividad metablica).

4. Estudio de los efectos de inhibidores de estructura qumica similar al fucoidan pero de

menor tanialio y con una mayor capacidad de distribucin en los tejidos.

5 . Estudios para la caracterizacin de los sitios de interaccin entre el DM64 y las

miotoxinas PLAz del gmpo 11, con la finalidad de aumentar el conocimiento de la
relacin estructura-funcin de estas miotoxinas.
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Zapopozhets TS, Besednova NN, Loenko IN (1995). AntibacteriaI and immunomodulating activity of
fucoidan. Antibiot. Khimioter. 40, 9-13.
APEND
ARTICULO
 Tfic Ititernalional Journal of Biochernistry Sr Ccll Biology 32 (2000) 63-71
www clscvicr corii/locatc/ijbcb

Isolation and characterization of myotoxin TI from

Atropoides (Bothrops) nummifer snake venom, a new Lys49
phospholipase A2 homologue
Yamileth Anguloa7b, Timoteo Olarnendi-Portugalc, Lourival D. Possanic,
Bruno Lomonteay*
"Instituto Clodomiro Picrrdo. Fncirllnd de Microbiologcl, Escuelcr de Medicina, Universidncl de Casrn Rica, Scln Josi, Corra Riccr
b~rpcrrtcinientoclr Bioqlrir~licn,Esct~elcrde Medicina, Univevrid(~~
(le COSICI
Rica, So11 J o F ~Co.~tcl
, R~c~I
'Df>parrn~ent o/ Molccukar Recognilion ar~dStrlr~tirral Biology, Instifitto de Biote~nolo~n,Ncrtional Autonnmnir.~Uriivcrsity ?/ Mexico.
Cir~rnnvclca,Me x i ~ o
Rcccivcd 1 1 March 1999; acccptcd 9 July 1999

Abstract

Myotoxic phospholipases A2 of class I I are commonly found in the veiloms of crotalid snakes As an approach to
iinderstanding their structure-activity relationship, diverse natural variants have been characterized biochemically
and pharmacotogicatly. This study describes a new myotoxic phospholipase A2 homologue, isolated frorn the venom
of Atropoides (Bothrops) nurnrnjfer from Costa Rica. A . tiuntmifer myotoxin 11 is a basic protein, with an apparent
subunit molecular mass of 16 kDa, which migrates as a dimer in sodium dodecylsulfate-polyacrylamide gel
electrophoresis under nonreducing conditions. It is strongly recognized by antibodies generated against Bothrops
nsper myotoxin 11, by enzyme-immunoassay. The toxin induces rapid myonecrosis upon intrarnuscular injection in
mice (evidenced by a n early increase in plasma creatine kinase activity), and significant edema in the footpad assay
It also displays cytolytic activity upon cultured murine endothelial cells The toxin (up to 50 pg) has no detectable
phospholipase Ai itctivity on egg yolk phospholipids, and does not show an anticoagulant cffect on sheep platelct-
poor piasma in vitro N-terminal sequence determintion (53 amino acid residues) dcmonstrnted that A ntrmmifrt
myotoxin II is 21 riew Lys49 variant of the family of myotoxic, class 11 phospholipases A2 Sequence co~nparison
with othei phospllolipases A2 revealed Asn53 a s a novel substitiition In addition, this myotoxin is the first Lys49
variant prescnting Asn in its N-terrninus Consequently, these finditigs suggest that neither Ser1 or Lys53, usually
found in this faniily of proteins, are essential alnino acid residues for their myotoxjc, cytolytic, or edema-inducing
eflects 0 1999 Elsevier Science Ltd Al1 rights reserved

Keyivorrl~ Snakc vcnom; Myotoxin; Phospholipasc A?; Atropoidl:~n~tn~nli/i~r;

Rotl1rop.r

* Coi rcspoi,ding author Fax: -t. 506-292-0485

E-n~ailcrtlnriw hlomonte@>cariariucr ac cr (B. Lomontc)

1357-2725/99/5- sce froiit rnattcr O 1999 Elsevier Scicncc Ltd Al1 rights tcscrvcd
PII: S 1357-2725(99)00099-O
1. Introduction
Snake bite envenomations in Latin America
are caused most frequently by species of the
genus Bothrops [1,2] In addition to systemic
alterations, these envenomations are character-
ized by prominent local tissue darnage including
myonecrosis, hemorrhage and edema [3]. Acute
rnuscle darnage induced by these venoms is
caused mainly by basic phospholipases A2
(PLA2s) which initialty affect the integrity of the
plasma n~embraneof rnuscle cells 141.
During the last years, severa1 myotoxins
fronl Bothrops venoms have been isolated,
and some progress has been made towards
iinderstanding their structure, mechanism of
action, and pathogenesis of myonecrosis
[4,5]. The class 11 PLA2 myotoxins found in
the venoms of crotalid snakes can be divided into
two groilps, namely, those with an aspartic acid
residue at position 49 (Asp49) and those in which
a lysine substitutes a t this position (Lys49).
Although the Lys49 proteins lack (or may have
extremely low) catalytic activity, they display
myotoxic activity comparable to that of Asp49
counterparts, both quantitatively and qualitat-
ively [4]. Biochemical characterization of myotox-
ins has demonstrated considerable inter- and
intraspecific variations of primary striicture,
which niight provide relevant clues for delineat-
ing their niolecular determinants of toxicity [6,7]
Tlierefore, the search for new niyotoxin variants
in the natural biodiversity, and their striictiiral
and pharrnacological characterization, are of
in terest.
Arropoirles nunlri~ifer (previously Bothrops nimz-
nz@r) is distributed in the tropical rain forests of
Costa Rica [8], and its venom induces drastic
local effects, including myonecrosis [93. From this
venom, a myotoxin was previously purified and
pharmacologically characterized [lo, 1 11. The
toxin described in this study, named niyotoxin 11,
represents a second isoform present in A . nzrnznti-
fer venom, belonging to the tys49 family of class
11 PLA2.
2. Materials and methods
A. rzrrmrnfir venom was a pool obtained from
more than 15 specimens collected in Costa Rica.
After centrifi~gation,venom was lyophilized and
stored at -20C. Sampies of 500 mg were dis-
solved in 0.05 M Tris-HC1, 0.1 M KCI, pH 7.0
buffer, and applied to a CM-Sephadex C-25
(Pharmacia) colunin (30 x 2.5 cm), equilibrated
with the same buffer. After coltecting unbound
proteins, a linear gradient from 0.1 M (400 ml)
to 0.75 M KCI (500 ml) was used to elute basic
fractions, a t a Aow rate of 0.4 ml/min [12]. Peak
4 screened positive for niyotoxic activity, and
was selected for further analyses It was rechro-
n-iatographed identically, dialyzed against water,
and 1yophiIized.
2.2. Homogeneity
Protein homogeneity was assessed by (a) poly-
acrylamide gel eiectrophoresis in the presence of
sodium dodecylsuffate (SDS-PACE) [13f, (b)
PAGE in the presence of urea, using a cathodic
buffer system [14], and (c) reverse-phase high per-
formance liquid chromatography (RP-HPLC) on
a C4 column, eluted at 1.0 ml/min with a gradi-
ent from O to 60% acetonitrile in 0 1% trifluor-
oacetic acid, using a Waters instrument.
Prior to sequence deterrnination, A . riunlmfer
myotoxin 11 was reduced and carboxymethylated
as described 1153. The automatic Edman degra-
dation procediire was used with a Beckman
sequencer, model L F 3000.
Sarnples of 0.1 ml, containing either 6, 12, 25
or 50 pg of toxin, were incubated with 1.0 m1 of
egg yolk suspension (diluted 1:5 in 0.1 M Tris-
HC1, 0.01 M CaC12, pH 8.5 buffer) containing
1 % Triton X-100, for 30 min at 37C. Then, the
 Y Attgtrlo ~l al / Tlie Intcrnnlioncil Joinncrl o j Biocfier,~is/ry& Cell Biology 32 (2000) 63-71
free fatty acids were extracted and titrated
according to tlie method of Dole [16]
2.5. Myotodxic irctivity
The toxin (either 20, 40 or 80 pg), dissolved in
50 pl of 0.12 M NaCl, 0.04 M sodium phosphate,
pH 7.2 buffer (PBS), was injected into groups of
four Swiss mice (18-20 g body weight), in their
right gastrocnemius muscle. A control group
received 50 111 of PBS. After 3 h, bIood sarnples
were collected from the tail, and the plasma cre-
atine kinase (CK; E C. 2.7.3.2) activity was deter-
mined by a colorimetric assay (Sigma No. 520).
Activity was expressed as U/ml, one unit corre-
sponding to the phosphorylation of 1 nmol of
creatiiie per min at 25OC. In addition to CK ac-
tivity measurements, formalin-fixed muscle tissue
s;imples were obtained from the injected site after
24 h, and processed for histological evaluation of
muscle damage, usirig toluidin blue-stained sec-
tions.
2.6. Arzficuagrrlnrrt (lctivity
Platclet-poor plasma from sheep was prepared
by centrif~igingcitrated blood twice a t 1000 x g,
at 5C. For the assay, 0.5 m1 of plasma was incu-
bated with toxin (either 1.2, 2.5, 5, 10, 20, 40 or
80 ~ i g )dissolved in O 1 m1 of PBS, for 10 min at
37C. Then, O 1 m1 of O 25 M CaCI2 was added,
and the coagulation time recorded. As a control,
plasma aliquots were iiicubated with O 1 mi of
PBS, and the coagulation tin.ie determined identi-
cally.
2 7 . Ecieina-forrnirrg nctivity
Groups of four mice (18-20 g) received a sub-
cutaiieous injection of toxin (either 5, 25 or 50
pg), dissoIved in 50 p1 of PBS, in their right foat-
pad. After 1 h, edema was estinlated by nieasur-
ing the increase in footpad thickness witli a low-
pressure spring caliper [17]. Control mice received
50 p1 of PBS.
65
O
c:
m
2 -
O 0 '", I 1 I I I 1
O 50 100 150 200 250 300
Elution volume (ml)
Fig l . lsolation of Atropnide.9 ntrmmifer myotoxin II by ion-
exchatige chromalography on CM-Scphadex C-25. (a) Eliition
pattern of crude venom (500 mg) equitibrated with O 1 M
Tris-HC1, 0.1 M KCI, pH 7.0 buffer, and elutcd with a linear
gradieiil (G, indicatcd by the arrow) towards O 75 M KCI, pH
7 O buffer (b) Rcchromatography of peak 4 from (a) on tbc
CM-Sephadex column (30 x 2 5 cm)
An enzyme immunoassay was utilized to test
antigenic cross-reactivity between A . numrnifer
myotoxin 11 and B. nsper myotoxin 11. Each
toxin (0.4 pg/l00 p1 PBS) was adsorbed onto
ImmuIon-2 microplates (Dynatech) overnight.
After five washings with 0.05 M Tris, 0.15 M
NaC1, 20 pM ZnC12, 1 mM MgC12, pH 7.4 buf-
fer, plates were blocked with 150 1~lJwellof PBS
containing 2% BSA, for 1 h. After decanting,
100 pl/welI of varying dilutions of rabbit anti-
serum to B. crsper inyotoxin II was incubated fol
I h, followed by five washings, and by 100 pl/
well of anti-rabbit immunoglobulin conjugated to
aikaline phosphatase (Sigma, 1:2000) for 1 h.
Color was developed with p-njtroplien ylpho-
sphate and recorded at 410 nrn on a Dynatech
MR 5000 microplate reader. As a control, nor-
mal rabbit serum was assayed in paralle
Cytutoxic activity was determined as pre-
viously described [18,19], using the murine endo-
thelial cell line tEnd. Cytolysis was estimated
 Y At~giiloel al / Tlze Interricrtiotinl Jotrnirrl
O 20 40 60 80
Myotoxin (pg)
Fig. 5 Myotoxic and cytolytic activitics of A~ropoidesntrmmi-
/kr rnyotoxin II (a) Plasma crcatine kinase (CK) activity 3 h
after thc intramuscular injection of myotoxin in the gastrocna
mius o mice Each point represents the mcan IT S D. of four
animals (b) Lactic dchydrogcnase (LDH) rcleasc to supcr-
natarits of cndothelial cell cultures, 3 h aftcr cxposurc to niyo-
toxin (pglwcll) Valiics are exprcssed rclative to cultiircs
exposed lo O 1% Trtoti X-100 (100%) or culture mcdiurn
+
(0%). Each point represents the mean S D. of duplicate cul-
tures.
further characterization, and rechrornatographed
iinder the same conditions (Fig. 1b). This protein
appeared as a single band in cathodic urea-
PAGE (Fig. 2a), as well as a single 16 kDa band
in SDS-PAGE, after reduction with 2-mercap-
toethanol (Fig 2b). By analyticaI RP-HPLC, the
piirified toxin eluted as a single peak (Fig. 3).
This myotoxin was strongly recognized by rabbit
antibodies genera ted against B. asper myo toxin
11, by enzyme-immunoassay (Fig. 4).
A . nurnmifer myotoxin 11 (up to 50 pg) had no
detectable PLA2 activity on egg yolk phospholi-
pids, and did not show an anticoagulant activity
(up to 80 pg) on sheep platelet-poor plasma
(data iiot shown). Iii vivo, the toxin induced
dose-dependent myonecrosis upon i.m, injection,
as evidenced by the significant increase in plasma
CK activity at 3 11 (Fig, 5a). Histological evalu-
(lfBio~lzv17ii~try 32 (2000) 63-71
& Cvll Binlog~~ 67
-
-
-
I I I 1 I
O 6 12 18 24
Time (hr)
Fig 6 Edcma-forming activity of Atropoides nirtr~ntij>rinyo-
toxin 11 in mice Time-course of thc incrcase in footpad thick-
ncss induccd by thc injeclion of 5 pg (a),25 ~ i (A)
g iir 50 ktg
(V)of myotoxin, or phospliatc-biiKc~edsalinc alonc (e),rc-
spectively, by subcutaneous route Each point represcnts the
nicaii 3- S D of four animals
ation of formalin-fixed muscle tissue (iiot shown)
confirmed the myotoxic effect. 111 addition, injec-
tion of A . ntrrnikzifer myotoxin 11 iri the footpad,
at doses as low as 5 pg, jnduced significant
edema, which was of immediate onset (Fig. 6). A
clear cytolytic effect was observed upon incu-
bation of myotoxin II with cultured endothelial
cells (Fig. 5b).
The results of automatic sequencing of ~ediiced
and alkylated toxin indicated that Ihe protein
was horiiogenoiis, and aii iriieqiiivocal sequence
for the first 53 aiiiino acid residues at the N-
terminal scgincnt was obtaiiied Fig 7 s h o u ~ sthis
sequence in coinparison with other rela tcd myo-
toxic PLAzs The 1iighest identity (88 7%) was
observed with Cet r-ophillia~igoiijncrr~imyotoxiii 11.
identity scores were hjgher in cornpalison to
Lys49 ( > 77%) than to Asp49 ( < 64%) myotox-
ins (Fig. 7)
The purification and characterization of a new
myotoxic PLA2-homologue from the venom of
A. nul?znz$er are presented. Previous repurts
68 Y Angrrln rt o1 / T/ICInt~rnatioricrlJoirr,rnl of' Biochernis~ry& Crll Biology 32 (2000) 63-71

Anum- 11 NLYQLWKMlLQETGKNAnPSYGFYECNCGVGSRGKPKDATDRCCHKCCY b identity

Cgod-II SMY..... . . . . . e . ..V a . . . ~ . . L ~ ~ . . . * . # . . . . . * , . . * - . a * . . . ... K

Brnoo- 1 J. S . F E + G. . . . . - . . . . P . K ...V. t . . . . - + G G + . . * - . . + - - . y . . . . . . .
Basp-11 S.FE.G ..a...v...P+K..*A....b..LG...ttttt+*--y..
S . F E . G .....-....P.K...A A . . . . . . L G * # . - ~ * - . + * . . Y . . * .
. K. . , .
B j su- D +K,
~ f l ~ - b ~S.V
I ....-.F...--E..KN*.LL L . . . . L L R . .. . . * . . * S . . y h h a -.K a.
Ac1 -K4 9 S.LE.G+ ..........IT ...S. . . . . . w . H . , Q . . . . . . . . . . . . . . . . . K
Batr S.VE.G ~ . . . . . . . ~ . P L T . . . ~ . . . . . . . . H . . H ~ ~ ~ ~ D . ~ ~ ~ ~ Y ~ ~ ~ ~ ~ ~
App-K49 SVLE.G . . . . . . . . ...IT...Sm.....WwH..Q......---.-.~.. .K
Abil PLA-11 S . L E . G * * ....*.I.IT.
* ~*SSS..W.H..W-H..R~...
Rsch- 'I SM.E.G.+..L..-....T..IA.,
Basp-IV S.VE.G..........PVT-..A.
B j SU-11 D.W.WGQ. . - K . . . . ~ P F . Y Y T T . . . Y . . . .~K V-
Bjsu-PLA D.W.FGQ ..+K..*.LPF.Y-TT...Y..W WGQ.Q......*..*..D-..GK
Vam-S4Cj
Basp-111
SVIEFG . . . Q E . . D . - P L T . . S . . . . ~ . . ~ . . - - - . - - s * . AK
S-IEFA...-E..KRLPF.Y*TT.. T Y..W-GQ.Q-....*----..D*m.GK
.
App- D4 9 ..F.FE.L.KKM.. .SGMLW.SA...Y..W W G Q . R R R R R . . . . . . . . ~ - * ~ G ~

Fig. 7 N-terminal sequcnce o f Atropoides numrnfer myotoxin 11 compared to related myotoxic class 11 phospholipascs A? Amino
acids idcntical to thc A riumniijkr myotoxin 11 sequcnce (first 53 residues; Anum-11) are represcnted by dots. Sequences correspond
lo: Cgod-11, C goclmcrni myotoxiri 11 (291; Bmoo-11, B moojeni myotoxin II [30); Basp-11, B cisper rnyotoxin 11 [21]; Bjsu-1, B jc~rcrr-
acltssu bothropstoxin 1 [3 I 1; Tflv-bpI, Trir?ieresitrrrsJclvoviridis basic protein 1 [32]; Acl-K49, Agkistrodon conlnrrrix lclticincttrs myo-
toxiii [6]; Batr, B nrrox PLA? [33J; App-K49, A piscivorur piscivorus K49 PLAz [27]; Abil PLA-11, A hilinecrtu~.basic PLA? (341;
Bsch-1, B schlegelii myotoxin 1 [35];Basp-IV, B nsper myotoxin IV [36]; Bjsu-11, B jcircrraciiwir bothropstoxin 11 [37]; Bjsu-PLA,
B jrrrarocrrssu PLA? [38J; Vamm-S49, Vipera errnniodyte~S49 PLAp [39]; Basp-111, B cisper myotoxin 111 [40]; App-D49, A pirci-
i t ~ PLAz [27]
vorirs p i ~ ~ i v o rD49

described a myotoxin of this venom [lo, 111, here
referred to as rnyotoxin 1. Cation-exchange chro-
matography at pH 7.0 confirmed the presence of
several basic proteins in A . iiurnm!:fervenom, in
addition to myotoxin 1 (peak 5). The slightly less
basic peak 4 screened positive for myotoxic ac-
tivity, and was further rechromatographed Tt
Inay correspond to the 'peak IV myotoxin'
rcported earlier by Bruss et al 1201, who studied
its in vitro cytolytic effcct towarcls rnuscle cells,
neurons and ibroblasts. Our purified protein
appeared homogeneous by severa1 analytic cri-
teria, including cathodic urea-PAGE, SDS-
PAGE, RP-HPLC, and N-terminal sequence de-
terrnination, and was named A . nummfer myo-
toxin 11. It showed a strong antigenc cross-
reactivity with B. asper nlyotoxin 11, a well
characterized Lys49 PLA2 hornoiogue [12,2 1,221.
GeI etectrophoresis indicated that A. rzttnznziJer
inyotoxin 11 occurs as a dirner, with an apparent
subunit molecular mass of 16 kDa after re-
duction Stable hornodimers in solution have
been observed in myotoxins isolated from
Borhrops spp. venoms [lo,12,231, but highly hom-
ologous myotoxins fronl Agkistrodon spp occur-
ring as monomers have also beeii described [6].
The pharmacological activities of A . ntrmm$er
rnyotoxin T I are qualitatively and qilantitatively
similar to tliose described for other basic PLA2
from crotalid snake venoms [4], and include the
ability to induce. (a) rapid skeletal muscle necro-
sis at the site of injection, (b) an irnrnediate
increase of local microvascular permeability, and
(c) rapid in vitro cytolysis. The latter effect has
been observed using several cell types as targets
[18,24], and emerges as a common attribute of
class 11 myotoxic PLA2s, both of the Asp49 and
the Lys49 types [ 191
Enzyrna tically active Asp49 myotoxins fre-
quently display potent an ticoagulan t effect jn
vitro, probably reIated to the hydrolysis of
plasma phospholipids [4] I n the case of A . iztrm-
mfer myotoxin TI, the iack of an anticoagulant
effect is in agreeinent with the lack of PILA2 ac-
tivity observed. Botli findings suggested that this
inyotoxin would be a Lys49 PLA2 hon~ologue.
This was confirnied by N-terminal sequencing,
which additionally showed the high homology (in
their first 53 residues) between A. ritmzm$er myo-
toxin 11 and several retated Lys49 myotoxins
from the genera Bolhrops, Agkistrodon, and
Trinieresurus. Structural and pharmacological
data on the family of class 11 PLA2 myotoxins is
expanding rapidly, and should lead to a better
uriderstanding of their mechanism of action,
gradually being iinraveled [4,5,25,26].
Comparison of the N-terminal sequence of A .
ntlrnrnifer rnyo toxin I I wi th o ther PLA2s reveaied
one novel arnino acid substitution, at position 53
(Asn), which is occupied by a Lys in atl other
Lys49 PLA2s described to date. 1n addition, this
niyotoxin is the first Lys49 variant presenting
Asn in its N-terminus. This position is occupied
by Ser in a11 other Lys49 PLA2s, and Asnl has
only been previously found in Asp49 PLA2s such
as t he Agkistrotloiz piscivorzcs piscivorzis D49
enzyme 1271, and the human class 11 secreted
PLA2 [28] Since A nlrninzij2r inyotoxin II dis-
plays a toxic/pharmacological activity profile
common to that of class II myotoxic PLAZs, our
rcsults suggest that neither Ser1 or Lys53 are
essential amino acid residues for myotoxic, cyto-
toxic, or edema-inducing effects of this family of
toxins.
Acknomlcdgernents
This work was supported by Grants from the
Interna tional Founda tion for Science (F/2766- 1),
Secretaria de Relaciones Exteriores de Mxico,
Bilateral Scieiltific Cooperation Program Costa
Rica-Mexico (302CR046), Vicerrectora de
Investigacin, University of Costa Rica (741-98-
5 18), and Consejo Nacional para f nvestigaciones
Cientficas y Tecnolgicas (CONICJT, 98-0 13-
FO) of Costa Rica
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1211 B Francis, J M Gtitirrcz, B Lomonte, I J Kaiser,
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Biochcm 26 (1994) 43-48
[35] Y Angulo, E. Chavcs, A Alape, A Rucavado, J M
Gutirrez, B Lomonte, lsolation and characterization
of a niyotoxic phospholipasc A2 from the vcnom of thc
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Costa Rica, Arch. Biochem Biophys 339 (1997) 26&
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venom, Nstt Toxins 3 (1994) 26-31
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ARTICULO 11
PERGAMON The Tntemationai Journal of Biochemistry & CeIl Biology 34 (2002) 1268-1278
www elscvicr corn/iocatc/jjbcb

Structural characterization and phylogenetic relationships of

myotoxin II from A tropo ides (Bothrops) nummifer snake
venom, a Lys49 phospholipase A2 homologue
Yamileth Angulo Timoteo Olamendi-Portugal ', Alberto ~ l a ~ e - ~ i r n " ~ ,
Lounval D. Possani ', Bruno Lomonte
a Instituto Clodomiro Picado, Facultad de Microbiologa, Universidad de Costa Rica, Sari Jos, Costa Rica
Departamento de Bioqumica, Esctrela de Medicina, Universidad de Costa Rica, San Jos, Costa Rica
Deparhnent of Molecular Recognition and Structural Biology, Instituto de Biotecnologa,
Universidad Nacional Atrt6noma de Mxico, Cuernavacu, Mexico
Received 12 February 2002 ; received in revised form 30 March 2002 ;accepted 17 April2002

Abstract
In order to analyze its structure-function relationships, the complete amino acid sequence of myotoxin II from Atropoides
(Bothrops) numrnifer from Costa Rica was determined This toxin is a Lys49-type phospholipase A2 (PLA2) homologue,
devoid of catalytic activity, stnicturally belonging to class IIA. In addition to the Asp49 -+ Lys change in the (inactive)
catalytic center, substitutions in the calcium-binding loop suggest that its fack of enzymatic activity is due to the loss of
ability to bind caz+.The toxin occurs as a homodimer of basic subunits of 121 residues. Its seqtience has highest similarity
to Lys49 PLA2s from Cerrophidion, Trinreresurus, Bothrops and Agkistrodon species, which form a subfamily of proteins
that diverged early from Asp49 PLA2s present in the same species, as shown by phylogenetic analysis. The tertiary structurc
of the toxin was modeled, based on the coordinates of Cerrophidion godmalzi myotoxin 11. Its exposcd C-terminal region
115-129 shows severa1 differences in comparison to the homologous seqiiences of other Lys49 PLA2s, i e. from Agkstrodon
p. piscivorus and Bottzrops asper. Region 1 15-129 of the latter two proteins has been implicated in myotoxic activity, on the
basis of the direct membrane-damaging of their corresponding synthetic peptides. However, peptide 1 15-1 29 of A nummfer-
myotoxin 11 did not exert toxicity upon cultured skeletal muscle cells or rnature muscle in vivo, Differences in severa1 amino
acid residues, either critica1 for toxicity, or influencing the conformation of free peptide 115-129 from A. ntlmmifer rnyotoxin
11, may account for its Iack of direct membrane-damaging properties.
O 2002 Elsevier Science Ltd Al1 rights reserved.
Keywords. Snake venom; Myotoxin; Phospholipase A2; Atropoides nuntmifer; Bothrops

1. Introduction tribute to prey digestion and cause significant tissue

Numerous snake species secrete toxins that induce
skeletal rnuscle necrosis (rnyotoxins), which con-
* Corresponding author Fax: +506-292-0485
E-mail address. blomonte@cariari ucr ac.cr (B. Lomonte)
damage in accidentally envenomed humans 11J. A
rnajority of myotoxins are basic phospholipases Az
(PLA2; EC 3.1.1.4) [2], which have been classified
into groups IB (from Elapidae/Hydrophidae) or IIA
(from CrotalidaeNiperidae), on the basis of their pri-
mary structure and disulfide bond pattern [3,4]. The

1357-2725/02/$ - see front matter O 2002 Elsevier Science Ltd Al1 rights reserved.
PII: S 1 3 5 7 - 2 7 2 5 ( 0 2 ) 0 0 0 6 0 - 2
 Y Angulo et al. /The hternational Jourrtal of Biochenlistry & Cell Biology 34 (2002) 1268-1278
group IIA PLAzs are further divided into two major
subgroups, namely, those with an aspartic acid residue
at position 49 (Asp49 PLA2s) and those in which
a lysine substitutes this position (Lys49 PLAzs), A
third, rninor subgroup has been described in which
the Asp49 residue is substituted by Ser, in the cases
of ammodytin L, a myotoxic/neurotoxic PLA2 from
Yipera ammodytes ammodytes [ 5 ] , and ecarpholin
S, a platelet-aggregating PLA2 from Echis carinatus
sochureki 16J .
Many observations indicate that the Asp49 PLA2
myotoxins are catalytically active, whereas, the Lys49
PLA2 homologues are either catalytically inactive,
or might display very low levels of enzymatic activ-
ity. It is still unclear whether the activity observed
in Lys49 PLA2 preparations should be attributed to
contarninant Asp49 PLA2s [5-8 ] or, alternatively, be
considered intrinsic [9,1 O]. In spite of their striking
difference in PLAz activity, Lys49 myotoxins have
molecular weights, isoelectric points, amino acid se-
quences, and inmunochemical properties that are sim-
ilar to those of their Asp49 counterparts, and induce
skeletal muscle alterations that are histopathologi-
cally indistinguishable from those caused by the latter
[ l l:I2].
Protein or cDNA sequences of toxic PLA2s have
been compared as an approach to predict the amino
acid residues involved in specific effects within this
functionally diverse protein family. In the case of my-
otoxic group 11 PLA~s,biochemical characterization
has demonstrated considerable inter- and intra-specific
variations of primary structure, which might provide
relevant clues for delineating their molecular determi-
nants of toxicity [13,14]. Therefore, the structural and
pharmacological characterization of novel myotoxic
PILA2 isoforms found in the tropical biodiversity has
been of interest over the recent years, for the purpose
of solving their complex structure-function relation-
ships.
Alropoides numrnfer (previously Bothrops num-
rnifer) is a terrestrial snake distributed along the cen-
tral American isthmus, commonly known as "jumping
viper" or "mano de piedra" [15]. Its venom causes
severe tissue damage, which involves hemorrhage,
edema, and myonecrosis [ 1 6J. Two myotoxic PLA2s
have been punfied from this venom. A. nurnrnifer
myotoxin 1 was characterized as a basic, dimeric, cat-
alytically inactive PLA2, suggesting that it belongs
1269
to the Lys49 subgroup [ 17,183. Although this toxin
has been crystallized, with preliminary X-ray diffrac-
tion data reported at 2.3-2.4 A resolution [19,20], its
complete amino acid sequence is lacking. A second
isoform, A. nurnrnfer myotoxin 11, was subsequently
isolated and characterized as a new Lys49 PLA2
variant, according to its N-terminal amino acid se-
quence [21]. In this report, the complete primary
structure, phy Iogenetic relationships, 3D model, and
properties of synthetic C-terminal peptide 115-1 29 of
A. numrnlyer myotoxin 11 are presented.
2. Materials and methods
2.1. Isolation of A. nummfer myotoxin 11
A. numrnfer venom was a pool obtained from more
than 15 specimens collected in Costa Rica. Myotoxin
II was purified by cation-exchange chromatography
on carboxymethyl-Sephadex C-25 (Pharmacia), as
described [21]. Homogeneity was assessed by poly-
acrylamide gel electrophoresis in the presence of
sodium dodecylsulfate (SDS-PAGE) f223, urea-PAGE
for basic proteins [23], and reverse-phase high per-
formance liquid chromatography (RP-HPLC) on a C4
column (Vydac), eluted at 1 .O ml/min with a gradi-
ent from O to 60% acetonitrile in 0.1% TFA, using a
model 6OOE Waters instrument.
2.2. Reduction and carboxymethylation
An aliquot (usually 2 rng of myotoxin 11) was dis-
solved in 1 m1 of 0.2 M Tris-HCl buffer, pH 8.6,
containing 6 M guanidine chloride plus 1 mglml
of EDTA. One drop of 1-octano1 was added and
the solution purged with a stream of nitrogen for
IOmin. Freshly prepared dithiothreitol (200mM)
was added to a final concentration approximately 10
times higher than that of the expected thiol groups
of the protein, and incubated for 45 min at 55 "C.
Alkylation with iodoacetic acid (at 10-fold excess to
the thiol groups available) was thereafter conducted
in the same buffer 0.2M Tris-HCI, pH 8.6. The
protein was then desalted on a Biogel P30 column
(Bio-Rad) previously equilibrated with 20% acetic
acid, eluted with the same solvent, and lyophilized
[34].
1270 Y Angrclo et al. /The lnternationul Journul of Biocherrtistry & Cell Biology 34 (2002) 1268-1278
2.3. Digestion with arginine-C, aspartate-l\r
lysine-C and chyrnotrypsin proteinases
The reduced and carboxymethylated protein was
digested with severa1 endopeptidases (Boehringer-
Mannheim, Gerrnany). About 0.5-1 .0 rng of reduced
and alkylated toxin were incubated with the following
endopeptidases at a ratio of 20: 1, and incubated in the
buffers and times indicated: Clostridium histulyticum
arginine-C (100mM Tris-HC1 buffer, pH 7.6, con-
taining lOmM CaC12, for 4 h at 37 "C); aspartate-N
proteinase (50mM phosphate buffer, pH 8.0, for 4 h
at 37 "C); lysine-C (50 m M Tris-HC1, I M EDTA
buffer, pH 6.5, for 3 h at 37 OC); and chyrnotrypsin
(100mM Tris-HC1 buffer, pH 8.0, for 4 h at 37C).
Purification of the resulting peptides was performed
by HPLC on a reverse-phase C18 colurnn (Vydac,
Hisperia, CA), previously equilibrated with 0.12%
trifluoroacetic acid (TFA) in water and run with a
linear gradient from this solution (solution A) to 60%
solution B (acetonitrile in 0.10% TFA), for 60 min, at
a flow rate of 1 ml/min.
2.4. Amino acid sequence
The complete amino acid sequence determina-
tion of myotoxin II was carried out by a combina-
tion of direct Edman degradation, using a Millipore
6400/6600 automatic sequencer with native and re-
duced and alkylated protein, plus the sequencing of
rnany sub-peptides obtained by speci fic endopeptidase
cleavage, as mentioned in the previous section. Po-
sitions of cysteines were al1 confirmed by the identi-
fication of the carboxymethylated residues. Once
the sequence was completed, isoelectric point and
molecular weight o the protein were calculated us-
ing the Compute pI/MW tool at ExPASy (http://ca.
expasy.osgltools/pi_tool.htiitl) [25].
A model of the 3D structure of A. nummlifer myo-
toxin II was constructed, using as a starting geometry
the crystal coordinates of Cerrophidion godmani my-
otoxin 11 obtained at 2.8A resolution (Protein Data
Bank code 1GOD; [Zh]), which shares 90.9% se-
quence identity. The toxin sequence was submitted to
the Swiss-rnodel server (http:li'www e~pasy.~h/spdb\l/
rnainpagc.htn-il), and the resulting structure file was
examined with Swiss-PDBViewer v.3.5 1 (Glaxo Well-
come Experimental Research) and WebLabViewer
v.4.0 (Molecular Simulations Inc.).
2.6. Sequence alignrnents and phylogenetic
analyses
Similarity searches for A. nummfer myotoxin II
were performed using FASTA [27], and amino acid
sequences were aligned using the program CLUSTAL
W E281 with a few gap adjustments. Phylogenetic
trees were calculated using PROTDIST [29] and the
neighbor-joining algorithm [3O], and the tree dia-
grams were generated with DRAWTREE 1291.
2.7. Peptide 115-129 synthesis
The 13-mer peptide KNYKIYPKPLCKK, corre-
sponding to residues 105-1 17 of A. nummifer myo-
toxin 11 (referred to as 115-129 in the numbering
system of Renetseder et al. [31]) was synthesized
using F-moc strategy, with free amino and carboxyl
endings (Mimotopes, Australia). Its molecular mass
(I623.0), as estimated by mass spectrometry, was in
agreement with the theoretical value (1623.1), and its
final purity was 97% by RP-HPLC analysis. The pep-
tide was kept dry at -20 "C, and dissolved in 0.12 M
NaCl, 40 mM sodium phosphate (PBS), pH 7.2, irn-
mediately before being tested for pharmacological
activities.
2.8. Pharmacological activities
The ability of peptide 115-1 29 of A. nurnmifer myo-
toxin 11 to induce cytolysis of C2C 12 skeietal muscle
cells in culture was evaluated as previously described
[32], using the release of lactic dehydrogenase (EC
l . 1.1.27) after 3 h, as a marker of irreversible cell dam-
age. A. nummifer peptide 115-129 was tested at con-
centrations up to 150 pg/well(l mg/ml). A. nummijier
whole myotoxin II or a synthetic peptide 115-1 29 of
B. asper myotoxin 11 were utilized as positive controls
for cytotoxicity. Assays were canied out in duplicate
cultures, in two independent experiments. In addition,
the in vivo effect of peptide 115-129 upon mature
skeletal muscle was evaluated by its jntramuscular
injection (up to 250 y g in 100 pl of physiologic saline)
 Y Angttlo et al /The Internationul Joirrnul of Biochenistry Le Cell Biology 34 (2002) 1268-1278 127 1

into the gastrocnemius of mice (1 8-20 g body weight;
n = 3), and subsequent detennination of plasma
creatine kinase (EC 2.7.3.2) activity after 3 h [ 3 3 ] ,
Control animals received a similar injection without
peptide.
3. Results
The amino acid sequence of A. nummifer myotoxin
11 is as shown in Fig. f . The first 53 residues were
determined by direct N-terminal sequencing, whereas,
overlapping peptides generated by proteolysis with
Arg-C, Asp-N, Lys-C and chymotrypsin proteases
allowed cornpletion of the primary structure of this
toxin (Fig. 1). Most of the peptides obtained from
enzymatic cleavage with endopeptidases were se-
quenced, although Fig, 1 only includes the results of
peptides needed for unequivocally positioning the futl
sequence, for clarity. The calculated molecular weight
and pI of A. nummfer myotoxin II are 13,712 and
8.7, respectively. The sequence was subrnitted to the
SwissProt database, and assigned the code P82950.
The amino acid sequence of A. nzrmmifer myotoxin
II contains all the conserved residues of Lys49 vari-
ants, including Leu5, Glnl 1, Asn28, Arg34, Lys49,
Lys53, Thr56, Ser74, Trp77, Lysll5, Lys122 and
Lys128, and al1 14 Cys residues (Fjg. 2). Position 53
had been earlier reported as Asn [21], and is here
conected to Lys. A conservative substitution is found
at position 108, where most Lys49 variants have
Glu, while A. nzlrnmifer rnyotoxin II has Asp. How-
ever, at position 86 (Fig. 2), where most Lys49
proteins have an acidic residue, this toxin has Lys.
The sequence identity of this toxin with other Lys49
variants ranged from 90.9 to 58.0%, whereas, the
sequence identity with Asp49 enzymes ranged from
58.0 to 35.0%.

5 1O 15 20 25
Asn Leu Tyr Gln Leu Trp Lys Met IIe Leu Gln Glu Thr Gly Lys Asn Ala Ala Pro Ser Tyr Gly Phe Tyr Gly
N-terminus------------------------------------------------------------
-----------------*-------------------------------------

30 35 40 45 50
Cys Asn Cys Gly Val Gly Ser Arg Gly Lys Pro Lys Asp Ala Thr Asp Arg Cys Cys Phe Val His Lys Cys Cys
---------------------------------N-terminus-k-------------L-------------------------------

SS 60 65 70 75
Tyr Lys Ala Leu Thr Asp Cys Ser Pro Lys Thr Asp Ser Tyr Ser Tyr Ser Trp Lys Asp Lys Thr Ile Val Cys
/
-m------------ D----------- /

80 85 90 95 100
Gly Lys Asn Asn Pro Cys Leu Lys Gln Glu Cys Glu Cys Asp Lys Ala Val Ala Ile Cys Leu Arg Asp Asn Leu
/
---------------"--*---4----------DDD------------------------------ ------ - K---- -- --- --- -- - -- --- - ---

105 110 115 120

Asp Thr Tyr Asn Lys Asn Tyr Lys Ile Tyr Pro Lys Pro Leu Cys Lys Lys Ala Asp Asp Cys
--------------------/ K- / ---------m-

Fig 1. Amino acid sequence of A. nurnmger myotoxin 11. The first 53 amino acid residues were determined by direct seqiiencing by
Edman degradation from the N-terniinus Overlapping peptjdes were generated by enzyrnatic cfeavage of the toxin with the following
proteinases: (D) aspartate-N, (R) arginine-C, (K) lysine-C, and (C) chymotrypsin, as described in Sectioii 2
1272 Y Angulo et al /The Inten~ationalJournal of Bioche~?iistry& Cell Biology 34 (2002) 1268-1278

An initial phylogenetic analysis of 73 class 11
PLA2 sequences available at SwissProt and TrEMBL
databases revealed that the Lys49 variants constitute
a monophyletic group, having a closer relationship
with a subset of class II Asp49 PLA2s (not shown).
A multiple sequence alignment of that subset and
the Lys49 variants (Fig. 2) was utilized to build a
phylogenetic tree, delineating the relationships of A.
nurrzmifer myotoxin II (PA22-ATRNU) with other
proteins of the PLA2 family (Fig. 3). The tree indi-
cates that al1 Lys49 PLA2s are divergent from Asp49
counterparts present in old world (Viperidae) and new
world (Crotalidae) snakes, and shows that Lys49 vari-
ants are more closely related to the two known Ser49
variants (ammodytin L and ecarpholin S), sharing a
common ancestor with them. A. nummger myotoxin
11 and C. godmani myotoxin II are closely related,
and diverged before the branch which contains the
sequences of the Lys49 variants present in several
Bothrops species (Fig. 3 ) .
The highest protein similarity for A. nzrmmifer my-
otoxin 11 was obtained with C. godmani myotoxin 11
(90.9% identity). Based on the crystal structure of the
latter, a model of the tertiary structure of A. nzcm-
mger myotoxin 11 monomer was constructed (Fig. 4).
Only very small deviations from the C. godmani toxin
a-carbon atorns were predicted (total rmsd = 0.08 A).
The model indcates that the C-terminal region of
the A. nummiJer protein, combining cationic and hy-
drophobic residues, is highly exposed, in similarity
with the structures of Lys49 PLA2s so far character-
ized. The 3D structure of the C-terminal region, which
is the most variable in this group of proteins, appears
virtually superimposable to that of the C. godmani my-
otoxin 11 template (backbone rmsd = 0.08 A). The se-
quence 11 5-1 29 is 100% identical between these two
proteins.
Interestingly, the free synthetic peptide 1 15-1 29
of A. nummlfer myotoxin 11 did not induce cell lysis
in the assay utilizing C2C12 cells (Table i), even at
doses as high as 150 pglwell (1 mg/ml). Microscopic
observation of the cultuxes confirmed the lack of mor-
phological celI alterations. In this assay, A. nzrmrnijier
myotoxin II caused 40 f 5% toxicity at 10 pglwell,
and 102 f 4% toxicity at 20 pglwell, while the syn-
thetic peptide 1 15-129 of B. asper myotoxin 11 in-
cluded as a control, reached 93 -t 6% toxicity at the
dose of 100 pglwell. On the other hand, the i.m. in-
jection of 250 k g of A. nurnmifer peptide 1 15-129 in
mice did not increase plasma creatine kinase values,
in cornparison to control animals, indicating its lack
of rnyotoxic activity (data not shown).
4. Discussions
The amino acid sequence of myotoxin II from A
nummifer snake venom shows that it is a PLA2 homo-
logue of the Lys49-type, which belongs to the struc-
tural class IIA of this protein family. It was previously
reported that this myotoxin lacks enzymatic activity
[21], explained by the critica1 substitutions in both the
(inactive) catalytic center of the rnolecule (Asp49 -+
Lys), and the calcium-binding loop, precluding its

Fig. 2. Sequence alignment of A nzrrnnlifer myotoxin 11 with other Lys49 variants and a subset of Asp49 phospholipases A2 from
viperid/crotalid venoms. Sequence numbering at the top refers to [3I]. Accession numbers in trEMBL (tr) or SwissProt (sp) databases
are indicated on the Iefi. Residues conserved arnong the Lys49 variants are printed against a black background Residues conserved in al1
sequences are indicated by an asterisk at the top. Positions conserved in al1 sequences are printed against a gray background: FA-ATRNU,
A nummger myotoxin 11 (present study); PAX-TRIOK, Trimeresurus oht~avensisK49; PA2BWTRIMU, Trinzeresilnrs mucrosquamrrfta
K49; PAZHAGKPI, Agkistrodon p piscivorus K49; PA2M-AGKCL, Agkistrodon con2ortrix lnficinctrts K49; PA2B_TRIFL, Ti.i~nerestt-
rus Jlavoviridis basic protein-I; PA22-CERGO, Cerrophidion godmani myotoxin 11; PAZ-BOTAS, Bothrops asper K49; PAZ-BOTMO,
Bothrops moojeni myotoxin-1; PA22_BOTAS, Bothrops osper rnyotoxin 11; PAZ-BOTPI, Bothrops pirujui piiatoxin 11; PAZ-BOTJA,
Bothrops jararacussu bothropstoxin 1; PAAGKAC, Agkistrodon acuius K49; PAY-TRIGR, Trimeresurus grarninezrs K49; PA25-TRIGA,
Trimeresurus gramineus PLA2-V; PAW-TRIGR, Triniereslrnrs gramineus PLA2-V'; PA2N_ECHCA, Echis cannntus ecarphoiin S;
PA2L_VIPAA, Vipera a~nmodytesammodytin L; PAZ-TRIMU, Trirneresttrus mucrosquariiutus D49; PA23_AGMP,Agkistrodon halys pullus
agkistrodotoxin; PA2C_CRODU, Crotalus durissus terr$cus crotoxin subunit B; PA2B-CRODU, Crotalus durisslrs terr[ficus crotoxin
subunit B'; PA2N-CROSS, Crotalus scutulutus scutztlatus mojave toxin subunit B; PA2C_VIPAA, Kperu antnroclyres crrnmodytes ammody-
toxin C; PA2A-VIPAA, Kpercr nntmolyfes ammodytes amrnodytoxin A; PA2B_VIPAA, fipera ummorlytes mnrnodytes ammodytoxin B;
PA21 BOTAS, Bothrops asper myotoxin 111; PA21-BOT.JR, Bothrops jurar~zctrsstrD49; PA2 1 -AGKHA, Agkistrodon halys bkornllofi D49;
PAZX-TRIFL, TrimeresurtcsJnvoviriciis PL-X; PA2Y_TRIFL, Trintereslir~rs flavovir-idis PL-X'; PAZ1 -BOTJA, Bothrops jm-umca BJ-PLA2;
PA21+TRIFL, Trinteres~trusflavoviridis isozyine 1; PAZ-TRIFL, Trir~rerestvtrsflavoviridis Amami-Oshima PLA2.
ir PAJTRNU E T G K N A A P S
o
. Y G F Y G
. a * *
G K
1

P K
.
D
m
A
.
T
.
D R
..
C C F V H
m
. - - D C S + - +
Ir PAX-TRIOK E M G K G A A K K . Y G L Y G G R P Q D A T D R C C S V H .- D C D - - .
sp PA2B-TRIMU E T G K N P V K N . Y G L Y L Q K P V D A T D R C C F V H - - . G C D - - .
sp PAZH-AGKPI E T G K N A I T S - Y G S Y G G Q P K D A T D R C C F V H - -. D C N - - -
sp PADMJGKCL E T G K N A I T C Y G S Y G G ~ P K D A T D R C C F V H . - D C N - -
.. . G C D - - .
sp PA20-TRIFL
sp PA22-CERtO
E T G K
E T G K
E A A K
N A V P
N
S
-
-
Y G
Y O
L Y G
L Y G
O
Q
K
K
P
P
K
K
D
D
A T D S C
A T D R C
C
C
Y
F
V
V
H
H 1 - -- D C S -
- . . G C N -
-- -.
ir PAZ-BOTAS E T G K N P V T S . Y G A Y G G K P K D A T D R C C Y V H
V PAZ-BOTMO E T G K N P A K S - Y G A Y G G K P K D A T D R C C Y V H - .. N C D - - .
sp PM2-BOTAS E T G K N P A K S - Y G A Y O G K P K D A T D R C C Y V H G C N - - .
't PALBOTPI E T G K N P A K S - Y G A Y G G K P K D A T D R C C Y V H
tr PAZ-BOTJA E T Q K N P A K S - H O A Y G Q K P K D A T D R C C Y V H - - - G C D - - -
b PA-IGUC E T G K N P V K N - Y G L Y G G E P L D A T D R C C F V H - . . D C Q - - .
tr PAY-TRlGR E M G K N A L T S - Y S L Y G R K P M D A T D S C C Y V H . . D C ( J - . .
sp PA25,TRIQA E T G K N P A T S - Y G L Y G R K P K D A T D R C C Y V H - . D C D - - .
tr PAW-TRIGR E T G K N P A T S - Y G t Y G R K P K D A T D R C C Y V H 1-. . D C O - - .
sp PA2N-ECHCA E L 0 E T G K S P F P S - Y T S Y G C F C G G G E R G P P L D A T D R C C L A H .- . D C S - -
rp PA2LVIPAA E F G E T O K N P L T S . Y S F Y G C H C G L G N K G K P K D A T D R C C F V H S C C . - . D C S . - .
r PAZ-TRIMU O F R A T G K E P L T T - Y L F Y A C Y C G W Q G R G E P K D A T D R C C F V H D C C - - - A C S - - -
sp PA23JGKHP Q F N E T G K N A I P F - Y A F Y G C Y C G G G G Q G K P K D O T D R C C F V H D C C . N C N - - .
sp PA2C-CRODU a F N E T R K N A V P F - Y A F Y G C Y C G W G G Q G R P K D A T D R C C F V H D C C - . - K C N - - -
sp PA25-CRODU Q F N E T R K N A I P F - Y A F Y G C Y C Q W Q Q R G R P K D A T D R C C F V H D C C . . - K C N - . -
sp PA2N-CROSS a F N E T R K N A f P F Y A F Y G C Y C G W G G R C R P K D A T D R C C F V H D C C - - - K C N - - -
sp PAiC-VIPAA E F G E T G K N P L T S . Y S F Y G C Y C G V G G K G T P K D A T D R C C F V H D C C . . - D C S . . .
sp PA2A-VIP.44 E F G E T G K N P L T S ~ Y S F Y G C Y C G V G O K G T P K D A T D R C C F V H D C C . - - D C S - - -
sp PA28-VIFA4
Sp PA21-BOTAS
E F G
E F A
E
E
T
T
G
K
K
R
N P L T
L P F P
S - Y S F Y G C Y C G V G G K O T P K D A T D R C C F V H D C C
Y p Y T T Y G C Y C G W G G Q G U P K D A T D R C C F V H D C C
- -
- -
- .
- D C S - -
- N C K -
- N C K -
.- .-
sp PA21-BOTJR Q F G E T G K L P F P Y - Y T T Y G C Y C G ~ V G G ~ G Q P K D A T D R C C F V H D C C +
sp P.421-AGKHA O F R M T G K E P V I S - Y A F Y C C Y C G S G G R G K P K D A T D R C C F V H D C C - . - G C K - - -
sp PA2XTRIFL Q F R M T G K E P I V S - Y A F Y G C Y C G K G Q R G K P K D A T D R C C F V H D C C . . - Q C D . -
sp PMY-TRIFL O F R M T G K E P I V S - Y A F Y G C Y C G K G G R G K P K D A T D R C C F V H O C C - - - G C D . . .
sp Pul-BOTJA a F G V M R E Y V V F N Y L Y Y G C Y C E W G G I G K P R D A T D R C C F V H D C C - - - G C N - -
SP PA21-TRIFL Q F E V V K K S G t L S ~ Y S A Y G C Y C G W Q Q R ~ K P K D A T D R C C F V H D C C - . Q C N - - -
P PAZTAIFL Q F E V V K K S G I L .- . G C N - - .
. -
S - Y S A Y G C Y C G W G G R G K P K D A T D R C C F V H D C C

10 ,m 1D
sp PA-AYRNU C - G K N N P - C C K O E C E C D
b PAX-TRIOK C G G D D P - C T K E V C E C D
sp PUB-TRIMU C G E K N P P C L K O V C E C D
sp PA2H-AGKPI C - E E K N P - C L K E M C E C D
sp PAZM-AGKCL C - E E K N P - C L K E M C E C D
op PA2B-TRIFL C - G E K N P P C L K O V C E C D
sp PA22-CERCO C G D N N P - C L Q E M C E C D
ir PAZBOTAS C G E N N S - C L K E L G E C D
Ir P ~ B O r n O C - G E E N P - C L K Q L C E C D
sp PAZ-BOTAS C - G E N N S - C L K E L C E C D
tr PAZ-BOTPI C - G E N N P - C L K E L C E C D
R PAZ-BOTJA C . G E N N P - C C K E L G E C D
b PAJGKAC C - G K N Q P - C M O E M C E C D
ir PAY-TIGR C - G E K N P - H L K E L C E C D
sp PA25TRIGA C - G E D N P - C L K E M C E C D
ir PAW-TRIGR C G E O N P - C C K E M C E C D
sp PAZN-ECHCA C - E N S T S - C K K R I C E C D R K N L N T Y N K K - Y T - Y Y P N F W - C K G D
sp PAZLV1PAA T N R Y E Y H R E - - N G C - G S S T P - C K K Q I C E C D R E N L K T Y N K K - Y K - V Y L R F K - C K G V
ir PAZ-TRIMU 1 D M Y T Y S Q K . - N E C . G G D D P - C K K E I C E C D L N N L G T Y N - E - E Y N N Y R K S R - C I E E
sp PA23AGKHP S D I Y S Y S L K - - E Q C G K G T N - C E E Q I C E C D R R N L D T Y N N G - Y M - F Y R D S K - C T E T
sp PMC-CRODU W D I Y R Y S L K - - S O C G K O T W - C K E Q I C E C D R R S L S T Y K N E - Y M - F Y P D S R - C R E P
sp PA28-CRODU W D I Y P Y S L K . - S G C - G K G T W - C E E Q I G E C D R R S L S T Y K Y G - Y M - F Y P D S R - C R G P
sp PA2N-CROSS W D I Y P Y S L K - - S G C - G K G T W - C E E Q I C E C D R R S L S T Y K Y G - Y M - F Y P D S R - C R G P
sp PA2C-VI PAA T D R Y K Y H R E - - N G C - G K G T S - C E N R I C E C D R K N L K T Y N Y I - Y R - N Y P D I L - C K E E
sp PA2A-VIPAA T D R Y K Y H R E - - N G C - G K G T S - C E N R I C E C P R K N L K T Y N Y I - Y R - N Y P D F L - C K K E
sp PAPB-VIPAA T D R Y K Y H R E - - N G C - G K G T S - C E N R I C E C D R K N L K T Y N H I - Y M - Y Y P D F L - C K K E
s p PA21-BOTAS T D R Y S Y S R K - - S G C - G E G T P - C E K Q I C E C D R E N L R T Y K K R - Y M - A Y P D L L - C K K P
sp PA21-BOTJR T D R Y S Y S R E - - N G C - G E G T P - C E K Q I C E C D R E N L R T Y K K R - Y M - A Y P D V L - C K K P
sp PA21JGKHA W D D Y T Y S W K - . N O C G G D D P - C U K E I C E C D R D N L K T Y K K R - Y M - A Y P D I L - C S S K
sp P A X T R I F L W S Y Y T Y S L E - " N G C - G G D P Y - C T K V K C E C D R D N L K T Y K N R - Y M - T F P D I F - C T D P
sp PA2Y-TRIFL W D Y Y T V S S E - - N O C . G G D N P - C T K E V C E C D R D N L K T Y K K R - Y M - T F P D I F - C T D P
sp PA21-BOTJA T D S Y T Y T Y S E E N G C G G D D L - C K K Q I C E C D R D N K D T Y D T K - Y - W C Y Q A K N - C O E E
sp PA2I-TRIFL L G K Y T Y S W N - - N G C E G D G P - C K - E V C E C D R D N L D T Y D R N - K Y W R Y P A S N - C Q E D
b PAZ-TRIFL L G K Y T V S W N - - N G C E G O G P - C K - E V G E C D R D N L D T Y D R N - K Y W R Y P A S N - C D E D
1274 Y Anglrlo et al /The ln~ernationnlJolirnul of Biochenzistry & Cell Biology 34 (2002) 1268-1278

ability to bind the caz+ cofactor. This toxin occurs
as a honlodimer of basic siibunits of 121 amino acid
residues, displaying cytoiytic activity in vitro, as well
as myatoxic and edema-forming activities in vivo E2 13.
The toxin has highest sequence identity to other
Lys49 PLA2s from Cerrophidion, Trimeresurus,
Bothrops and Agkistrodon species. PhyIogenetic anal-
yses indicate that the Lys49 PLA2s form a subfamily
of proteins that diverged early from that of Asp49
PLA2s present in the venoms of the same species. A
higher seqiience identity exists between Lys49 PLA2s
isolated from the venoms of different genera, than be-
tween the Lys49 and the Asp49 PLA2 isoforms found
in a single species, as observed by Francis et al. [34].
Interestingly, the phylogenetic tree constnicted with
a group of 34 class IIA PLA2s in the present study,
places the Lys49 variants in closer relationship with
the Ser49 PLA2s from the old world genera Epera and
Echis, suggesting a common ancestor that branched
after their divergente from the ancient Asp49 PLA2s.
DetaiIed analyses on the genes encoding Asp49
and Lys49 PLA2s of Asian Trimeresurus species
have disclosed an accelerated evolution mechanism
[35--371, The adaptive value of the fast evolution from
catalytically-active, class IIA myotoxic Asp49 PLA2s,
to catalytically-inactive, and still myotoxic, Lys49 or
Ser49 PLA2s is ciirrently unknown, Studies with a
number of basic Asp49 and Lys49 PLA2 myotoxic
isoforms isolated from various Bothrops species have
shown that their potency and spectrurn of toxic activ-
ities are similar [ l 11, making it unlikely that the latter
would have evolved to acquire enhanced toxicity.
A. numrnqer myotoxin II shows the dosest phy-
logenetic relationships with myotoxin 11 of C. god-
ntani, aiso distributed in Central America, and with
the Lys49 PLA;! isoforms, BP-1 and BP-11, fiom
the Asian pit viper T Jiavoviridis. Its relative sepa-
ration from the cluster formed by Lys49 PLA2s of
both Central American and South American Both-
rops species (.e. B. asper myotoxin II, B. moojeni
myotoxin 1, B. jararacussu bothropstoxin 1, and B.
pirajai piratoxin 11) in the phylogenetic tree, would
be in agreement with the taxonomical division that
proposed the change from its forrner genus Bothrops
to the new genus Atropoides [3 81.
The high sequence identity of A. nurnrnifer my-
otoxin 11 with C. godmani myotoxin IJ allowed the
construction of a reliable modef of the 3D stmcture
of the forrner. The predicted stmcture of the toxin
monomer follows almost identically that of the tem-
plate protein, with an rmsd for a-carbon atoms of
0.08 A. Even at the C-terminal region, which shows
greatest sequence variability among different Lys49
PLA2s, the predicted stmcture of A. nummfer my-
otoxin 11 is virtually superimposable to that of the
C. godmani protein, due to their high identity. The
C-terminal region is of particular interest in Lys49
variants, as previous studies have shown that synthetic
3-mer peptides corresponding to residues 115-1 29
exert direct rnembrane-darnaging activities in the case
of two myotoxins of this group: B. asper myotoxin IT
1393 and Agkistrodon p. piscivorus Lys49 PLA2 [333.
Since the sequence 115-1 29 of A. nzrrnrnifer myotoxin
TI showed several differences with those myotoxins,

Table 1
Sequence 115-129 of A nurnrnifer myotoxin II compared to other myotoxic Lys49 phospholipases A2 for which corresponding synthetic
peptides display toxic activiy
Myotoxic PLA2 Residue numbeP Peptide References
toxicityb
115 116 117 118 119 120 121 122 123 124 125 128 129
-- - -- - -- -. -

A p piscivorus Lys49 PLA2 M K Y K A Y F K L K C K K High E331

B asper myotoxin 11 K K Y R Y Y L K P L C K K L o w [33,J I ]
A nrinrmijkr myotoxin 11 K N Y K I Y P K P L C K K None Present
study
"esidue nurnbering according to [Li 11; identical residues in the three proteins are shown in bold.
Synthetic peptide 1 15- 129 of A p piscivortrs Lys49 PLAz causes half-maximal cytotoxicity at approximately 12 pgwell (-45 pM)
iipon C2C12 cells in vitio, and induces myonecrosis in mice. Synthetic peptide 115-129 of B asper myotoxin 11 displays lower cytotoxic
activity (half-maximal effect at -250 pM),and does not induce myonecrosis in vivo. N o toxic effects were recorded after testing synthctic
peptide 115-129 of A nuninliJer myotoxin II under the same assay conditions (as described in Scction 2), either in cell culture or in vivo.
 Y A~rguloet al. /Tire /nlernational Journul of Biocheniistry & Cell Biology 34 (2002) 1265-1278

0UfgTOl.l P
r PAZ CAEEL
PAZ0 VlPAA
PA2C VlPAA
PA2A VIPAA
PA21 BOTAS
PA21 BOTJR
FA23 AGKH P
PA2C CRODU
PA2B CRODU
PA2N CROSS
PAZ1 AGKHA
PA2X TRlFL
PA2Y TRIFL
PAZ TRIMU
PA21 BOTJA
PA21 TRlFL
PAZ TRlFL
PA2L VIPAA
PA2N ECHCA
PAX TRIOK

- PA28 TRlMU
PA2H AGKPl
- PA2M AGKCL
PA AGKAC
I PAY TRlGR
PAW TRlGR

- b
PA25 TRlGA
PA2B TRlFL
PA ATRNU
PA22 CERGO
b
b PAZ BOTMO
i PAZ BOTAS
P

- PA22 BOTAS
PAZ BOTPl
PAZ BOTJA

Fig 3. Phylogenetic tree relating A nummfer myotoxin TI to other Lys49 variants from viperid venoms, and a subset of Asp49 phospholipases
Az; the prirnary structures (legends as described in Fig. 2) were aligned with the program CLUSTAL W, and the tree was built with
PROTDIST, using the sequence of Cuenorhabditis elegans PLA2 as an outgroup. The length of branches is proportional to the mutational
distance

it was of interest to evafuate the possible pharrnaco- skeletal muscle cells or mature muscle tissue in vivo.
logical activities of its corresponding synthetic pep- Therefore, this Lys49 PLA2 presents a case where the
tide. Interestingly, the peptide 1 15-129 ofA. nunzmi$er toxic actions of the whole moIecule are not reproduced
myotoxin TI was devoid of toxic effects upon cultured by its free C-terminal peptide. Moreover, since the
Fig. 4. The 3D model of A. nummger myotoxin 11. The rnodel was built with the Swiss-model server, based on the structure of C. godmani
myotoxin 11 (PDB code IGOD), which shares 90.9% sequence identity: (A) ribbon representation of the A nummifer myotoxin 11 model;
(B) a-carbon backbone representation (gray) showing the amino acid side-chains of the cationichydrophobic region 1 15-1 29 (black).
region 1 15-1 29 of A. numrnifer myotoxin 11 is iden-
tical in C. godmani myotoxin II, the present observa-
tions can also be extrapolated to the latter protein.
As summarized in Table 1, severa1 arnino acids
differ between the non-toxic peptide 115-129 of A.
numrnifer rnyotoxin 11 (and C. godmani rnyotoxin 11),
the moderately toxic homologue peptide of B. asper
rnyotoxin II, and the more potent peptide of A. p. pis-
civorus Lys49 PLA2. AIthough there is a difference
in the net positive cbarge of these three peptides (+5,
+6, +7, respectively) that would appear to correlate
with their toxic ability, the net charge alone should not
be sufficient to explain the variations in toxic activity.
In support of this assumption, it was earlier demon-
strated that peptide 115-129 of B. asper myotoxin II
increases its toxic potency by one order of magnitude
after substituting its cluster of three Tyr residues by
Trp, without altering its net positive charge of +6 [41)].
The lack of direct toxic effects of peptide 115-1 29
of A. numrnifer myotoxin 11, which is in contrast to the
activity of corresponding peptides of two other Lys49
myotoxins 133,391, may orginate on differences in
amino acid residues that could either be critica1 for
the toxic mechanicm itself, or that could significantly
influence its free confonnation in solution. There-
fore, the present findings do not necessarily exclude
a participation of the C-terminal region of A. num-
mfer myotoxin 11 (and C. godmani myotoxin II) in
the toxic mechanism exerted by these proteins. It
should strictly reflect that their free segment 115-1 29
is unable to mimick the actions of the whole toxins.
Further analyses and approaches will be necessary to
conclusively establish whether the C-terminal region
of A. numrnifer myotoxin II (and C. godmani my-
otoxin 11) is involved in their toxicity, as would be
expected in the light of evidence obtained with the
Lys49 myotoxins of B. asper [ 3 9 4 3 ] and A. p. pis-
civorus 1331 venoms, or if other molecular regions
are responsible for their toxic actions.
Acknowledgements
We thank the International Foundation for Sci-
ence (F/2766-1), Secretaria de Relaciones Exteri-
ores de Mxico, Bilateral Scientific Cooperation
Program Costa Rica-Mexico (302CR046), Vicer-
rectora de Investigacin, University of Costa Rica
(VI-741-99-269), and Consejo Nacional para In-
vestigaciones Cientficas y Tecnolgicas (CONICIT
98-013-FO) of Costa Rica, for supporting these stud-
ies. We also acknowledge the technical assistance of
Dr. Fernando Zamudio duxing amino acid sequence
determination. This work was perforrned in partial
fulfillment of the doctoral degree of Y. Angulo at the
University of Costa Rica.
 I: Angulo et u1 /The Internationul Journal of Biocheniistry & Cell Biology 34 (2002)
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[la] J M. Gutirrez, F Chaves, J A. Gen, B. Lomonte, Z
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[19] R K . Arni, J.M Gutirrez, CrystaHization and prelirniiiary
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[20] W.F. de Azevedo, R J Ward, J.M Gutirrez, R K Ami,
Structure of a Lys49-phospholipase A2 homologue isolated
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[ 2 1 j Y Angulo, T. Olamendi-Portugal, L D Possani, 3
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[22] U K. Laernmli, Cleavage of structural proteins during the
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[23] P Traub, S. Mizushima, C.V. Lowry, M. Nomura, Recons-
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[24] B M. Martin, E Carbone, A Yatani, A M Brown, A N.
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[25] B Bjellqvist, G J. Hughes, C Pasquali, N. Paquet, F Ravier,
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[263 R IIK Arni, M R M Fontes, C. Barberato, 3 M Gutikrrez, C.
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1321 B Lomonte, Y Angulo, S Rufini, W. Cho, J.R Giglio,
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1331 C E. Nez, Y. Angulo, B. Lomonte, Identification of the
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[38] S Werman, Phylogenetic relationships of central and south
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Maccarana, Neutralizing interaction between heparins and
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Biophys Acta 1461 (1999) 19-26.
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J P. GorveI, E Moreno, Bactericidal activity of Lys49 and
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snake venom: synthetic Lys49 myotoxin II-(115-129)-peptidc
identifies its bactericidal region, Eur J Biochem 253 (1998)
452-46 1
CULO
 Available online a t www.sciencedirect.com
DIOCtIIMICA I
X iI1OPtIYSICA ACTA

scILNcE @ D I A E O i e

ELSEVIER Biochimica et Biophysica Acta 1703 (2004) 87-89

http://www elsevier corn/locnte/bba

Short crystallization paper

Crystallization of the Lys49 PLA2 homologue, myotoxin 11,

fi-om the venom of Atropoides nummifer
Leandra Watanabea, Lisandra M. Gavaa, Yamileth ~ n ~ u l o ~ ~ ' ,
Bmno ~ o r n o n t e Raghuvir
~, K. ~ r n i ~ . *
"Departmentof Physics, IB/LCE/UNESe R Cristovo Colornbo 2265, Sfio Jos do Rio Preto, SF: CEP 15054-000, Rrazil
b~nstitutoClodorniro Picado, Facultad de Microbiologia, San Jos, Costa Rica
cDepartamento de Bioqumica, Escuela de Medicina, Universidad de Costa Rica, San Jos, Costa Rica

Received 25 May 2004; rcceived in revised fom 9 September 2004; accepted 10 September 2004
Available online 18 September 2004

Abstract

Myotoxin 11, a Lys49 catalytically inactive phospholipase A2 homologue from Atropoides nummifer venom, was purified, characterized
and crystallized. The crystals belongs to the tetragonal system, space group P432i2, with unit cell parameters (a=b=68 66 and c=63.87 A).
Diffraction data were collected to a resoliition of 2.32 A. The crystal structure is currently being determined using molecular replacement
techniqiies
O 2004 Elsevier B.V. All rights rcserved.

Keyrvords Myotoxin; Snake venom; Phospholipase A2; Lysine-49; X-ray diffraction

1. Introduction determined, accurate stnrcttiral data are unavailable. The

Atropoides nirrnnillfei- (previously Bothrops nummfer)
is a crotalid snake species distributed along the Central
Arnerican isthmus [I], and frequently found in the tropical
rain forests of Costa Rica [2]. As characteristic of
crotalids, its venom induces drastic local effects, including
hemorrhage, inflammation, and myonecrosis [3]. The
latter effect is mainly caused by a group of basic
phospholipase A2 (PLA2) homologues, such as myotoxins
1 [4,5] and 11 [6a,b], previoiisly isolated fiom this venom
These two toxins belong to the Lys49 family of catalyti-
caIly inactive PLA2s [7,8], which cause irreversible
damage to skeletal muscle fibers through a yet poorly
unders tood rnechanism.
A. nz~mnzifei-myotoxin 1 has been crystallized, and the
structure has been reported at 2.4-A resolution [9,10];
however, since the amino acid sequence has not been
* Coiiesponding alithor Te1 tfax: -t-55 17 22 1 2247
E-rnnil address arni@df.ibilcc unesp.br ( R K Arni)
1570-9639/$ - see front rnatter O 2004 Elsevier B V Al1 rights reserved
doi: I 0 10 1 6/j bbapap 2004 09 006
complete sequence of A. nurnnzi$er n~yotoxin11 has been
determined (SwissProt code P82950) [6b], and this toxin
has been well characterized in terms of its biological
activities [6a].
It has been proposed that the C-terminal region corre-
sponding to amino acids 95-1 17 includes the rnyotoxic site
of Lys49-PLA2s [ l 1-1 31, and some synthetic peptides
representing this sequence are capabIe of reproducing the
toxic actions of their corresponding proteins [8,15]. How-
ever, an interesting feature of A. ntimmrfer myotoxin 11 is
that its C-terminal synthetic peptide KNYKNPKPLCKK
(sequence 115-1 29 in the numbering system of Renetseder
et al. [14] which is based on the structural similarity to
pancreatic PLA2 or amino acids 95-1 17 using the linear
numbering scheme) is devoid of direct toxic effects in vitro
and in vivo [6b3, in contrast to hornologous synthetic
peptides of some related Lys49 PLAz myotoxins [12,15].
Therefore, in-depth structural studies of A nummifer
myotoxin 11 may provide valuable information to clariQ
the molecular basis of toxicity in this family of PLA2
homologues.
88 L Nhtonabe et nl / Biochirnica et Biop~~ysica
2. Materials and methods
2.1 Isolition of A nummifer myutoxin II
A. nunzmijkr venom was a pool obtained from more than
15 specimens collected in Costa Rica Myotoxin 11 was
purified by cation-exchange chromatography on carboxy-
methyl-Sephadex C-25 (Pharmacia), as described [&J.
Homogeneity was assessed by polyacrylamide gel electro-
phoresis in the presence of sodium dodecylsulfate (SDS-
PAGE) [16], urea-PAGE for basic proteins [17j, and
reverse-phase high performance liquid chromatography
(RP-HPLC) on a C4 column (Vydac), eluted at 1.0 rnl/rnin
with a gradient fiom 0% to 60% acetonitrile in 0.1%
trifluoroacetic acid, using a model 600E Waters instrument.
2 2 Crystallization and X-ray analysis
A lyophilized sample of myotoxin 11 was dissolved in
distilled water at a concentration of 10 mg ml-'. Crystal-
lization was performed by the hanging-drop vapour-diffu-
sion method using 24-well tissue culture plates. Initial tria1.s
were carried out using a crystallization screening technique
similar to that described by Jancarik and Kim [181.
Typically, 1-pL drops of protein solution were mixed with
an equal volume of the screening solution and equilibrated
over 1 mL of the latter as reservoir solution. The conditions
were refined by trial-and-error, and the single crystal f o m
was obtained when a 2-pL protein droplet was mixed with
an equal volume of reservoir solution consisting of 0.1 M
sodium acetate (pH 4.6), 20% PEG 3350 and 0.2 M
amrnonii1n.i siilfate (jnset Fig. 1). The crystal of myotoxin 11
Fig l . X-ray diffraction pattern obtained using a MAR 345 imaging plate
detector Photomicrograoh of the cnstals used for X-ray diffraction
G. 4
experiments (inset)
Acta 1703 (2004) 87-89
Table 1
X-my diffraction data collection statistics of A ritrrnrnfer rnyotoxin 11
crystal
Space group P432 2
Unit Cell parainetcrs (A) u=6=68 66 and c=63 87
Resolution of data set (A) 300-2 3
Percentage solvent (%) 54 83
Number of observations 37,306
Nuinber of iinique reflections 12,626
R,,,,, ('/o)" (2.40-2.32) 7 6 (50.3)
Completeness (%) (2.40-2 32) 96.6 (99 2)
V , (A3 ~ a - ' ) 2 74
Number of molecules per asymmetric unit 2
(Ila(I)) (outermost shell) 13 97 (1 98)
" R,,,,,,=~lZ(h)i-(I(h))[a(l(h)i); I(h) is the observed intensity of the
i I h rneasutement of reflection h and (I(h)) is the mean intensity of reflection
h calculated after scaling
was transferred to a mother-Iiquor solution containing 15%
glycerol and was flash-fiozen.
Three-dimensional X-ray diffraction data were collected
from a single crystal (maximum dimension of 0.3 mm) at
the Department of Physics (USPISio Carlos). Cu KCY
radiation was generated by a Rigaku RU300 rotating anode
generator operating at 50 kVand 100 mA and equipped with
Osmic mirrors. The diffraction intensities were measured
utilizing a MAR345 imaging plate detector (MAR
Research). The diffraction data were collected in 78 irnages
using the oscillation method range of l o per image. The
crystal diffracted to a nominal resolution of 2.3 A (Fig. 1).
The X-ray diffraction data were processed, scaled and
integrated using DenzoIScalepack [19]. Data-processing
statistics are presented in Table 1.
3. Results and discussion
The crystal diffiacted to 2.3 A and beiongs to the space
group P4,2,2, with unit-cell parameters (a=b=68.66 A and
c=63.87 A) as judged fiom autoindexing and consideration
of the systematically absent reflections (Tabfe 1). Assurning
that there are two rnolecules of myotoxin 11 in the
asymmetric unit, a Matthews parameter value [20] of 2.7
Da-' was obtained, corresponding to a solvent content
of 54.8%, values that are wjthin the expected range for
typical protein crystals. Processing of the 37,306 measured
reflections led to 12,626 unjque refiectjons with an R,,,,,, of
7.6% for the data at 2.3-A resolution.
The crystal structure has been deterrnined using molec-
ular replacement techniques as impIemented in the program
AMoRe [21] and using a homology-based model for
rnyotoxin 11 which was obtained from the crystal stnicture
of the Lys49 PLA2 fiom the venom of Cerrophidiorr
(Bothrops) godmani (PDB code lGOD [22]) which shares
9 1% sequence identity. Data in the resolution range 20-3.5
A Were used. A solution obtained for the rotation and
translation functions for the two molecules in the asym-
metric unit, resulting in a final correlation coefficient of
53.9% and R-factor of 48.8% afler rigid-body refinement
iising data in the same resolution range. The refinement of
the stnrcture is currently in progress.
Acknowledgrnen ts
Financia1 support by FAPESPBMOLBNet, CAPES/
DAAD, CNPq, FUNDUNESP, NeTropica network, and
CONICIT (FV-058-02) is gratefu1Iy acknowledged. LMG is
recipient of a CNPq fellowship and LW is recipient of a
FAPESP fellowship. Part of this work was performed in
fulfillment of the doctoral degree of Y. Angulo at the
University of Costa Rica.
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885 - 90 1
[9] R K Ami, J M Gutirrez, Crystallization and preliiniiiaiy cliffraction
data of two myotoxins isolated from thc venoms of Bothrops usper
(terciopelo) and Botlirops nlcmmifer (jumping viper), 'Toxicon 31
(1993) 1061-1064
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Lys49-phospholipase A2 homologue isolated frorn the venom of
Bothrops nunrmfer Cjurnping viper), Toxicon 37 (1999) 371 -384
[l 11 B. Lomonte, E Moreno, A Tarkowski, L A Hanson, M Maccaiana,
Neutralizing interaction between heparins and myotoxin 11, a Lys-49
phospholipase A2 from Bothrops asper snake venom Identification
of a heparin-binding and cytolytic toxin region by thc use of
synthetic peptides and inolecular modeling, J Biol Chem 269
(1994) 29867-29873.
E121 C.E. Nitez, Y Angulo, B. Lomonte, Identification of the myotoxic
site of the Lys49 phospholipase A2 from Agkistrodon piscivorus
yiscivonts snake venom: synthetic C-terminal peptides froin Lys49,
but not from Asp49 myotoxins, exert membrane-damaging activities,
Toxicon 39 (2001) 1587- 1594.
1131 L. Chioato, A H.C. de Oliveira, R. Ruller, J M. S, R J Ward, Distinct
sites for myotoxic and membrane-damaging activities in the C-
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[14) R. Renctseder, S. Brunie, B W Dijkstra, J Drenth, P B Sigler, A
comparison of the crystal stnictures of phospholipase A2 from bovine
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11627- 11634
[15] B Lomonte, Y. Angulo, C Santaniaria, Comparative stiidy of
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[16] U K. Laemmli, Cleavage of structural proteins during the assembly of
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ribosomes from subribosornal components, Methods Enzyrnol 20
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[18] J Jancarick, S.H. Kim, Sparse matrix sampling: a screening
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[19] Z. Otwinowski, W Minor, Processing of X-ray diffraction data
collected in oscillation modc, Methods Enzymol 276 ( 1997) 307 - 326
[20] B W, Matthews, Solvent content of protein crystals, J Mol Biol 33
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[21] J. Navaza, Collaborative Comptitational Project, Nlirnber 4 "The
CCP4 Suite: Programs for Protein Crystallogaphy", Acta Crystallogr ,
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[22] R K Ami, M R M Fones, C Barberato, J M. Gutierrez, C Diaz,
R.J. Ward, Crystal structure of myotoxin 11, a monomeric Lys49-
phospholipase A2 homologue isolated from the venom of Cerro-
phidion (Bothrops) godmani, Arch. Biochein Biophys 366 (1999)
177- 182
ARTICULO
 Toxicon 42 (2003) 307-31 2

Comparative study of synthetic peptides corresponding

to region 115- 129 in Lys49 myotoxic phospholipases A2
from snake venoms
Bruno ~ o m o n t e ~ *Yamileth
*, ~ n ~ u l oCarlos
~ ? ~Santarnaraa
,
"Facultadde Microbiologa, bisiituro Clociorniro Picado, Utiiversicforlrle Costa Rico, San Jos, Costa Rico
h~epartame~rrode Bioqunrico, Escuela de Medicitia, Uriiversidad de Costa Rica, Sarz .Tos&,Costo Rico

Received 1 April 2003; nccepted 12 June 2003

Abstract
Lys49 phospholipase A2 homologues constitute a group of catalytically-inactive proteins, present in the venoms of many
crotalid snakes, which induce rnyonecrosis Current evidence supports the mapping of their toxic site to the C-terminal region,
where amino acids comprised within the sequence 115- 129 appear to play a central role in toxicity This study evaluated the
possible toxic effects of several synthetic peptides coriesponding to the sequence 115- 129 of different Lys49 inyotoxins, using
in vitro cytotoxicity and in vivo myotoxicity assays Peptides varied widely in their activities, ~angingfioni fully toxic to
harmless Thus, the toxic actions of Lys49 myotoxins cannot always be reproduced by their free pcptidcs 1 15- 129 Peptides
from Agkistrodon p. piscivo~us (AppK) and A contorttix laticinctus Lys49 myotoxins exerted both cytotoxicity and
myotoxicity Random scrainbling of peptide AppK resulted in complete loss of toxicity, demonstrating that its specific
sequence of residues, rather than ther simple presence or frequency, confers its ability to daniage muscle Peptide AppK
synthcsized with D-amino acids retained botli activities of the natural 1--enantiomer, suggesting that its inechanism of action
does not involve the tecognition of a proteic receptor/acceptor site on muscle cclls, but possibly the binding to other structuies,
such as negatively-charged membrane phospholipids
O 2003 Elsevier Ltd. Al1 rights reserved
Keywords: Myotoxin; Snake venorns; Phospholipase A2; Synthetic peptidcs; Cytotoxicity

Muscle necrosis is a dramatic effect induced by a variety of
snake venoms, potentially leading to permanent sequelae
such as tissue loss and disability (Nishioki and Silveira.
1992; Waii cll, 1996). Veiioms of snakes within thc family
Crotaiidae possess a number of basic phospholipases A2
(PLA2; EC 3.1 1.4) which have a prominent role in the
pathogenesis of myonecrosis (Mcbs oticl (1wiil)y. l990),
These rnyotoxic PLA2s are classified as group IlA, on the
basis of their primary structure and disulfidc bond patteln
(Six anll Llcniiis 2000) Within the group H A PLA?
myotoxins, there is a subgroup of catalytically-inactive
variants (or PLA2 homologues), first discovered in
* Corresponding nuthor Fax: +506-292-0485
E-niail atidress: blomonte@cariariucr ac cr (B Lornonte)
0041-0101/03/$ - see front mtter O 2003 Elsevier lJtd A11 rights rcservcd
tloi: I O 1016/S0041-0101(03)00149-1
the venom of the North American water mocassin
Agkistrodon p. piscivorus, in which the conserved Asp at
position 49 is replaced by Lys (Mninganoie el al , 1083;
hlaiag;~noie:ind FIeini iksnri, 1986: Scott e t nl 1992) Such
'Lys49 myotoxins' have been subsequently found in the
venoms of many crotalid species (Hu~iisi-U~aritlcburgo .
et al
1088: C j ~ t j E r l c zaiid Lomonre, 1995: Soales ct al 109S;
Owiihy c t id., 1990; Chijiwii ct al , 2000; Thoi et al . 2001)
The Lys49 PLA2 myotoxins constitute an inteiesting model
for the study of catalytically independent mechanisms of
muscle darnagc, and several strategies have been utilized to
determine their inolecular deterininants of toxicity
1 ,nnionrc ct al ( 1993) first described that hepatin binds to
a site comprising residues 115- 129 of the Lys49 inyotoxin
II from Bothrops osper, resulting in the neutralization of its
308 B. hniotite et al. / Tuxicori 42 (2003)307-3 12

toxic actions Moreover, it was reported that the synthetic
peptide 115- 129 of this protein mimics its cytotoxic effects
in vitro (Loiiionte et i d , 1994; Nlirz et al . 2001) Further
studies with B asper myotoxin 11 confirmed the functional
relevance of region 1 1 5 - 1 2 9 for its toxic activities
(Cildern and Lonlonce, 3!)98. 1999; Piramo ct al . 1998;
1,oiiionrc cr al . 1999a). More recently, it was demonstrated
that a synthetic peptide corresponding to region 1 1 5 - 129 of
A. p piscivonts Lys49 PLA2, induces a full pattern
of myonecrosis in vivo, reproducing al1 the toxic activities
.
of this protein (Nez et al 2001) In agreement with these
findings, recent site-directed mutagenesis studies of the
Lys49 PLA? bothropstoxin 1, from Bothrops jararacussu,
demonstrated the functional relevance of residues com-
prised within its C-terminal region (Warcl c~ 211, 2002;
Chioato ct a1 . 2002)
The observation that two different synthetic peptides,
conesponding to thc sequence 115-129 of two Lys49
myotoxins, respectively, are capable of exeiting direct toxic
actions (to~iiontcct ril . 19'34; NLiic7 ct al.. 2 0 0 1 ) , prolnpted
the present study, aiming to iiivestigate if this would
represent a general rule for proteins within this PLA2 fainily
Therefore, a group of sequences 115-129 frorn various
Lys49 myotoxins was selected, and the possible toxic
activities of their corresponding synthetic peptides were
comparatively evaluated, in vitro and in vivo.
The peptides studied are described in T:tble I They were
synthesized using Fmoc strategy (Wilkci, IC)C)4), either
iiianually in our laboratory, or automatically by coiiiinercial
Table 1
Synthetic pcptides 115- 129 of Lys49 inyotoxic phospholipases Al studied
providers (Chiron Inc., or SynPep Inc , USA). Their final
purity was at least 95% by RP-HPLC analysis, and their
observed mass spectrometry values corresponded to their
expected formula values. In addition to the natural
sequences of peptides 115- 129 of the different myotoxins
studied, some of the peptides correspond to rnodified
sequences, seIected to assess specific issues ('l'able 1).
In order to evaluate the toxic activity of the different
peptides in vitro, a cytotoxicity assay using the C2C12
murine myoblast cell line (ATCC CRL-1772) as a target
was perforrned (Lomoiitr et a l . 199C)b).Three hours after
exposure to the peptides, celI darnage wrts quantified based
on the reiease of lactacte dehydrogenase (LDH) to
supernatants, determined by a colorimetric procedure
(Sigma No, 500) Previous studies have shown that
cytotoxic activity towards C2C 12 ce1ls is a common
property of al1 class 11 myotoxic PLA,s analyzed to date,
and that this in vitro assay for myotoxins and myotoxin-
derived peptides is more sensitive than the in vivo
myotoxicity assay performed in mice (Loiiionte et u1 ,
199%: Niez et al , 200 1). Synthetic peptides were
dissolved in 0.12 M NaCI, 0.04 M sodium phosphate, pH
7 2 buffer (PBS) immediateiy before the experiments, and
diluted in culture medium. Then, cells were exposed to
different peptide concentrations, in a volume of 150 p1,
ranging from 25 to 150 pglwell (approximately 9 5 -
580 pM)
As summarizecl in Fig 1. peptides corresponding to
region I 15- 129 of Agkistrodari cor~tortrix laticinctus

Lys49 rnyotoxidsnake species Sequence 1 15- 129 Peptide code Protein referente

K49 phospholipase A? Agkistruriuti KKYKAYFKLKCKK APPK M:~Iag:iiiciic 311d Hciiil ikson

piscivorus piscivorrrs ( 1986)
K49 phospholipase Al Agkistruclotr LKFKYKKACKKYK AppK (scramblecl) -

piscivorus pis6 ivorlrs

K49 phospholipase A? Agkistrorlon KKYKAYFKLKCKK AppK (all-D) -
piscivorrts piscivor us
ACL myotoxin Agkistrorlorz KKYKAYFKFKCKK Acla
corrlortrix /oficirictus
Myotoxin II Borhrups asper KKYRYYLKPLCKK Basp-11
Myotoxin 11-alternative forrn" KKY RYYLKPFCKK Basp-11-F
Burlirops asper
Myotoxin 11-modified Botllrops asper KKWRWWLKALAKK Basp-11-pEM-2 -
Myotoxin II Bothrops moojeni KKYRYNYLKPFCKK Brnoo-II Soares rt al ( 1 908)
Bothropstoxin 1 and piratoxin 11 KKYRYHLKPFCKK Bjsu-11Bpir-11 Cintra ct a1 (1993) zind
Buthrcps jrirat acirssu iind B pirajai 'roy;1111;1et al (2(H)O)
Myotoxin 11 Atropoides tiunimifer ttnd KNYKIYPKPLCKK Anum-l11Cgod-11 Angiilo ct al. 12002) aiid
Cert ophidion goclnicrrii clc Sotis:~ct :i1 ( 1998)
Myotoxin 11-tnodifred Atropoides KKYKIYPKPLCKK Aniim-I11Cgod-Il (mod) -
rlitmnzifer
Amrnody tin L~ Vipero ~lrtlnio(Iyte~ KKYKVYLKFKCKG
ittnnlodytcs

" A microheterogeneity ai position 124 of either Lcii/Phe was described.

This protein is i Ser49 variant of PLAI with myotoxic activity
 o APPK
O AppK (scrambled)
A AppK (atl-D)
v Acia
O Anum-11 / Cgod-ll
O Anum-ll / Cgod-H (mod)

Peptide (pg/well)
Fig 1 Evaluation of the in vitro cytotoxic activity of synthetic peptides corresponding to the seqtience 115- 129 of Lys49 phospholipase A2
myotoxins, towards C2C12 skeletai muscle cells Cell dainage was quantified by the rclcase of lactate dehydrogenase (LDH), 3 h after exposure
to 25- 150 pg of peptides, in 150 p1 of rnedium (95-580 KM) Each point represents mean -t- SD of triplicate cultures Peptide abbreviations
are dcscribed in f;ihlr: I

myotoxin (Acla), A p. piscivorus Lys49 PLA2 (AppK), B.
nsper myotoxin 11 (Basp-II), and its alternative natural form
(Basp-11-F), respectively, displayed a clear cytotoxic
activity, demonstrated by the dose-dependent release of
LDH, and by microscopical observation of cell darnage
Peptides from the two Agkistrodon species were more active
than those dcrived from B nsper in this assay (Iiig 1).
Although the cytotoxic activity of peptides AppK and Basp-
11 has beeii repoited (Loinonte ct al , f 993: Niiei ei al
700 1 ), they were included here for comparative purposes.
On the other hand, peptides corresponding to B. nroojeni
myotoxin 11 (Bmoo-Il), B. jararacussu bothropstoxiri 1
(Bjsu-1)lB. yirajai piratoxin 11 (Bpir-II), Atropoides nummi-
fer myotoxin II (Anum-1I)ICerropltidion godmarzi myotoxin
11 (Cgod-111, and Vipera ammodytes anznzodytes ammodytin
L (Vaniin), did iiot cause cell damage, at concentiations as
Iiigli as 580 pM (150 pglwell; I;ig. 1)
The most active peptide, AppK, was sefecred to assess
two specific questions by the use of variants. in order to
evaluate if the cytotoxic activity of this peptide could be
attributed to a non-specific effect of its numerous positively-
charged amino acid residues, its sequcnce was scrambled at
randoin, as specified in T;ible I . Peptide AppK-scrambled
was unable to damage the cell cultures (Fig 1), demonstrat-
ing that the specific seqiience of residues in the natural
AppK peptide, rather than their simple presence or
frequency, is the factor that confers its ability to damage
muscle cells in culture. In order to evaluate if peptide AppK
acts upon the cell cuitures by recognizing a configiiration-
dependent structure (such as a proteic receptorlacceptor
. site), an analogous peptide synthesized with D-amino acids
(AppK all-D; Tiible 1) was tested This D-enantiomer was
clearly cytotoxic, with a potency comparable to that of its
natural L-counterpart (Fig. l), strongly suggesting that the
cytotoxic rnechanism of peptide AppK does not involve the
recognition of a proteic acceptor site on the C2C12 skeletal
muscle cells. This observation, if relevant to the whole
parent molecule, would indicate that the mechanisin of
action of myotoxic Lys49 PLA2s may not involve the
recognition of PLA2 receptor proteins (L,ariihe:i\i aiid
Lazcfiiiiski, 1999), but possibly the binding to negatively
charged menibrane phospholipids (Daz et ;iI . 2001)
The sequence of B. asper myotoxin 11 was originally
reported with a microheterogeneity at position 124.
 .
occupied by either Leu or Phe (Fraiicis et al I9!)L). Since
previous studies with peptide 115- 129 of this toxin (Basp-
11) have been performed with Leu124, the alternative
peptide form with Phel24 (Basp-11-F) was tested, observing
that it also has cytotoxic activity (Fig 1) Thus, a LeuPhe
exchange at position 124 of this peptide does not affect its
toxic ability On the other hand, a modified peptide (Anum-
IIICgod-11-mod; Tiible I ) in which AsnlI6 of the original
sequence of Anum-IIICgod-11 was changed to Lys116, as an
attempt to restore this relatively conserved positive residue
among many Lys49 myotoxins, did not show cytotoxic
activity (Fig. 1). Finally, a modified peptide (Basp-11-pEM-
2) based on the sequence of B. asper myotoxin 11, was
evaluated in the cytotoxicity assay. This peptide contains a
previously characterized triple Tyr* Trp substitution
(Loiriolitt: cf 31 , 10901), in addition to the substitutions
Pro -. Ala and Cys -.Ala ('l'ul~le I ) The lack of Cys in
Basp-11-pEM-2 allowed to evaluate if the possible covalent
dimerization of this peptide (that might occur due to
intermolecular oxidation of Cys residues), would be
necessary for its toxic activity Peptide Basp-11-pEM-2
exerted cell damage (Fig l), demonstrating that dimeriza-
tion is not required for its cytotoxic activity.
The activity of peptides was subsequently evaluated in
vivo, by injecting 250 pg/50 p1 intramuscularly in mice,
and assessing myonecrosis through quantification of the
plasma creatine kinase (CK) activity after 3 h (Sigma No
520). This dose was selected according to previous studies
of myotoxin-derived peptides (I,oiriontc c t al . IY99a;
Niiic~ct a l , 2001) Results showed that, in addition to
peptide AppK, peptide Acla was also capable to exert a full
myotoxic activity, significantIy increasing plasma CK levels
in vivo (Fig 2) This finding dernoiistrates that the region
1 15 - 129 of A contortrix latici~ictusLys49 PLA, encloses
its myotoxic site, in agreement with previous observations
with A p. piscivorus Lys49 PLA2 (Ncz et ii1 , 2001). Both
peptides differ only at one position, namely, Leu123 in
AppK vs. Phe123 in Acla, showing that this LeuPhe
exchange (in similarity with the Leu124Phe124 exchange
of peptides Basp-1IBasp-11-F) does not cause drastic
changes in toxic activity.
fn agreement with results obtained in vitro, peptide
AppK-scrambled was devoid of myotoxicity in mice,
whereas AppK-all-D was clearly myotoxic (Ilg 3). Thus,
the same conclusions on (a) the specific toxic effect of the
natural AppK sequence, and (b) the lack of a stereoisomer-
sensitive receptor requirement in its toxic mechanism, also
apply to the in vivo situation, which involves fully
differentiated skeletal muscle fibers as targets.
Peptides other than those of Agkistrodon species,
including the B. asper myotoxin 11-derived peptides that
were cytotoxic in vitro, did not induce myotoxicity in vivo
(Fig 7). Thus, these results show that the toxic activities of
the Lys49 PLAz proteins cannot always be reproduced by
their corresponding synthetic peptides 115- 129. Such
peptides may express the full myotoxic and cytotoxic
AppK (scrambled)
AppK (all-D)
Acla
Baspll
Basp-ll-F
Varnm
Plasma CK actkity (Ulml)
Fig 2. Evaluation of the in vivo myotoxic activity of synthetic
peptides corresponding to the sequence 115- 129 of Lys49
phospholipase A2 myotoxins, in mice. Skeletal muscle necrosis
was quantifiedby detemining plasma creatine kinase (CK) activity,
3 hr after i m injection of 250 pg of peptides, in 50 y1 of PBS Each
bar represents mean it SD of three animals PBS: phosphate-
buffered saline, pN 7 2 Peptide abbreviations are described in
Tnble 1.
activity of the parent protein (i e AppK and Acla), only a
weaker cytotoxic effect in vitro (Basp-11, Basp-11-F), or no
detectable toxic effects at a11 (Bmoo-11, Bjsu-IIBpir-11,
Anum-IVCgod-11) The apparent discrepancy between the in
vitro and in vivo activities of the B asper myotoxin 11-
derived peptides might be explained by a higher sensitivity
of the cytotoxicity assay, in cornparison to the in vivo
myotoxicity test, as suggested earlier (Lomonte et a l ,
1999a: Nez et i ~ 1, 2001). It may be assumed that the
number of target sites for :he peptides should be
significantly lower in the cell culture model, than in the
larger mass of muscle tissue in vivo. This would aliow the
peptides to concentrate at higher densities on the surface of
the cultured cells, in contrast to a dispersion along the large
muscle fibers
Finally, should the observed Iack of toxic activities, for
several of the peptides studied, exclude the possible
participation of sequence 115-129 as a toxic region of
their corresponding Lys49 PLA2s? From an evolutionary
perspective, it would seem unlikely that the Lys49 PLA2
myotoxins, forrning a closely related phylogenetic group
(Angirlo ct al.. 3002). would have developed markedly
different active regions in different snake species. The direct
evidence of toxicity exerted by synthetic C-terminal
peptides of some Lys49 myotoxins (i e. from B asper
(Loniontc ct al , t 904), A p. piscivor~ds(Nicz cr al , 200 1 ),
and A. c laticirictus (present work)) suggests that this region
might also play a central role in the toxic mechanism of
other members of this piotein family. But this would have to
be evaluated through other experimental approaches, in
each particular case, due to significant sequence variations
in the C-terminal region of these proteins. The reasons for
the lack of activity of severa] peptides 115-129 are
presently ~inclear.A possible explanation could be that,
free in solution, some peptides might adopt an altered
conformation, unfavorable for the toxic mechanism. The
segment 115-129 forrns a loop at the C-terminal rcgion of
the Lys49 myotoxins (Aiiii and Waid. 1996), but confor-
mation of the corresponding free peptides in solution would
have to be determined. In addition, the participation of other
protein regions (adjacent or distant) in the toxic mechanism
cannot be excluded. Independently of the reasons, it is
proposed that, while a positive observation of direct toxicity
by a particular C-terminal peptide demonstrates the
involvement of its conesponding protein region in the
toxic mechanism of action, the opposite is not necessarily
true, .e. a negative result does not exclude the participation
of the corresponding protein region in toxicity In support of
this proposal, recent studies with recombinant B jarar-
acussu bothropstoxin 1 mutants clearly demonstrated the
functional relevante of residues within the sequence 115-
125 in the toxic actions of this protein (Chioato et aI., 2002).
Yet, in the present work, its free synthetic peptide 115- 129
was unable to mirnic the actions of the whofe toxin.
Therefore, even for the Lys49 PLA2 myotoxins whose
synthetic peptides 1 15- 129 tested negative in this study, the
possibility of the involvement of their C-terminal regions in
toxicity remains still open to evaluation by other exper-
imental approaches.
Acknowledgements
We thank Drs Georgina GurroIa and LourivaI Possani
(Instituto de Biotecnologa, UNAM-Cuernavaca, Mexico),
for expert advice with peptide synthesis; Dr Igor Krizaj
(Josef Stefan Institute, Ljubljana, Slovenia) for providing
purified Vipera ammodytes ammodytin L (as parent protein
of peptide Vamm); and Javier Nez, Xinia Porras, and
Rodrigo Chaves for excellent laboratory support Financia1
aid was received from Vicerrectora de Investigacin,
University of Costa Rica (VI-741-99-269), International
Foundation for Science (F/2766-2), Consejo Nacional para
Investigaciones Cientficas y Tecnolgicas (CONICIT 98-
013-FO), the Embassy of Japan in Costa Rica. and
NeTropica Swedish-Central American Network for
Research and Training (G-02) This work was performed
in partial fulfillment of the doctoral degrce of Y. AnguIo at
the University of Costa Rica.
.
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ELSEVIER Toxicon xx (xxxx) 1-6 60
www elsevier coi~~/locate/toxicon6 1
62
63
Myotoxic and cytolytic activities of dimeric Lys49 65
64
phospholipase A2 homologues are reduced, but not abolished, 66
67
by a pH-induced dissociation 68
69
70
Yamileth ~ n ~ u l o " ' Jos
~ ~ *Mara
, Gutirreza, Andreimar M. Soaresc, 71
Wonhwa chod,Bruno Lomontea 72
73
"Fucult~dde Microbiologa, Instituto Clodomiro Picado, Universidad de Costa Rica, Sun Jos, Costa Rica 74
b~epartumet~to cit. Biogurnicu, Escuela de Meicina, Universidad de Costa Rica, San J o ~ Costa
, Rica 75
"Departumerttode At~lisesClnicas, Toxicol6gicas e Bromatolgicas, Fuculdade de Ciencias FarmucEuticus 76
de Ribeiro Preto, Universidade de Seo Paulo, Ribeiro Preto, SP, Brazii
77
d~epartrnentof Chemistry, University of Illinois at Chicugo, Cbicago, 115, USA
78
Received 3 February 2005; revised 30 March 2005; accepted 31 March 2005

Abstract

Lys49 phospholipase A2 (PLA2) homologues are myotoxic proteins devoid of catalytic activity. Their toxic determinants
rnap to the C-terminal region 115-129, which plays an effector role in membrane damage. The dimeric state was reporte6 to be
essential for a Lys49 PLA2 which lost its fiposome-dismpting activity after dissociating into monomers at pH 5.0. This study,
evaluated the effects of a pH-induced dissociation on the toxicity of four Lys49 PLA2s, using biological targets instead. Both
their cytolytic and myotoxic activities were lower at pH 5.0 than at pH 7.2. However, in contrast with experiments using
artificial bilayers, toxic effects upon biological targets were not abolished at pH 5.0. Importantly, C-terminal synthetic peptides
of two Lys49 PLA2s also showed Iower cytolytic action at pH 5.0 than at pH 7.2, indicating that factors other than the
dirnerictmonomeric state of the proteins may also be involved in these differences of toxicity. Results support the view that the
dimeric state of Lys49 PLA2s could play an enhancing, although not essential role, in their C-terminal region-mediated
rnechanism of rnyotoxicity.
O 2005 Published by Elsevier Ltd.

Keywords. Snake venoin; Myotoxin; Pbospholipase A2; Dimeric protein; Muscle

1. Introduction venoms induce prominent local tissue alterations, among

Crotaline snakes (family Viperidae, subfamily Crotali-
nae) are widely distributed in America (Campbell and Larnar,
1989), being responsible for the majority of snakebite
envenomations in this continent (Gutii-rez, 1995). Their
" Correspwnding author. Address: Facultad de Microbiologa,
Instituto Clodomiro Picado, Universidad de Costa Rica, San Jos,
Costa Rica Te1 : $506 229 0344; fax: +506 292 0485.
E-rnnil aldress- yangulw@icp iicr ac cr (Y Anguio)
which acute muscIe damage, i.e. myonecrosis, is comrnoii
(Guti rrez and Lomonte, 2003). The most important
myotoxic cornponents in crotaline venoms are phospho-
lipases A2 (PLA2; EC 3.1.1.4), ubiquitous enzymes that
catalyze the hydrolysis of the sn-2 position of glyceropho-
spholipids (Kudo and Murakami, 2002). In snake venoms,
PLAzs have evolvedinto potent toxins with diverse activities,
such as neurotoxicity, myotoxicity, anticoagulant, hypoten-
sive, cardiotoxic, edema-inducing (Kini, 2003), and bacteri-
cidal (Pramo et al , 1998) effects.

0041-0101/$ - see front matter O 2005 Published by Elsevier Ltd

doi:lO 1016/j toxicon 2005 03 025

TOXCON 2482-2015/2005-13: 1 1-SWAPNA-148824-XML MODEL 4 - pp 1-6

 PLA2s have becn classified into eleven groups (Six alid
Deiiiiis, 2000), amorig which the enzymes from snake
veiionis belong to groups 1 (elapid PLA2s) and 11 (viperid
PLA2s). The latter are further, divided into two main
subgroups, namely catalytically-active enzymes pi esenting
a conserved aspartic acid residue at position 49 (Asp49
PLA2s), or homologues with Iysine substituting at this
position (Lys49 PLA2s) (Maraganore et al., 1984) which are
catalyticaily-inactive (Scott et al., 1992; Lee et al., 2001;
Ward et al., 2002).
Despite their lack of enzyrnatic activity, the Lys49 PLAz
homologues induce conspicuous rnyonecrosis in vivo, as
weli as a series of in vitro effects such as cytolysis, liposome
disruption, neuromuscular blockade, and bactericidal action
[reviewed by Loinonte et al. (2003b), and Soares et al.
(2004)l. Since, their effects cannot be attributed to
phospholipid hydrolysis, a number of investigations have
focused on the elucidation of the molecular basis of their
toxicity. Evidence based on studies with synthetic peptides,
mapping of interaction sites with neutralizing molecules,
and site-directed mutagenesis [see reviews by Lomonte
et al. (2003b) and Chioato and Ward (2003)], identified the
C-terminal region of Lys49 PLA2s as essential for their
myotoxic, cytolytic, bactericidal, and liposome-disrupting
activities. The strongest evidence for a key efiector role
of this region derives from (a) the observation that severa1
13-mer synthetic peptides, representing rhe sequence
115-129 of Lys49 myotoxins, are capable of reproducing,
albeit with lower potency, the toxic actions of their parent
moIecules (Lomonte et al., 1994, 1999a, 2003n; Nez
et a l , 2001); and (b) the demonstration that specific
C-terminal mutant; of a Lys49 PLAz display reduced
toxicity (Chioato et al., 2002).
Structural studies on bothropstoxin 1, a homodimeric
Ly s49 PLA, homologue from Bothrops jnrnracussis venom,
demonstrated that this protein presents two conformations,
'open' and 'closed' forrns, which differ in the angle formed
by the monomers. On this basis, it was proposed that the
dimer interface might function as a hinge, allowing a
relative displacement of the C-terminal region that would
contribute to disorganization of phosphoiipid bilayers
(da Silva Giotto et al., 1998). Subsequent analyses examined
the effect of pH on the monomer-dimer equilibrium of
bothropstoxin 1 (de Oliveira et al., 2001), leading to the
conclusion that the dirneric state of the protein was essential
for its ability to disrupt liposomes, since the toxin
completely lost this activity at pH 5.0, concomitantly with
its dissociation into monorners. Therefore, the present study,
was uridertaken to assess if the dimeric state is functionally
relevant for the toxic activities of Lys49 PLA2s upon
biologically relevant targets. The effects of a pH-induced
dissociation on the cytolytic and rnyotoxic activities of four
Lys49 myotoxins, as well as of two C-terminal synthetic
peptides, using C2C12 myoblast ceils in vitro, and rnature
skeletal rnuscle in vivo, respectively, were evaluated to gain
TOXCON 2482-2015/2005-13: 1 1-SWAPNA- 148824-XML MODEL 4 - pp 1-6
insights into the functional role of their dimeric state iii the
mechanism of rnuscle damage.
2. Materials and methods
2.1. Isolation of Lys49 PLA, myotoxins
Lys49 myotoxins were purified from their corresponding
crude venoms by cation-exchange chromatography as
described for Bothrops asper myotoxin 11 (Lomonte and
Gutirrez, 1989), Atropoides nummifer myotoxin II (Angulo
et al., 2000, 2002), B. jararacussu bothropstoxin 1
(Andriao-Escarso et al., 2000), and Agkistrodott p, pisci-
vorus (Maraganore et 1 , 1984), respectively. Toxin
homogeneity was assessed by urea-PAGE for basic proteins
(Traub et al., 1971), and by RP-HPLC on a C4 colurnn (25 X
4.6 mm;Vydac), eluted at 1.0 mLfrnin with a gradient from
O to 60%acetonitrile in 0.1 % trifluoroacetic acid (v/v), using
an Agilent 1100 system.
2.2' Chernical cross-'inking
Solutions of 2 lg/pL of purified PLA2s in phosphate-
buffered saline (PBS; 0.12 M NaCI, 0.04 M sodium
phosphate), either at pH 5.0 or 7.2, were incubated done
or in the presence of 2 pg/pL bis-(sulfosuccinimidy1)
suberate ( B S ~ ;Sigma Chemical Co., USA) for 30 rnin at
25 "C. Then, al1 samples were heated at 95 "C for 5 min in
the presence of SDS and [3-mercaptoethanol, and analyzed
by SDS-PAGE using 12% gels, at 100 V for 2 h. Proteins
were visualized by Coomassie blue R-250 staining.
-- -
2.3. Cytolytic activity in vitro 202
203
The cytotoxic activity of PLA2s was assayed at either pH 204
5.0 or 7.2, on rnurine C2Ci2 skeletal muscfe myoblasts 205
(ATCC CRL-1772) grown in 96-well plates, as described 206
(Lomonte et al., 1999b). Myotoxins were dissolved in assay 207
medium ar the corresponding pH,which consisted of either 208
DMEM supplemented with 1% fetal calf serum at pH 7.2, or 209
the same solution previously titrated with 2 M citric acid to 210
pH 5.0. Myotoxins were incubated at roorn ternperature for 21 1
30 min, and then added to the cell cultures (after aspirating 212
their growth medium) in a total of 150 pL/well, containing 213
doses of 5, 10,20 or 40 pg. After 3 h of incubation at 37 "C, 214
lactate dehydrogenase (LDH; EC 1.1.1.27) released to 21 5
supernatants was determined by a kinetic assay (Wiener 216
LDH-P UV). Reference controls for O and 100% cytolysis 217
consisted of cells incubated with medium alone, or medium 218
containing 0.1% Triton X- 100, respectively . Assay S were 21 9
carried out in triplicate. 220
Two synthetic peptides, corresponding to the bioactive 221
C-terminal sequences 115-129 of B. asper myotoxin JI 222
(KKYRYYLKPFCKK; (Lomonte et al., 1994)) or 223
A. p. piscivorus Lys49 PLA2 ( K K Y K A Y F K L K C K K ; 224
(Nez et al., 2001 )), respectively, were also evaluated for
cytolytic activity. These peptides (150 pg/150 1iL) were
dissolved in assay media of either pH 7.2 or 5.0, as described
above, and applied to the myoblast cultures for deterrni-
nation of cell fysis.
r-
,
9 4
M -

a
L
- -
6$*~:

, .:*A
?
pH 7.2 pti 5 O NR 281
282
283
284
285
1 67
1
286
2.4. Myvtoxic activity in vivo f 43 287

Myotoxic activity of the PLA2 homologues was

evaluated by determining the plasma activity of creatine
kinase (CK; EC 2.7.3.2) in groups of four CD-1 mice
(18-20 g body weight), after an intramuscular injection of
75 pg of each toxin in 75 pL of PBS, either at pH 7.2 or 5.0,
in the gastrocnemius. The toxins were preincubated in each
solution for 30 min at roorn temperature, before injection.
Control groups received an identical injection of pH 5.0 or
7.2 buffer, respectively. After 3 h, blood sampies were
collefted into he~afinizedc a ~ i l l a rtubes, ~ and plasma CK
activity was by a kinetic assay (CK-NAC ''9
~ i e n e r ) ~. c t i v i t ywas ex~ressedas U/L (1 unit defined as
[he amount of enzyme producing 1 of NADH/min at
30 "C).
296
Fig. I . Coomassie-stained SDS-PAGE (1 2%) comparing the 297
chemical cross-linking of Bohrops asper myotoxin II by 13s3
298
dunngincubationat pH 7.2 or 5.0. Toxin sampleswere incubaledjn
the presence (+) or absence (-) of BS), at the indicatcd pH vahes. 299
and then reduced with 2-rnercaptoethanol at 95 'C and analyzed A 300
small proportion of covalently stabilized rnyotoxin 11 dirner is 301
obtained at pH 7 2 (arrow), but not at pH 5.0. MolecuIar weight 302
msirkers (M) are indicated at the left. For comparison, the migration 303
of dimeric myotoxin II under non-reducing conditions is shown to 304
the right (m).

The pH-induced dissociation of rnyotoxins was sup- 307

ported by electrophoretic analysis after their incubation with 308
the homobifunctional cross-linker bis-(sulfosuccinirnidyl)
suberate (BS~),which links primary amino groups separated
A
by up to 11.4 to form stable amide bonds, in both intra-
and intermolecular reactions (Staros, 1982). Fig. 1 shows a
representative gel with B. nsper myotoxin II under reducing
conditions, where B S covalently
~ linked a proportion of the
dimeric form at pH 7.2, whereas at pH 5.0 only the
manomeric form was observed. Similar results were
observed with the other myotoxins (not shown). Thus,
chemical cross-Iinlung results with the Lys49 myotoxins
here studied, are in agreement with previous spectroscopic
and electrophoretic evidence showing that the dimeric state
is favored at pH 7.2, while dissociation into monomers
occurs at pH 5.0 (de Oliveira et al., 2001).
When the four Lys49 PLA2s were tested on C2C12
myoblasts in vitro, a significantly lower cytolytic effect was
observed at pW 5.0 than at pH 7.2 in al1 cases, at various
toxin concentrations (Fig. 2). The highest difference in
activity under these two conditions was found for the Lys49 Myotoxin (pg) 327
PLA2 homologue from A. p. piscivorus. Incubation of 328
control cultures with assay medium alone at pH 5.0 did not Fig. 2. Effect of pH on the cytolytic activity of Lys49 phospholipase 329
cause cell damage in the time period utilized, as evaluated Az myotoxins upon C2C12 skeletal muscle myoblasts in vitro. 330
Cytotoxicity was determiiied by the release of lactate dehydrogen- 33
by both LDH release and microscopical observation.
ase (LDH) to cell supernatants, 3 h after exposure to variable
l n similarity wi th resul ts obtained with their parent 332
amounts of toxins at pH 7.2 ( m ) or 5.0 (0). ( A ) Bothrops usper
proteins, when the synthetic C-termino1 peptides were myotoxin 11; (B) Atropoides numtni$er myotoxin 11; ( C ) Bothrops 333
assayed against C2C12 cells, their cytolytic effect was jurarucussu bothropstoxin 1; (D) recornbinaiit A g k i s t r o h p 334
also significantly lower at pH 5.0 than at pH 7.2 (Fig. 3). In piscivorus Lys49 PLA2 Each point represents mean+SD of 335
vivo experirnents performed in rnice to assess muscle triplicate cultures. 336

TOXCON 2482-20/5/2005-13: 1 1-SWAPNA-148824-XML MODEL 4 - pp. 1-6


Fig. 3 Effect of pH on the cytolytic activity of synthetic peptides
corresponding to the C-terminal regions (sequcnce 115-129) of
Bohtrups asper myotoxin 11 (p-Ba-mt-11; 150 pg1150 pL) and
Agkisirorlon p piscivorus (p-AppK49; 150 pg1150 pL), upon
C2C12 skeletaI muscle myoblasts in vitro. Cytotoxicity was
determined by the release of jactate dehydrogenase (LDH) to cell
supernatants, 3 h after exposure to peptides at pH 7 2 (filted bars) or
5 0 (empty bars). Each bar represents meanf SD of triplicate
cultures.
An mt-ll
Ba mt-ll
PBS
O 1000 2000 3000 4000 5000
Plasma CK (U/L)
Fig. 4 Effect of pH on the myotoxic activity of Lys49
phospholipases A2 in mice. Toxins (75 pg/50 pL) were injected
intramuscularly, at pH 7.2 (filled bars) or 5.0 (empty bars), as
described in Section 2 After 3 h, plasma creatine kinase (CK) levels
were detennined, and cxpressed as U/L. Control groups received
vehicle alone (phosphate-buffered saline; PBS) at pH 7.2 or 5 0.
BthTx-I, B. jarlirncussu bothropstoxin-1; An mt-11, Atropoirles
nicmrnifer myotoxin 11; Ba mt-11, Bothrops asper myotoxin 11; PBS,
phosphate-buffexed saline. Each bar represents rnean-1-SD of four
animals
TOXCON 2482-201512005-1 3; 1 1-SWAPNA-14882GXML MODEL 4 - pp !-6
dainage also evidenced that myotoxins preincubrtted at pH
5.0 before injection induced a significantly weakei- effect
than those incubated at pH 7.2, as estimated by determiiiing
plasma CK levels (Fig 4).
4. Discussion
Lys49 PLAzs characterized to date occur as homodimers
(Lee et al., 2001; da Silva Giotto e&al., 1998; Arni et a1 ,
1995; de Azevedo et al., 1998,1999; Magro et al., 2003) and
few cases, where these proteins have been crystallized as
monomers might be related o the use of acidic pH
conditions (Arni et al., 1999). A previous study, demon-
strated that bothropstoxin-1, a dimeric Lys49 myotoxin from
B. jarartrcrrssu, dissociates into monoiners at pH 5.0,
concomitantly with a complete loss of its ability to disrupt
artificial bilayers (de Oliveir et al., 2001). In the present
work, the functional consequences of such pH-induced
dissociation were further assessed, using a set of four
punfied Lys49 PLA2 myotoxins acting upon skeletal muscte
cells and mature muscle tissue as biologically relevant
targets.
Results support the view that the dimeric state of the
Lys49 myotoxins may contribute to their toxic mechanism,
in agreement with the conclusions of de Oliveiia et al
(2001), since the biological effects here, evaluated were
higher at pH 7.2 than at pH 5.0. However, in contrast with
findings using liposomes as targets (de Oliveira et al., 200 1),
our data using cultured rnyoblasts deinonstrate that the toxic
action of Lys49 PLA2s is not nbolished at pH 5.0. This
would imply that a dimeric state is not an absolute
requirement in the muscle-damaging mechanism of Lys49
PLA2 myotoxins, with rnonomers stil1 being able to exert
cell darnage. Moreover, results of paralle1 experiments with
synthetic peptides in the pi-esent work open additionai
interpretations for findings obtained with their parent 429
proteins, because these short peptides do not have a 430
quaternary structure. The lower cytoly tic activity of 43 1
peptides at pH 5.0 cannot be attributed to variations in the 432
degree of protonation of side chain groups which could 433
affect their interaction with the plasma rnembrane. This 434
possibitity can be ruled out on the basis of the known pKa 435
values of arnino and carboxy groups yresent within these 436
peptides, which should not change their net charge by 437
lowering the pH from 7.2 to 5.0. Therefore, it is conceivable 438
to consider that pH 5.0 could instead alter acceptor 439
structures on the cell rnembrane, or that the increased 440
extracellular proton concentration may have a protective 441
effect against mernbrane permeabjlization. While the former 442
possibility is only speculative, because the acceptor 443
structures for Lys49 PLA,s are still unknown (Lornonte 444
et al., 2003b), the Iatter phenomenon of proton-mediated 445
mernbrane protection has been previously dernonsti ated 446
for several cytolytic molecules (Bashford et al., 1989; 447
Rostovtseva et nl , 1994) Thus, despite the fact that a low 448
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Ptostaglaiidins Other Lipid Mediat. 68, 3-58.
Lec, W H , de Silva Giotto, M T , Marangoni, S., Toyama, M H ,
Polikarpov, 1 , Ganat. R C , 2001 Structural basis for low
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crystal stiucture of piratoxin 11 cornplexed to fatty acid.
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Lomonte, B., Gutirrez, J M., 1989 A new muscle damaging toxiri,
myotoxin TI, from the venoln of the snake Bothrops asper
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Lomonte, B , Moreno, E , Tarkowski, A , Hanson, L A ,
Maccarana, M , 1994. Neutrafizing interaction between heparins
aiid myotoxin II, a Lys-49 phospholipase A2 from Bothrops
nsper snake venom: identification of a heparin-binding and
cytolytic toxin region by the use of synthetic pcptides and
molecular modeling. J. Biol Chein. 269, 29867-29873.
toinonte, B , Pizarro-Cerda, J., Angulo, Y , Gorvel, J P.,
Moreno, E , 1999a. Tyr -tTrp-substituted peptide 1 15-129 of
a Lys49 phospholipase A2 expresses enhanced inembrane-
dainaging activities and reproduces its in vivo myotoxic effect.
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Lonionte, B , Angulo, Y , Rufini, S., Cho, W , Giglio, J R ,
Ohno, M , Daniele, J.J., Geoghegan, P , Gutirrez, J.M.,
1999b. Comparative study of the cytolytic activity of inyotoxic
phospholipases A2 on mouse endothelial (tEnd) and skeletal
muscte (C2C12) cells in vitro Toxicon 37, 145-158
Lomonte, B , Ang~ilo,Y , Santamara, C , 2003a. Coinparative
study of synthetic peptides corresponding to region 115-129 in
Lys49 myotoxic phospholipases A2 from siiake venoms.
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Lomonte, B , Anguio, Y , Caldern. L . 2003b. An overview of
lysine-49 phospholipase A2 myotoxins frorn crotalid snake
venonis and their structural determinants of myotoxic action
Toxicon 42, 885-901
Magro, A.J, Soares, A M , Giglio, 3 R , Fontes, M.R.M. 2003
Crystal structures of BnSP-7 and BiiSP-6, two Lys49-pho-
spholipases A2: quatcrnary structure and inhibition mechanisin
insights Biochein Biophys Res Commun. 31 1, 7 13-720.
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Maraganore, J M , Merutka, G ,Cho, W , Welches, W , Kezdy, F J ,
Heinnkson, R L , 1984 A new class of phospholipases A2 with
lysine iri place of aspartate 49. 3 Biol Chein 259, 13839-
13843
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myotoxic site of the Lys49 phospholipase A2 froin Agkistroclori
pis~ivorns p i s c i i ~ r w snake venon-i: synthetic C-terminal
peptides from Lys49, but not from Asp49 myotoxins, exert
membrane-damaging activities. Toxicon 39, 1587- 1594
Pramo, L , Lomonte, B., Pizarro-Cerd, J., Bengoechea, f A ,
Gorvel, J.P , Moreno, E , 1998. Bactericidal activity of Lys49
and Asp49 myotoxic phospholipases A2 from Bothrqx usper
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identifies its bactericidal region. Eur. J. Biochem. 253,452-461.
Rostovtseva, T.K.,Bashford, C.L., Lev, A.A, Pasternak, C A ,
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Scott, D.L., Achari, A , Vidal, J.C., Sigler, P.B , 1992. Crystal-
lographic and biochemical studies of the (inactive) Lys-49
phospholipase A2 from the venom of Agkistrodon piscivorus
piscivunts J B iol. Chem. 267, 22645-22657.
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phospholipase A2 enzymes: ciassification and c h a acterization 637
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myotoxins from Rothrops snake venoms: structure-function 640
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64 1
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642
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cheinistry 21, 3950-3955 644
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Reconstitution of ribosomes from subribosomnl components. 646
Methods Eiizymol. 20, 39 1-417. 647
Ward, R.J., Chioato, L., de Oliveira, A H C , RuIIer, R , S, J M , 648
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65 1
CEI-I, HlOCHEMIS 1 KY A N I I FUNC'I ION
Cell Biocheln F l l ~ t (in
~ t press)
Published online in Wiley Intei-Science (www.interscience.wiley.com). DOI: 10.1027/cbf 1208

Differential susceptibility of C2C 12 myoblasts and myotubes

to group 11 phospholipase A2 rnyotoxins from crotalid
snake venorns
' ? Bruno
Ymileth ~ n ~ u l oand ~ ~ o m o n t e*'
'I/istituto Clodotniro Picado, Fcicultacl de Microbiologcr, Urtiversiliad (le Costa Rica, San Jos, Cosfa Rica
'~epnrt(urzento(le Bioqzrrica, Esc.ltela de Medicina, Universidad de Cosfa Rica, Sarz Jos, Costn Riccr

Group 11 phospholipase A2 (PLA2) rnyotoxins isolated from Viperidaelcrotalidae snake venoms induce a rapid cytolytic
effect upon diverse cell types in vitro. Previous studies suggested that this effect could be more pronounced on skeletal
muscle myotubes than on other cell types, including undifferentiated myoblasts This stitdy utilized the rnurine skeletal miis-
cle C2C12 cell line to iiivestigate whether diffcrentiated rnyotubes are more susceptible than inyoblasts, and if this chaiac-
teristic is spccific for thc group 11 niyotoxic PLA2s. The release of lactic dehydrogenase was quantified as a measure of
cytolysis, 3 h aftcr ccll expnsure to dif'ferent group 11 PLA2s purified froni Bothrops cisper, Atropoides n~a>unifer, Cerrophi-
dion gadfnatti, and Bothr-iechisschlegelii vcnoms. In ddition, susceptibility to lysis induced by synthetic mefittin and group
111 PLA2 fsoni bcc (Apis rnellifer-ci) venoin, as well as by aiiionic, cationic, and neutral detcrgents, wris comparatively eval-
uated on the two cultures Myotubes were significantly more susceptible to group 11 PLA2 myotoxins, but not to the othcr
agcnts tcstcd, undcr the s:imc conditions. Moieovcr, the increased siisceptibility of lnyotubes over niyoblasts was also
demonstrated with two cytolytic synthetic peptides, derived from the C-terminal region of Lys49 PLA2 myotoxins, that
reproduce the action of their parerit proteiils. These results indicate that fusion and differeiitiation of myoblasts into myo-
tubes induce changes that render these cells more susceptible to the toxic niechanism of groiip 11 PLA2 rnyotoxins, but not to
general perturbations of mernbrne hoineostasis. Such changes are likcly to involve myotoxin acceptor site(s), whicti
iernaiii(s) lo be identificd. Copyright KJ 2005 John Wiley & Sons, Ltd.

KEY WORDS-niyoblast; myotiibe; skeletal muscle; phospholipase AZ;myotoxin; snake venom

INTRODUCTJON dae are classified as group IJ, together with rnamrna-

Phospholipases A2 (PLA2; EC 3.1.1.4) are major com-
ponents of snake venoins that have acquired a variety
of toxic activities during evolution, including myo-
toxic, neurotoxic, cytotoxic, anticoagulant, and
'"
il~flammatogeniceffects. Two structural groups of
PLA2s have been ctistinguished in snake venoms:
those from species of the Elapidae farnily belong to
group 1, which also includes the pancreatic PLAzs
of inammals, whereas those from ViperidaelCrotali-
* Coircspondcnce to: Di B. Lomonte, Instituto Clodomiio Picado,
Faciiltad (le Miciobiologa, Univcisidad dc Costa Rica, San Jos,
Costa Rica Fax: (+506) 292-0485
E - m i l : bloiiionte@carixi.ucrac cr
lian secreted inflammatory P L A ~ S . ~ . ~
Skeletal muscle necrosis is a drastic and frequent
consequence of ~nakebites.%~otoxic PLAzs have
been shown to play a rnain role in the pathogenesis
of this effect, inducing a commoii pattern of degenera-
tive effects leading to cell death.'"' Current evidence
indicates that PLA2 myotoxins act prirnarily on the
sarcolemrna, rapidly altering its permeability by either
catalytically-dependent or -independent mechan-
isms. 'o-'2 Howevei; the nature of the nlernbrane
acceptor site(s) jnvolved in these niechanisrns, and
the detailed molecular events that follow toxin bind-
ing, are still unknown.
Studies using a variety of cell liiies in culture have
shown that myotoxic group 1 PLAzs are not cytolytic,

Keceii~ed17 Augu~t2004
Xeilisecl4 October 2004
Copyiight (.fi 2005 John Wilcy & Soiis, Ltd. Accepted 14 Octobe~2004
 DIFF;EREN'I'IAI, SUSCEFIJBII,IrrY OF MYORLASTS A N D M Y O I U13I:S

exposed to variable anlounts of the different

inyotoxic/cytolytic agents (purified groiip II nlyotoxic
PLA2s, syiithetic peptides, detergents, group 111 bee
venom PLA2, and synthetic melittin), in order to gen-
erate dose-respanse curves. A11 agents were diluted in
DMEM containing 1% FCS, and added to the cells
(xnyoblasts or myotubes) after aspirating their med-
ium, in a total volume of 15Opl per well. After 3 h
of incubation at 37"C, supernatant aliquots were
obtairied for quantification of Iactate dehydrogenase
(LDH; EC 1.1.1.27) released into the mediurn, using
a colorinietric end-point procedure (Sigma N0500).
Reference controls for O and 100%cytolysis consisted
of medium done or mediurn containing 0.1%(vlv)
Triton X-100, respectively. A11 assays were carried
out in triplicate.

RESULTS
Under the culture conditons described, a large pro-
portion of C2C12 myoblasts fused into myotubes
within a few days of lowering the FCS concentration
in the nieclium to 1%. The morphology of cells uti-
lizecl as targets in this stiidy, rnyoblasts and inyotubes,
is shown in Figure 1. Dose-response curves showed
that the cytolytic effect of the diffesent group 11 myo-
toxic PLA2s tested, in al1 cases, was significantly
higher on differentiated myotubes than on rnyoblasts,
as summai-ized in Figure 2. Differences in susceptibil-
ity to lysis were more evident at submaximal doses of
inyotoxin, i.e. 5-10pg per well, corresponding to
approximate protein concentrations of 2-4 PM
(Figure 2).
In contrast to the group 11 PLA2 myotoxins from
snakes, both the bee venoin group 111PLA2 and melit-
tin had similar cytolytic activities on inyoblasts and
myotubes, resulting in nearly superimposable dose-
response curves (Figure 3). 111 similarity with these
two rnyotoxic proteins from bee venom, al1 three types
of detergents tested (neutral, anionic, or cationic)
caused the same Iytic effect on both types of cell cul-
tures (Figure 4). On the other hand, the two synthetic
peptides corresponding to the C-terminal regions
Lys49 myotoxic PLA2s from B. asper and A. c. lati-
cinctus, respectively, were significantly more toxic
to myotubes than to ~nyoblasts (Figure 5), thus
mimicking the action of the whole proteins. Figure l . Light microg~aphsshowing the cell morpliology of
iindiffeletitiated C2C12 myoblasts (A), and differentiated myotubes
(B) utilizcd as iargets in the expcriments Aii cxainple of the
DISCUSSION cytolytic effect induced on niyoblasts after exposiire to the
C-terminal synthetic peptidc (scquence 1 15-1 29) r>I A~kistrocloii
The use of skeletal iniiscle myobIasts/myotubes as lar- ronrorrrix lutici>rc~trsinyotoxin (100 ~ i gper well) is shrnvvn in C
gets for group II PLAzs has been proposed as a usefiil
ir1 vitro rnodel to study their inyotoxic mecllanisrn(s),

Copyiight O 2005 John Wiley & Sons, Ltd.


Figure 2 Comparison oC the cytolytic activify of diffeierit grorip 11
phospholipasc A2 myotoxins on C2C 12 myoblasts (e)or myotubes
( O ) (A) Bothrops ispet rnyotoxjii 1; ( R ) R nsper n~yotoxin11; (C)
R. aspej myotoxin 111; (D) H iiqwr myoioxiii IV; (E} Atropoirfes
tzummifer myotnxin 1; (F) A nirrtlin(fir inyotoxin 11; (G)
Cerr.opki~lliorigodt~tarii iiiyotoxin JI; ( H ) Rntlrr-iechis ~rlzlegelii
myotoxin T Cytolysis was estimated by the release of lrictic
dehydrogenase (LDI I) into siipcrnataiits aftcr 3 h Maxiiiial
inyotoxin doses (20 pg per well) coi respond to approx in~ateproteiri
coiicetitrations of 8 Eacli point Jepresents niean f SD of
triplicatc cultures
as it correlates with the muscle-dainagiiig activity
observed in v i i ) ~ . ~ " ' ~~ h~ i' ~
sstudy investigated
whether the fusiori and differentiation of C2C12 myo-
blasts, renders them more susceptible to the cytolytic
actiorl of gi-oup 11 inyotoxic PLA2s
An ii~creasedsusceptibility of the myotubes was
clearly observed using a panel of eight different snake
myotoxins, including both Asp49 PLA2 and Lys49
PLA2 variants. Interestingly, other types of proteins
for which myotoxic action has been documentecl, such
as the bee venom (group 111) PLA2, and the cationic
peptide me~ittin,'~ affected botli iypes of cells iii cul-
Copyright ;r.l 2005 John Wiley 1(( Sons, Ltd
Apis PLA2 (kglwell)
O 1 2 3
Apis melitt in (pglwell)
Figure 3. Co~i~paiison of the cytolylic activity of Apis rnelliJern
groiip 111phospholipasc A2 (A) and synthetic rnelittir~(B)o1.i C2C12
inyoblasts (e) oi myotubes (0) Cytolysis was esti~natedby \he
release of lactic dehyclrogeriase (LDH) into supe1natants after 3 h.
Eacli point rcpiesenis mcan & SD of triplicate ciiltiiies
tirre to the same extent. The use of variuus detergents
also demonsti-ated a similar cytolytic effect in bot1.t
inyoblasts and inyotubes. Thus, the iiicreased susceyt-
ibility of inyotubes to the group 11 PLA2s cannot be
attributed to a possibly lower ability of these cells to
cope with general perturbations of mernbraile home-
ostasis. The differentiation of myoblasts into myo-
tubes must lead to changes that specifically favour
the action of group 11 PLA2 myotoxins It is likely that
these chariges involve the expression of either a higher
nurnber, or it higher affinity type, of acceptor sites for
the myotoxic PLA2s on the plasma inenibrane. How-
ever, the possible participation of intracellular pro-
cesses arising after rnyoblast fusion, as seen in the
enhanced dainage to myotubes, cannot be excluded
at this time.
Cell Kinchein F'icttct (iii piess)

Detergent (%)
Figuic 4 Cornpaiison of the cytolytic activity nf different
deteigent types on C2C12 niyoblasts (e)or myotiibes (0) (A)
Triton X- 100, neutral; (B) sodiuin dodecylsulfate, anionic; aiid (C)
cetyl-trimethyl ammonium bromide, catioriic Cytolysis was
estimated by the ielease of lactic dehyd~ogcnase (LDI-1) into
slipciiiatanis after 3 ti Each poirit reprcsents inean SD of triplicatc
cultuies
The selectivity of the myotoxic effect of venoln
PLA2s in i ~ i v ohas been ascribed to the existence of
specific binding sites on the pIasina mernbrane of ske-
letal niuscie cells, but their nature is completely
unknowil. A number of high affinity protein acceptors
for presynaptically active rleurotoxic PLAzs have
Ixen ideiltified in neurona1 tissue.""" On the other
haiid, a multidomain membrane protein of 180 kDa,
known as the M-type receptor, has been characterized
as ri binding site for sorne gi'oup 1 PLA2s i i i skeletal
Copyiight (<) 2005 John Wiley & Sons, Ltd
pt 15-129 ~115-129
B asper myotoxin II ACL myotoxin
Figure S Conipaiisoii of the cytolytic activity of two synthetic
peptides corresponding to thc C-terminal regions (sequence
1 15-1 29) of Bothrops o,c;pet niyotoxin 11 (KKYRYYLKPFCKK)
and Agkisfrodon cotirortrix !~rticiiicnis myotoxin (KKYKAY-
FKFKCKK). rcspectively, on C2CI2 inyoblasts (m) or myotiihes
(m) Cytolysis was estimated by the release of lactic dehydiogenase
(LDH) into supcriiatants aftcr 3 h Peptides were assayed at 100 ~ i g
per wcll, correspoiiding to appioximate pioteiii concenttations of
360 I ~ MBars represent mean fSD of thiee deteiminations
r n i i s c ~ e I-Iowever,
.~~ 170 evidence for the involveinent
of M-type receptors in the rnyotoxic effect of group
II PLA2s has been reported.
Alternatively, group II PLA2 myotoxins could be
actiiig through the recognition of membrane lipids,
such as glyceropliospholipids 01- glycolipids, that
might be differentially expressed, or differentially
clustered, in myoblasts and myotubes. Severa1 obser-
vations suggest that negatively-charged phospholi-
p i d s ' ~ a ybe iinportant acceptors for the inyotoxic
n-iechariisrn of these proteins, as recently
reviewed.' In particular, the cytolytic activity
denlonstrated for an all-D amino acid forn of a syii-
thetic peptide derived frorn Agkistrodorz piscivor-lis
piscivnr~ls Lys49 PLA2, strongly suggests that the
mechanism of action of these myotoxiris does not
involve 1-ecognition of a proteinaceous acceptor sjte
on muscle c e ~ l s . ~ '
In the subgroup of Lys49 PLA2 myotoxins, the
C-termiral region has been identified as central for
myotoxic/cytolytic activity.20-22,39 Synthetic peptides
corresponding to the sequence 115-129 of sorne of
these proteins can reproduce their mernbrane-darna-
ging actions and, therefore, it was also of interest to
compare the susceptibility of myoblasts and rnyotubes
to such peptides. The results demonstrated a higher
cytolytic effect of synthetic peptides from Lys49
PLA2s on the n-iyotubes thari on the inyoblasts. Thus,
Cell Uiocheni fiaic! (jn press).
 Y . ANGUL20 A N D B. LOMON J 1:
these C-terminal peptides behaved iri a qualitatively
sirnilar fashion to their parent proteins, in agreenxent
with their proposed effector role in the ineinbrane
diiiiiaging mechanisiii of Lys49 PLAz inyotoxins,I 2
and furthes supporting the notion of a specific
iiicreased susceptibility of rnyotubes to group 11
PLA2 inyotoxins.
ACKNOWLEDGEMENTS
This work was supported by the International Founda- 16. Lomonte B, Tarkowski A, Hanson LA Broad cytolytic speci-
tion for Science (F/2766-2), University of Costa Rica
(VI-74 1 -99269 and VI -74 1-A350 1 3), CONICIT-
FORINVES (FV058-02), and NeTropica Swedeii-
Central America network.
We thank Dr J. M. Gutirrez for critically reading 18. Andriao-Escarso SH, Sonres AM, Rodrig~iesVM, et al. Myo-
this inanuscript.
This study was perforined in partial fulfilment of
the doctoral degree of Y. Angulo at the University of
Costa Rica.
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 Available online at www.sciencedirect.com
ScIENcrz GDIREcT.
Biochemicl Pharrnacology 66 (2003) 1993-2000
ivww elsevier coiii/locate/biochcmphai in

Inhibitory effect of fucoidan on the activities of crotaline

snake venorn myotoxic phospholipases A2
Yamileth ~ n ~ u l o " ' ~Bruno
? * , Lomonte"
"bisrituto Clodoniiro Picnilo, Facttlrnd de Microbiologa, Ur~iversidudde Costa Rica, Son Jos, Costa Rico
b~eporranientore Bioqunzica, Esclrela de Medicirin, Urziversidad de Costa Rico, San Jos, Costa Rica
Received 25 June 2003; accepted 24 July 2003

Abstract

Myotoxic phospholipases A2 account for most of the muscle necrosis that results from envenenomation by crotaline snakes. In this study,
we investigated the protective effect of fucoidan, a natural sulfated polysaccharide obtained froin the brown seaweed Fucl~svesiciilosus,
against the cytotoxic and rnyotoxic activities of a group of phospholipase A2 myotoxins from crotaline snake venoms Bothrops asper
myotoxins 1, II, 111, and IV, Cerrophidio~igodmani myotoxins 1 and 11, Atropoides n~ttnmifermyotoxins 1 and 11, and Bothriechis schlegelii
myotoxin 1. All of the toxins tested were efficiently inhibited by fucoidan, in both their cytotoxic and inyotoxic effects The basis for this
inhibition appears to be the rapid formation of complexes between fucoidan and myotoxins, as evidenced by ti~rbidimetricanalysis. The
possible binding site of fucoidan on the inyotoxins was investigated using short synthetic peptides that represent the membrane-damaging
region (residues 115-129) for three of these toxins Fucoidan clearly inhibited the cytolytic activity of the peptides, indicating its ability to
interact with the C-tennirial inyotoxic region of these phospholipases A2 Fucoidan significantly inhibited inuscle damage in rnice, when
administered locally, iinmediately after experimental etivcnoination with cmde venom from B. asper These results encourage furtlier
studies of sulfated fucans as conipounds of potential use to iinprove the treatment of envenoinations by crotaline snakes
O 2003 Elsevier Inc. AIl rights reserved
Key,vol.ds. Fucoidan; Myotoxin; Phospholipiise A*; Snake venorn; Iiihibition; Polysaccharide

1. Introduction of crotaIine species from the genera Bothrops, Agkistrodon,

Phospholipases A2 (PLA2; EC 3.1.1.4) are ubiquitous
enzymes that catalyze the hydrolysis of the sri-2 position of
glycerophospholipids, leading to production of free fatty
acids and 1ysophospholipids [ 1 1 . Although al1 PLA2s cat-
alyze essentially the same reaction, their biologic activities
vary considerably. In snake venoms, PLA2s have evolved
into potent toxins with diverse specific activities such as
neurotoxicity, myotoxicity, stirnulation or inhibition of
plateIet aggregation, anticoagulant, hypotensive, cardio-
toxic, edema-inducing 121, and bactericidal [3] effects.
Crotaline snakes (family Viperidae, subfamily Crotali-
nae) are widely distributed in Amei-ica f4], comprising a
large number of species which are responsible for the
majority of snakebitc envenomations in this continent
[5]. Myotoxic PLAzs have been described in the venoms
* Corresponding author Te1 : +506-229-0344; fax: $506-292-0485.
E-rnail address yanguloC3caiiari ucr ac cr (Y Angulo)
Abbrevintions: PLA2, phospholipasc A?; CK, creatine kinase; LDH,
lactic dehydrogenase
Porthidiiinr, Trirrzeresurus, A tropoides, Crutalus, Borhr ie-
chis, CaEloseEasr~za,and Cerrophidiorz E6-101, as compo-
nents that have a prominent roe in the induction of skeletal
muscle necrosis. These PLA2s are structurally classified
into group IIA [ l 11, belonging either to the enzymatically-
active (Asp49-type) or enzymatically-inactive (Lys49-type)
subgroups.
The clinical relevance of myonecrosis in snakebite
envenomations has motivated the search for nonimrnune
neutralizing inolecules against rnyotoxic PLA2s, including
natural inhibitors of animal and plant origins [ 12- 161, that
could complement conventional antivenom therapy.
Among the different types of inhibitors studied, some
anionic polysaccharides such as heparin have been shown
to neutralize myotoxic PLA2s isolated from Bothrops
jarnrncussil [17] and B. asper [18,19] venoms. On this
basis, we decided to investigate if fucoidan, a compicx
sulfated polysaccharide extracted from the brown seaweed
Fucus vesiculosus, could inhibit the toxic activities of a
group of myotoxic PLA2s frorn crotaline venoms

0006-2952/$ - see front matier O 2003 Elsevier Inc. Atl rights ieserved
doi: 10 1016lS0006-2952(03)00579-3
1994 Y Arigir lo, B )mor,te /Llioc hemicril Plicirmucology 66 (2003) 1993-2000
The structure of fucoidan has been characterized in detaii.
The core region of the fucan is composed primarily of a
polymer of a l -3-Iinked fucose, with sulfate groups sub-
stituted at the 4 position on some of the fucose residues.
Fucose is also attached to this polymer to form branch
points, one for evc1.y 2-3 fucose residues within the chain
f20,211. This polysaccharide mediates a variety of biological
effects in niammals, including leukocyte recrujtment inhibi-
tion [22], revascularization of ischemic tissue [23], platelet
aggregation 17241, collagen contraction [25],and inhibition
of smooth muscle cell proliferatioii [26]. Fucoidan interacts
with severa1 components of the coagulation and fibrinolysis
systems, such as heparin cofactor 11, antithrombin 111,
thrombin, glutamic plasminogen, tissue plasminogen acti-
vator and low molecular weight-urokinase 127,281, and
exerts antitumor 1291, as well as antimicrobial activities 1307.
In this study, we report and characterize the inhibitory
effect of fucoidan on the cytotoxic and myotoxic activities
of different myotoxic PLA2s isolated from crotaline snake
venoxns, and evaluate its potential to reduce myonecrosis
when rapidly administered after the injection of Bothrops
usper venoni in a mouse model.
2. Materials and nietliods
2 1 lsolntinrz of niyotnxic PLA2s
Each venorn was a pool obtained froni specimens col-
lected in Costa Rica and kept at the serpentarium of the
Iristi tu to Clodomiro Picado. Myotoxic PLA2s were purified
by cation-exchange chrornatography on carboxymethyl-
Sephadex C-25 (Pharmacia, Sweden), as dcscribed: B. nsper
rnyotoxins 1 [3 1],11 [32], 111 [33] and IV 1341; C. go~lrrlnrzi
inyotoxins 1and 11 [353;A. numn@"ermyotoxins 1 [36] and 11
1371; and B. schlegelii myotoxin 1 /38]. Toxin homogeneity
was assessed by urea-PAGE for basic proteins [39], and
reverse-phase high performance Iiquid chromatography
(RP-I-IPLC) on a C4 column (25 mm x 4.6 mm; Vydac),
eiuted at 1 .O mLlmin with a gradient from O to 60% acet-
onitrile in 0.1 % trifluoroacetic acid (vlv), using an Agilent
model 1100 HPLC system.
2.2. Irihibition of cytoloxic activity in vitro
Cytotoxic activity of the purified PLAzs and its inhibi-
tion was assayed on murine C2C12 skeletal rnuscle myo-
bIasts (ATCC CRL-1772) grown in 96-weII plates, as
described [40]. Toxins alone, or mixed with fucoidan
(n-iolecularmass 135 kDa; Sigma) at different molar ratios,
were incubated for 30 rnin at room temperature. Then,
aiiquots of 150 pL (containing 30 pg of toxin in DME
medium) were applied to the cultures, after aspirating their
growth medium (DME with 15%, v/v fetal calf serum).
After 3 hr of incubation at 37", lactate dehydrogenase
(LDH; EC l . 1.1.27) reIeased to supernatants was deter-
mined with a colorimetric procedure (Sigma 500C). Refer-
cnce controls for O and 100% cytotoxicity consisted of
medium alone and mediurn containing 0.170 (vlv) Triton
X-100, respcctivcly. Additional controls consistcd of cells
iicubated with fucoidan in the abscnce of toxins Assays
were carried out in triplicate.
2.3, Time-course of the inhibition of B. nsper
niyotoxin II
In order to determine the time required for the inhibition
of cytotoxicity by fucoidan, B. nsper myotoxin 11 alone, or
mixed with fucoidan at a 1:1 molar ratio, was incubated for
5,10,15, or 30 min, at room temperature. Then, cytotoxicity
was quantified on the C2C 12 cultures, as described above.
2.4. Inhibition of the cytotoxic activity of synthetic
peptides of Lys49-PLA2s
Three synthetic peptides corresponding to the C-term-
inal regions (sequence 115-129) of B. asper myotoxin II
(KKYRYYLKPFCKK), Agkistrodon piscivorus piscivorus
Lys49-PLA2 (KKYKAYFKLKCKK), and A. contartrix
laticinctus myotoxin (KKY KAYFKFKCKK), respectively,
have been shown to exert direct cytotoxic activity upon
C2C12 cultures, therefore rnimicking the effects of their
parent toxins [4 1,421.These peptides, either alone (100 pg/
150 pL) or mixed with fucoidan at a molar ratio of 0.25
(fucoidanlpeptide), were incubated for 30 min at room
ternperature, and then applied to the C2C12 cultures to
assay for cytotoxicity, as described above.
2.5. Inhibition of lnyotoxic activity in vivo
Myotoxic activity of the PLA2s was estimated by deter-
mining the plasma levels of creatine kinase (CK; EC 2.7.3.2)
in groups of four CD-1 mice (1 8-20 g body weight), after an
intramuscular injection (in the gastrocnemius) of 75 pg of
each toxin, either alone, or preincubated with fucoidan at a
I :1 molar ratio for 30 min at room temperature. Control
groups received an identical injection (100 pL) of PBS, pH
7.2 afone, or fucoidan alone. In addition, inhibition of the
rnyotoxic activity of whole B. asper venorn by fucoidan was
tested similarly, using a venom challenge dose of 50 pg.
After 3 hr, blood samples were collected from the tail into
heparinized capillary tubes, and the plasma CK activity was
determined by a kinetic assay (Sigma CK- 10). Activity was
expressed as U/L ( 1 unit defined as the amount of enzyme
wllich produces 1 pmol of NADHlmin at 30").
2.6. Inhibition of myotoxicity in vivo by locally
advzinistered filcoidnn
Three groups of four mice received an intramuscular
injection of crude B. asper venom (50 pg) in the gastrocne-
mius. In two of the groups, this was followed immediately by
an injection of fucoidan (90 or 270 pg) at the sme site.
Assuming that the venom contriins 20% of myotoxins by
weight, these fucoidan amounts would represent approx-
imate molar ratios of 1:1 and 3: 1 (fucoidanlinyotoxin)
Control animals received PBS alone, or fucoidan alone.
Myonecrosis was estirnated as described, 3 hr after vcnoni
injection. Al1 in vivo experiments were approved by the
University Committee o11 Animal Use and Care.
2.7. Yhospholipase A2 activity
PLA2 activity of two Asp49-type myotoxins (B. nsper
myotoxins 3. and 111) was determined using micellar egg
yolk phospholipids (suspended in 0.1 M Tris-HCI, 0.01 M
CaC12, pH 8.5) as substrate, in the presence of 1 % (v/v)
Triton X-100. Toxins (10 pg), either alone or preincubated
with fucoidan at different molar ratios for 30 min at room
temperature, were added to 1 mL of substrate. After an
incubation of 15 min at 37", the free fatty acids were
extracted and titrated with 0.018 M NaOH, as described
by Dole [43]. Controls consisted of substrate with PBS,
or substrate with fucoidan. Assays were carried out in
triplicate.
absorbances were recorded on a microplate reader (Labsys-
tems Multiskan RC) at 405 nm. Normal rabbit serum was
utilized as a negative control.
3. Results
Fucoidail inhibited the cytotoxic activity of al1 the
myotoxic PLA2s tested, although with some quantitative
variations among the~n.B. nsper- myotoxins I I and IV, A.
nuntmifer myotoxins 1 and If, C. godrnani myotoxins 1 and
11, and B. schlegelii myotoxin 1 were completely inhibited
by preincubation with fucoidan, whereas the activity of B.
clsper myotoxins 1 and IU was reduced by 50-65%, at a I :1
molar ratio (Fig. 1). Toxin inhibition, as evaluated by LDH
release, was consistent with microscopic observations of
cell culture morphofogy. Fucoidan alone, at the maximal
concentrations utilized in inhibition experiments, did not
alter cell morphology, and did not cause LDH release.

2.8. Fornicztion of ~izncronioleculnrconlplexes between

f u c a i h n nnd R. nsper nzyotoxins

Complex formation between fucoidan and B. nsper

myotoxins 1-1V was assessed by turbidimetry, as prc-
viously described [19]. Two-hundred ~ t gof each toxin
were added to 2 mL of Tris-KC10.01 M, pH 7.0, followed
by consecutive additions of 1 pL of fucoidan (20 mg/mL)
to give different final ratios of fucoidan/myotoxin, After
each addition, the mixtures were incubated for 1 mi11
before reading the absorbance at 340 nm.

Competition between fucoidan and rabbit antibodies

against the C-terminal region 1 15-129 of myotoxic PLA2s
[44], for simultaneous binding to B. asper myotoxin 11, was
evaluated using an enzyme-immunoassay. Microplates
(Dynatech Laboratories) were coated with B. asper myo-
toxin 11 at 0.4 pg per well, by overnight incubation in O. 1 M
Tris 0.15 M NaCI, pH 9 0 buffer. After five washing with
solution A (O 05 M Tris, 0.15 M NaC1,20 pM ZnCl?, 1 mM
MgC12, pH 7.4), varying dilutions of the rabbit serurn to
region 1 15-129 were added to triplicate wells, diluted in Fucodanlmyotoxin molar ratio
solution A containing 2% (wfv) BSA, and incubated at 4"
Fig 1 Inhibition of the in vitro cytotoxic activity of myotoxic
for 24 hr. A parallel set of rabbit serum dilutions was phospholipases A2 by fucoidan Cytotoxicity was determined by the
incubated in the presence af 500 pg per well of fucoidan. release of Inctnie dehydiogeniise (LDH) from C2C12 skeletal muscle
After five washings with solution A, bound antibodies were myoblasts, 3 hr after exposuie to myotoxins (30 pg), either done or
detected with an antirabbit inmunogIobulin-alkaline phos- preincubated wi!h fucoidan at the indicaled molar ratios, as described in
Section 2. (A) B aspel myotoxins 1 (e), 11 (m), 111 (A),and IV (+). (B)
phatase conjugate (Sigma), diluted 1:2000 in solution C. godniani rnyotoxins 1 (0) and 11(u), A nummifer rnyotoxins I (A) and
A-BSA, and incubated for 1 hr. After washing as described, 11 (O), and B. schlegelii myotoxin 1 (O). Each point represents the
color was developed with p-njtrophenylphosphate, and mean k SD of triplicate culiures

O 5 10 15 20 25 30
Time (min)
Fig. 2. Timc-course of [he inhibition of the in vilyo cylotoxic activity ofB.
urpsr myotoxin 11 by fucoidan. Cytoloxicity \vas determined by the release
of Iactiite dehydrogenase (LDH) from C2C12 cells, 3 hr after exposure to
nlyotoxin 11 (30 klg) done (time 0) or preincubated with fucoidan at n 1:l
inolar ratio, for the iiidicnted periods of time Each point represents
mei\n -Ir SD of rriplicate cultures
Fucoidan pretreatment of the cells, folIowed by washing
with medium, and subsequent exposure to the toxins, did
not inhibit their cytolytic activity (data not shown)
B. asper myotoxin 11 was selected to study the time-
course of the inhibitory effect of f~icoidanupon its in vitr-o
cytotoxic activity. As shown in Fig, 2, tlie action of
fucoidan was very rapid, with a complete toxin inhibition
achieved within only 5 mjn of incubation at room tem-
perature, before the application of thc mixture t o ceil
cultures. Thcse results are in agreement with the rapid
forrnation of macromolecular complexes between fucoidan
and B. clsper myotoxins 1, 11, 111, and IV, as observed by
turbidimetry assays. Addition of fucoidan to these toxins in
solution resulted in a rapid increase of turbidity at 340 nm,
indicating the formation of insoluble complexes (Fig. 3).
These complexes caused maximal turbidity at a molar ratio
of approximateIy 0.1 :1 (fucoidan/myotoxin), and subse-
qucntly redissolved by the gradual addition of a polysac-
charide excess (Fig. 3).
The possible interaction site of fucoidan on the myotoxic
PLA2s was investigated by a solid-phase competition
binding assayy and by the use of cytolytic 'ynthetic pep-
tides- In the former test, the presente of fucoidan caused a
sjgnificant reduction in the binding of rabbit antibodies
towardS the C-terminal 1 15-129 of B. usper niyo-
toxin 11, to immobilized myotoxin II (Fig. 4). In addition,
cytotoxicity experiments showed that the activity of three
cytolytic synthetic peptides, corresponding to the region
115-129 of diffcrent myotoxic PLA2s, was completely
inhibitcd by their preincubation with fucoidan (Fig. 5 ) .
Using the two Asp49-type PLAzs from B. asper (myo-
toxins 1 and III), the possiblc inhibition of their catalytic
activity by fucoidan was iiivestigated When fucoidan was

Fucoidanlmyotoxin molar ratio

Fig. 3 Formation of macromolecular complexes between fucoidan and B osper myotoxins Two-hundred pg of B. asper rnyotoxins 1 (A), 11 (B), 111 (C) or IV
(D)were added to 2 rnL of Tris-KCI 0 01 M. pH 7 0, followed by consecutive sdditions of 1 ~ I of
L fucoidan (20 mg/mL) to give the indicated final ratios of
fucoidaiilmyo~oxin Afier each addition the mixtures were incubated for 1 min before reading the absorbance at 340 nm.

Serum dilution
Fig 4. Competition between antibodies tu peptide 115-129 and fucoidan
for binding to immobilized B. usper myotoxin 11. Binding of rabbit
nntibodies raised against the C-terminal region of B asper myotoxin 11
( 1 15-129) wns tested by enzyme-immunoassay, using microplates coated
with myotoxin 11, as described in Section 2. Varying dilutions of the rabbit
immune serum were tested eithcr alone ( O ) or in the presence of 500 pg
per well of fucoidan (O). Normal rabbit serum (A) was included as a
negative control Each point represents the mean SD of tripiicaie wells.
incubated with these toxins, up to a molar ratio of 2:l
(fucoidan/rnyotoxin), their PLA2 activity was identical to
that of the control toxins incubated without fucoidan,
evidencing that this enzymatic activity was not inhibited
by fucoidan (data not shown).
pAcl
LDH release (%)
Fig. 5. Inhibition of ihe in vifro cytotoxic activity of synthetic peptides
115-129 of myotoxic phospholipases A2 by fucoidan. Cytotoxicity was
determined by the release of lactate dehydrogenase (LDH) frorn C2C12
cells, 3 hr after exposure to synthetic peptides (100 pg) representing the
C-terminal iegion 115-129 of B. asper myotoxin 11 (pl15F). Agkistrodon
piscivorus piscivorus Lys49 phospholipase A2 (pAppk), and A. contortrix
luticirtctus myotoxin (pAcl), either alone (filled bars) or preincubated with
fucoidan (empty bars) at a 0.25 molar ratio (fucoidanlpeptide). Peptide
sequences are described in Section 2. Each bar represents the mean & SD
of triplicate cultures.
C. godmani M t 11
A. nummifer Mt II
A. nummifer Mt 1
B. asper Mt IV
B. asper Mt III
B. asper Mt 11
B. asper Mt I
B. asper venom
O 25 50 75 100 125
Plasma CK activity (%)
Fig. 6. Inhibition of the in vivo myotoxic activity of phospholipases A2
and crude B. asper venom by fucoidan Myotoxins (75 kig) or crude B
asper venom (50 pg) were injected intramuscularly, either alone (filled
bars) or preincubated with fucoidan (empty bars) at a I:I molar ratio, as
described in Section 2 After 3 hr, plasma creatine kinase (CK) levels were
determined Values are expressed as a percentage of the CK activity
resulting from the injection of each toxin (or venom) done One-hundreci
percent values varied from 2105 to 4223 U/L. The plasma CK values of
mice receiving a PBS injection done were subtiacted in al1 cases Eiich bar
represents the mean 3z SD of four ailimals
The inhibition of rnyotoxic PLA2s by fucoidan was
subsequently examined in vivo, by estimating skeletal
muscle damage through the increase of plasma CK leveis
in mice. Data summarized in Fig. 6 show a clear reduction
Fuc 1 V 3:l
Fuc / V 1:t
Venom
PBS
Plasma CK activity (UIL)
Fig, 7. Inhibition of the in vivo myotoxic activiiy of crude B asper venom by
independent local injection of fucoidan Venom (50pg) was injected
intramuscularly into three groupc of mice, two of which received irnmediately
an injection of fucoidan (90 pg, FuclV 1:l; or 270 pg, FucN 3:1) at the same
site A control group received an injection of PBS, pH 7 2 done After 3 hr,
plasma creatine kinase (CK) levels were determined Bars represent ihe
mean f SD of four animals. The values corresponding to FucN 1: 1 and
FucN 3:l are significantly lower ( P < O OS) than those of the venoin group
in the plasma CK activity induced by al1 toxins tested, after
their incubation with fucoidan at a 1:l molar ratio.
Although there are minor quantitative differences between
toxins, irihibitions varied between 70 and 95% (Fig. 6).
Accordingly, fucoidan also inhibited the rnyotoxic action
of crude B. nsper venom, in these preincubation experi-
ments (Fig. 6). Injection of fucoidan alone did not increase
plasina CK values above those of control animals receiving
a PBS injection (data not shown).
Finalfy, the protective effect of fucoidan was evaluated
jn independent administration tests, in which the polysac-
charde was injected locally, immediately after an intra-
muscular challenge with B. asper venom in mice. Fucoidan
administration significantly reduced the extent of myone-
crosis, resulting in CK values that corresponded to approxi-
rnately 50% of those of the control group receiving venom
done (Fig. 7). A 3-fold increase in the dose of fucoidan
administered in situ (from 90 to 270 pg) did not improve
the leve1 of protection (Fig. 7).
4. Discussion
Within the Iast decade, a growing number of inhibitors
of snake venom myotoxic PLA2s have been reported,
ranging from animal plasina proteins [12-141 and poly-
saccharides [17-191, to plant components [15,4S].
Together with the gradual advances in our understanding
of the molecular determinants and mechanisms of action
involved in the toxic effects of myotoxins, the search for
efficient inhibitors might lead to a significant improvement
of snakebite treatment, in addition to conventional anti-
venom therapy, in the future.
The discovery of enzymatically-inactive Lys49 PLA2
variants in the venoms of crotalines [46,47] prompted the
search for a toxic site in these proteins, not necessarily
related to the known catalytic site of PLA2s. Current
evidence indicates that the C-terminal region of Lys49
variants, combining cationic and hydrophobic amino acids,
constitutes a key determinant for their myotoxic activity
119,41,44,48,49]. Thus, polyanionic molecules with the
ability to bind to this toxic site constitute rational candi-
dates for evaluation of myotoxin inhibitory activity.
The present study focused on fucoidan, a complex
sulfated polysaccharide extracted Irom marine algae.
Although sharing some biological properties with heparin,
sulfated fucans constitute aIternative polyanionic mole-
cules with a range of different pharmacological profiles
[50]. Using a group of myotoxic PLA2s purified from
different crotaline venoms, it was demonstrated that fucoi-
dan efficiently inhibits both their cytotoxic (in vitro) and
rnyotoxic (in vivo) effects. Moreover, in the case of two
catalytically-active (Asp49) PLA2s, fucoidan inhibited
their toxic actions without affecting their ability to hydro-
lyze phospholipids. This result further exernplifies the
dissociation between toxicity and catalysis jn the family
of class 11 Asp49-type myotoxins, reported in a number of
previous studies [7,18,5 1,523.
The inhibition mechanism of fucoidan against myotoxic
PLAzs appears to be the rapjd formation of complexes,
probably mediated through multivalent, electrostatic inter-
actions between the anionic sulfates of the polysaccharide
and the niimcrous cationic residues of the toxins, which
have highly basic isoelectric points. Reversible cornplex
forrnation was evidenced by turbidimetry xnesurements,
where the Iight scatterng properties of the fucoidan/myo-
toxin mixtures varied according to the relative proportions
of these two interacting components, resulting in typical
bell-shaped absorbance curves.
The possible binding site of fucoidan on the myotoxins
was investigated using short synthetic peptides that repre-
sent the membrane-damaging region (residues 1 15-1 29)
for three of toxins 1423. Fucoidan clearly inhibited the
cytolytic activity of the three synthetic peptides, demon-
strating its ability to interact with the C-terminal myotoxic
region of these phospholipases As. Further support to this
conclusion was provided by cornpetition binding data
iising antibodies raised against the synthetic peptidc
1 15-129 of B. asper myotoxin 11 [ U ] .Binding of these
antibodies to the immobilized toxin was reduced in the
presence of fucoidan. The region 1 15-1 29 of Lys49 myo-
toxic PLA2s, such as B. asper myotoxin 11, was previously
shown to constitute a heparin-binding site, and to be
involved in its cytolytic action in vitro [19,41) In this
rcgard, fucoidan appears to act in a similar mode as heparin
[19], in agreement with their common polyanionic nature,
provided by a high density of sulfate substituents. How-
ever, it should be noted that the density of negative charges
on a polysaccharide backbone is not the only factor
determining jts ability to interact with the rnyotoxic PLA2s,
For example, the binding of chondroitin sulfate and der-
matan sulfate to B. nsper myotoxin II is weaker than the
binding of heparan sulfate, although the Iatter is less
sulfated 11 91, highlighting the contribution of the type
of polysaccharide backbone upon an optimal interaction
with proteins. Therefore, it was of interest to characterize
and evaluate the inhibitory activities of fucoidan against
myotoxic PLA2s.
Using the crude venom of B. nsper, the ability of
fucoidan to inhibit muscle damage when administered
locally, immediately after cnvenomation, was evaluated
in mice, Under these conditions, fucoidan injection par-
tially decreased the myonecrosis induced by the venom, to
a leve1 of approximately 50% of that of untreated animals.
Thus, its efficiency is clearly limited in experiments that
resernble the actual situation of a snakebite, although a
50% reduction in muscle damage is stiti a considerable
benefit. A major limitation for the development of any
clinically effective inhibitor for myotoxins is the dramatic
rapidity by which these proteins affect skeletal muscle, as
recorded by intravital miscroscopy techniques [53]. The
high molecular weight of the fucoidan (135 kDa) may also
be a factor that cou1d limit its distribution and diffusion in
the tissue. Furthcr characterization of fucoidan fragments
of lower mass, or of different types of sulfated fucans
obtained from a varicty of natural sources 150) might be of
interest in the search for a clinically usefui, optimaI
inhibitor of myotoxic PLA2s from snake venoms.
Acknowlcdgments
We thank the fnternational Foundation for Science (F/
2766-21, Consejo Nacional para Investigaciones Cen-
tficas y Tecnolgicas (CONICIT) of Costa Rica (FV-
058-02), Vicerrectora de Investigacin, University of
Costa Rica (VI-741-99269 and 741-98518), and the
Ernbassy of Japan in Costa Rica, for generous support
to these studies. We also thank Prof. Mats Wahlgren
(Karolinska Institute, Sweden) for introducing us to the
study of fucoidan, and Drs. Jos Mara Gutirrez and
Alberto Alape for critica1 review of the manuscript. This
work was performed in partial fulfillment of the doctoral
degree of Y. Angulo at the University of Costa Rica.
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