0022-1554/91/$3.

30 The Journal of Histochemistry Copyright 1991 by The

and Cytochemistry
Histochemical Society, Inc.

Vol.

39,

No.

4,

pp.

469-478,

1991

Printedin

USA.

Original

Article

Expression of Blood Group Heterochromatin in Mucous

MITSURU SHINGO
Department

A and B Antigens in Nuclear Cells of Human Cervical Glands

KATSUJI NISHI, YOSHIRO OKAMURA,

NAKAJIMA,1 KAWAHARA,
ofLegal Medicine,

NOBUAKI

ITO,

and TADAOMI
Nara Medical and University, in revised

HIROA
Nara, form Japan. October 23, 1990; accepted October 27, 1990 (0A2056).

Received

for publication

August

3, 1990

Using a post-embedding we found that monodonal

B antigen regions, (MAb-B) reacted as well as secretory

immunogold antibody

labeling procedure, against A (MAb-A) or

reactions
related

provide
antigens

evidence
exist in the

that certain
nudear glands.

types
In contrast

ofblood
to the

groupin mu-

heterochromatin

with nudear heterochromatin granules, in mucous cells of human cervical glands. Systematic and Critical observation of specimens from 24 individuals ofdifferent blood groups revealed that the labeling pattern with MAb was strictly dependent on the blood group (A,B, or 0) ofthe donors, i.e., MAb-A reacted with the heterochromatin from blood group A and AB but not with B and 0 individuals. Labeling with MAb-B was also specific for the heterochromatin from blood

secretory granules in which ABH antigens were recognized by blood group-specific lectins, heterochromatin regions had little or no affithty for these lectins. Furthermore, the secretory status ofindividuals affected the staining intensity with MAb in secretory granules but not in the heterochromatin. These results suggest that the blood group substances found
cous cells ofhuman cervical in the heterochromatin may have different molecular prop-

group B donors. On the other hand, MAb gen did not react with the heterochromatin
viduals examined, despite the fact that

against H antifrom any mdiwere de-

erties from those in the secretory granules, although both have the same determinant structures of ABH antigens. (J
Histodiem
KEY

Cytochem
Blood gland; group

39:469-478,
antigen; gold; Nucleus; Colloidal

1991)
Heterochromatin; microscopy. HuElectron

H antigens

WORDS:

tected

by the

MAb

in secretory

granules.

Such

specific

man

cervical

Introduction
The existence
in various positional precursors, high-mobility types analyses,

ofglycoproteins
of cells or metabolic with proteins

in the nucleus
by means of lectin radiolabeling subcellular HMG14

has been
binding procedures fractionation

demonstrated
analyses, with (15). comsugar The

mic exchanges through the nuclear pores (8,11,15,16), roles of nuclear glycoconjugates are not yet known. Blood
(43). types They

the precise or glycolipids Their antigenic

group and

ABH tissues

substances not only throughout

exist as glycoproteins on red blood the body (42).

are found

cells but also in various

in conjunction group

ofcells

and (36).

17 are among The oligosaccharide

the best side of (36). the struchas not

characterized
chains of these

nuclear mannose, the nature

saccharide

glycoproteins
have

determinants chains and gens

precursor

reside in the terminal portion of carbohydrate side the minimum immunoreactive structure of the H antito be a disaccharide,
A- and

glycoproteins

been

shown

to be composed

is known
ofthe

Fuc-(al-2)-Gal-R,

which respectively

is a (Fuc, obser-

galactose, However,
ture yet of the

glucose, fucose, and N-acetylglucosamine ofcarbohydrate-protein linkage and

chains of these nuclear glycoproteins

B-active

trisaccharides GaINAc, antigenic droplets,

and goblet

GaINAc-(al-3)-[Fuc-

(al-2)J-Gal-R L-fucose; GlcNAc, Gal,

and Gal-(al-3)-[Fuc-(al-2)]-Gal-R, D-galactose; N-acetyl-D-glucosamine) revealed that these apparatus, mucous (3). Electron

N-acetyl-D-galactosamine; microscopic determinants and brush

cells

been

elucidated between

except single

for the novel

(15,16,22).

type
Recently,

of protein-saccharide and serine

ultrastructural

linkage
nine

N-acetylglucosamine in different procedures. glycoproteins

(15,16,19,20)

or threolo-

hydroxyls

(O-GlcNAc)

vations have on the Golgi

are located border plasma

the secre-

calization been have

tors

of glycoproteins (25,26) that staining nuclear

processes

nuclear (1), lectin-gold

structures some

has also or facreports

analyzed suggested
in transcription

by autoradiography

(21,36,40), be important

membrane ofintestinal absorptive tory granules of salivary glands plasma vascular der (7), dence group membrane endothelia and and the Golgi corpuscles

(38,39),

(31,32) apparatus

and

cervical of kidney

glands tubules However,

(34), and bladcviblood

enzyme-gold

Although could
and

(17), the transitional that with

epithelia of thymus (30).

ofurinary

in nucleo-cytoplas-

the Hassalls

has not yet been presented specificity are associated study,

regions

glycoconjugates with the nuclear structure.

1 Correspondence

to:

Mitsuru

Nakajima, 840 Shijo-cho,

MD,

Department Kashihara-shi,

of Legal

In the present
heterochromatin

Medicine,
)apan.

Nara Medical

University,

Nara 634,

we found positive labeling in the nuclear of human uterine cervical glands with against A or B antigens at the electron mi469

monoclonal

antibodies

470

NAKAJIMA,

ITO, NISHI,

OKAMURA,

KAWAHARA,

HIROTA

croscopic evidence
in the

level. To our knowledge, this is the first report presenting that glycoconjugates with blood group specificity exist
nucleus.

each After with tron

for

5 mm);

(f) with

the

streptavidin-gold and (g) BSA-PBS water and and then

complex (three drying, examined were the using obtained

solution times, each aJEM from sections

diluted for 5 were 1200EX three

1:50

in BSA-PBS washing uranyl microscope. Monoclonal

for 30 mm; acetate for 5

mm).
dcc-

distilled

stained

mm

Materials
Specimens.
tamed bined at surgery. with 2.5%

and
Normal
Tissue

Methods
portions
blocks

anti-A (Seradone anti-A,

or -B antibodies

different

sources: ofhuman
were fixed

Immunohistochemicals

uterine

cervical glands were obcompH 7.4, embedded sections buffer UK). (PB), and Ultra-thin

Serum-Institute Product tems (Bioclone

(Lot. No. 098A) (Detroit, MI); Biotestanti-A, Product No. 611261 and Seraclone anti-B,
FRG); No. BAA1O9F-1, anti-H Monoclonal and Ortho antibody Diagnostic anti-B, was purchased SysProduct Bioclone

in 2 % paraformaldehyde ofethanols,

No. 611271)(Frankfurt-am-Main, Product N)).

glutaraldehyde dehydrated (London Resin

in 0.1 M phosphate in a graded Co; series London,

for 2 hr at 4C, in LR White

No. BBB5O6C-1)(Raritan,

resin

( pale gold in color) were collected

then stained.
Typing of blood groups ABO

on 200-mesh
and Lewis

uncoated
tissue

nickel grids and

donor was per-

from Immunohistochemicals(Lot. No. 098A). Monoclonal antibodies from Immunohistochemicals, Biotest-Institute and Ortho are hereafter referred
to as MAb[IJ, MAb[B], and MAb[O], absorbed type, followed respectively.

for each

Control
tions with responding

experiments
an MAb blood group

were performed

as follows:
with washed by incubation

(a) incubation
erythrocytes with ofits

of 5cccor-

formed by routine hemagglutination testing of peripheral blood, by use of human anti-A and anti-B serum, and by mouse monoclonal anti-Lewis a and anti-Lewis b antibody, respectively. Secretor status of the donor was determined by Lewis blood type, since the saliva ABH secretor types are
closely nonsecretor five from related one), blood shown to the eight group erythrocyte from blood Lewis group three, types (43). Of the 24 specimens

previously

biotinylated the trypanoof Parasilabeling

second antibody and streptavidin-gold ofprimary MAb(IgM class)by a mouse

some surface Nara step antigen Medical (a gift from University) of both solution with as: with
-

complex
MAb(IgM absence

solution;
class)against MD,

(b) replacement

Y. Takahashi, to verify

Department of nonspecific

of uterine

cervical

glands,

seven were from

blood

group
two),

A (secretor
and four

six,
three), from

tology,

B (secretor nonsecretor

five, nonsecretor

of mouse
bation tavidin-gold Intensity des

immunoglobulin
and application complex of labeling

M; and (c) omission

biotinylated according final different
+ , + + , +

of the primary
second antibody

MAb incuand of gold 12,000). degree particles streppartiThis stainof per

0 (secretor

blood
clinically the

group
time

AB (secretor

two, nonsecretor
proliferative

two).
stage

All of the donors

of the menstrual

were
at

alone. to the density magnification from

+ + ,

to be in the

cycle

was graded equivalent not significantly 21-50, background; 10-20,

of surgery. of Streptavidin-Gold with an average method particle and of Slot

on photographs

(x increasing SO gold

Preparation loidal cording 2% gold HAuCI4 water) to the

and Lectin-Gold
diameter Geuze water) added turned to the (41). of 15 nm The Au3 was heated Au3

Complexes. was prepared solution to 60C and

Colac(1 ml in a hot stirred

was represented ing; positive

-

background

/+

barely

above

labeling

and

greater

than

in 159 ml double-distilled

cm2 , respectively.
Lectin sections temperature. solution diluted and and glands, absorbed perature, terstained cervical ously

water bath. A reducing

distilled for 2 hr at 60C. After

mixture(8
quickly the solution

ml 1% trisodium
red and

citrate and 32 ml doublesolution sol formation was comaccording

Staining
were They rinsed

Procedure.
were transferred

Before treatment
of 0.1% onto water. a drop incubated After drying, above.

with lectin-gold
BSA-PBS ofthe for 5 mm lectin-gold the sections

complex,
at room complex tem-

was then

incubated

on a drop BSA-PBS,

pleted,

the solution

was heated
and reported

to the boiling
complexes (30-32). were The agglutinin used

point.
were lectins I-B4 in this prepared Helixpomati.a (GSAI-B4), They The pH g) 6.3) (12). UEA-I, (100-200 pH study.

1:30 in 0.1% in distilled examined sections with its

for 30 mm

at room

Streptavidin-gold to the method glutinin Ulex cific the 6.4) 0.1 solved solution
(MW

lectin-gold simplicifolia (UEA-I) to the 7.4; Streptavidin

previously Griffonia agglutinin-I group lectin

agand are speof (pH with gold glycol was dis-

were coun-

as described

(HPA), europaeus for blood solution

After confirming

positive
were

labeling
incubated corresponding negative

in secretory
with

granules
lectin-gold

of human
complex sugar

uterine
previ-

A, B, and pH M NaCI. After

H antigen, isoelectric GSAI-B4, To these

respectively point pH or lectin solutions, 5.0;

inhibitory

(N-acetyl.D-

gold

was adjusted (HPA, or 0.1 N HCI.

for streptavidin

galactosamine
a concentration tory above granules.

for HPA; D-galactose

of 0.1 M, and

for GSAI-B4;
labeling

L-fucose
was verified

for UEA-I)
in the

at

or each M K2C03

secre-

in 0.1 ml 0.005 were added. 20,000) was added.

5 ml of colloidal polyethylene

Labeling
in the

intensity
section

was expressed
on staining with

according
monoclonal

to the criteria
antibodies.

as described

1-2 mm,

0.15 ml 5% aqueous

were purified
40 mm

These crude streptavidinor lectin-gold complexes and concentrated by ultracentrifugation at 55,000 x g for
of5% azide. glycerol containing 0.05% about use. polyethylene 6.5 ml of water-

on a 2.5 ml cushion sodium

Results
1

glycol and 0.01% clear supernatant

ml of condensed HPA

Mtercentrifugation,

was carefully
sol to be stored

aspirated
at 4C

and discarded,
until

leaving

about

Identification

ofEuchromatin,

Heterochromatin,

was purchased

was from Louis,

from Vector Laboratories

Purified

EY Laboratories (San Mateo, CA), GSAI-B4 (Burlingame, CA), and UEA-I from Honen
streptavidin was purchased from Sigma (St

Interchromatin Cells
Fixation aldehyde

Granules, Uterine
paraformaldehyde M PB, pH 7.4, 2%

and Cervical
and

Nucleoli Glands
combined

in Secretory
with 2.5% glutarresin eneuand Both

ofHuman
with in 0.1

Oil Co (Tokyo,)apan).

MO).
Staining Procedure. Sections reagents were placed temperature: con-

embedding

in LR White

Immunocytochemical
secutively on top of a drop

of the following

at room

resulted in satisfactory preservation of the ultrastructure abled four distinct intranuclear regions to be identified. chromatin and heterochromatin examined in this study, but were not necessarily observed appearance the proliferative (14). of the nuclei stage

(a) 1% bovine serum albumin (BSA) in 0.01 M PBS, pH 7.4 (hereafter referred to as 1% BSA-PBS) for 5 mm; (b) undiluted or appropriately diluted mouse monoclonal antibodies (1gM class) against blood group A, B, and H antigen for 30 mm; (c) BSA-PBS (three times, each for 5 mm); (d) undiluted biotinylated goat anti-mouse immunoglobulins (supplied as the second antibody in the Stravigen biotin-streptavidin amplified system; BioGenex Laboratories, San Ramon, CA)for 30 mm; (e)BSA-PBS(three times,

regions were found in all nuclei interchromatin granules and nucleoli in every nucleus. The ultrastructural in this study resembled that reported at cycle as previously

observed

of the menstrual

BLOOD

GROUP

A AND

B ANTIGENS

IN

NUCLEAR

HETEROCHROMATIN

471

Table
Blood group 0 S

1 . Labeling
Donor no. 54-2 48-3

of

nu clear

heterochromatin
MAb

with

MA6

and lectin
MAb

to

to
[B]

B [0]
-

MAb

to

H H1A +)
(-) (-) (-) (-) (-)

Lectins
GSAI-B
-

[I]
-

[BJ
(-) (-) (-) (-) (-) -

[O
-/+ -

[I]
(-) (-) (-) (-) (-) -

LII
-

UEA-I -(+
)

(+

(-) ((-) (-) (-)

+) +) +)
(-) (-)

0 0
0 0

5 5
NS NS

( -)

-(+ -(+
-

56-2
55-3
75-1 51-3

(+
(-) (-)

+)

A
A

S
S

++ ++ + + +

(+)
(-)

+ +++

++

(-) (-) (-) (-) (-) (-) (-)

(++)
(-) (-)

(++)
(-)

(-) (-) (-) (-) (-) (-) (-)

-(++)
-

57-3

+#{247}
+++ +++ + + + +++ + + + + + +

-/+

(-)

A A A A A B B B B B B B B AB AB
ABNS ABNS

S S S S NS S S S S S NS NS NS S S

68-1 62-2 46-1

53-1 47-2

+(++) +(++) +(+

-/+
-/+

nd.
-

(++)

nd.
-(++) -(+ -(++)
(-)

(++)
(-)

(++)
+)

+)

+ ++ +

-/+(+
-/+

+)

(++) (+)
(-) (-) (-) (-) (-) (-) (-) (-)

(+)
(-)

(++)
(-)

60-1 67-2
73-1
44-1

+ -/+ + + + + -

(+)
(-)

+ + + -

+ + +

(-) (-) (-) (-) (-) (-) (-) (-)

(-) (-) (-) (-) (-) (-) (-) (-)

-(+
(-)

+)

-(+

+)

nd.
+) +) +) -(+ -(+ -(+
(-) (-) (-)

+ + +

+(-) +(-)

-(+ -(+ -(+

-

+) +) +)

-/+
+ -

+ -

66-1 63-1
72-1 74-1

+(+)
(-) (-) (-)

#{247}

(-) (-) (-)

-/+
+ + + + -

45-2

-/+
-

(+)

++ ++ +
-

+++ +++ + + +

(-) (-) (-) (-)

(-) (-) (-) (-)

-/+(++)
-

-(++) -(++)
-

-(++) -(++)
(-) (-)
-

80-2
65-1 58-3

+ + +

(++) (+) (+)

the labeling
[0],

(++)
(-) (-)

-/+
-

(-) (-)

a
from

Figures

in parentheses

indicate

intensity
MAb

ofMAb[I]

and lectins

in secretory

granules

(33).

S. secretor;

NS, nonsecretor;

nd.

. not done.

[I).

MAb

purchased

Immunon;

[B],

MAb

from

Biotest;

from

Ortho.

The results obtained Table 1. Labeling patterns tins are also presented

in the present study ofsecretory granules in Table 1.

are summarized with MAb and

in Icc-

Immunogold Antibody
When against

Labeling Against

with

Mouse in the

Monoclonal Nuclei

B Antigen

sections from group B donors B antigen, five out of eight

were incubated with MAb[I] cases showed mild to intense ofnuclei negative three same moderate MAb[B] positive
in other

Immunogold Antibody
Mter treatment with MAb[O]

Labeling Against

with

Mouse in the

Monoclonal Nuclei
group A or AB donors to intense labeling was

labeling in the heterochromatin ure 3). Among these five cases, ing in secretory barely secretory MAb[B] cases with tive labeling blood of MAb granules. group did or not granules labeled (Figure

regions and nucleoli three cases showed 3). The remaining of the from revealed cases
was

(Figlabel-

A Antigen

cases were as well as in cases and

cases.

of sections obtained from against A antigen, moderate

at all in any area Staining against regions (Figures sections B antigen in three 4a and 4b). examined, any regions of probable

nuclei, the

granules. or MAb[O] MAb[O] with not

with two
In

seen not only in heterochromatin regions but also in nucleolar regions from all individuals examined, regardless of their secretor status (Figures la and lb). No labeling was observed in euchromatin or interchromatin granules in every with case examined. Staining sections from group A or AB donors A antigen revealed that heterochromatin showed positive labeling (Table 2b).
did not

labeling or negatypes secretory with group A in nuclei con-

of heterochromatin

with
seen

Probable these three positive

these react the

two antibodies with

MAb[I] or MAb[B] to regions from some cases (Figures 2a and others were against
group

AB individuals exception

different or with labeling

of nuclei

whose labeling

intensity those from patterns of MAb

from nuclei

was mild

With

2c) to intense at all (Figure of the secretor

in this study

1), whereas These

react

were not labeled also independent A antigen

B and 0

MAbEl] in heterochromatin and one group 0 individuals,

was

regions of nuclei no incompatible used against

from three labeling B antigen.

status. experiments

Three
with

types

used
donors.

observed

with

the

MAb

Control

indicated

negative

labeling

in the nuclei.

No binding was observed in the nuclei after performing trol staining according to the procedure mentioned above.

472

NAKAJIMA,

ITO, NISHI,

OKAMURA,

KAWAHARA,

HIROTA

::
::.:stU
#{149}J#{149}__ .... -

:tU

1p_

t(_
ib.-f::..

-.::

#{149}

-.r
---.--- :-.-

_p?#{149}_ :-.
., . , .. -. .

La,-..

j
:

:., .

:#{149}:#{149}.

:.

:-

#{149} .

-:

..---.

;r#r

f!

vl
.-,

#{149};%,4

i*W;
:.

I-

!
I.S

#{149}.
-.

:- -..*.

c.

..-s--,
&

jP?
S.

#{149}S 459

I.
,,

#{149} #{149}#{149}

#{149}

..

.:

...

#{149}

S
#{149}5 5 #{149} S

,a

:,#{149};#{149}

#{149} :F
S

4,

,
m

4#{224}

#{149} ::
5 .

.%

....

#{149} #{149} {149} S #

5.

..

Figure 1 . Secretory cells from a blood group AB nonsecretor incubated with MAb(O] against A antigen. (a) Intense (+ + +) labeling is observed in the heterochromatin region, whereas no labeling is seen in euchromatin and interchromatin granules. (b) The nucleolar region is moderately labeled with the antibody. The paranuclear region ofthe cytoplasm is moderately (s- -i-) stained with the antibody. Original magnifications: a x 13,600; b x 30,800. Bars = 1.0 tm.

BLOOD

GROUP

A AND

B ANTIGENS

IN

NUCLEAR

HETEROCHROMATIN

473

Figure 2. Secretory cells from a blood groupAB nonsecretor (the same individual as in Figure 1) incubated with (a) MAbIII and (b) MAb(B] against A antigen, and (C) those from a blood group A secretor individual incubated with MAb[I]. Moderate (++) labeling is seen in the heterochromatin region after incubation of sections from (a) a blood group AB nonsecretor and (c)agroupAsecretor(photo C)with MAbEII. Littie or no labeling is seen in euchromatin, interchromatin granules and in mucous granules. b shows that MAbIBI cannot react with any regions of nuclei from the same individual as shown in a. Original magnifications: a x 21,000; b x 15,800; C x 16,100. Bars = 1.0 tm.

::

Ej

2a
-.

,
_4,
. -.-

..p

h- -

2c

474

NAKAJIMA,

ITO, NISHI,

OKAMURA,

KAWAHARA,

HIROTA

.9,

..

):
:

;.

9--

.1

;
t_

I:..

-L..
,

..

1
-#{149} I ,,. -.

I----...,,-#{149},_

II ,

-.4

. -

5_

,t
- -

,.

I;

s.9,s-

1tE.

..

.-

#{149},-.

.. -,-,

n#{149}.

-a.
-.

;
. .

,
#{149} Pt

#{149}
,

,c.
S

r
. -.

,,

is.

-ate
.-.r--

. 3sp9

--

,
intense (+ + +) labeling is seen in the heterochroand secretory granules. Original magnification

Figure 3. Secretory cells from a blood group B secretor incubated with MAb[l] against B antigen. Moderate (+ +)to matin regions and nucleolus of nuclei. Little or no labeling is observed in euchromatin, interchromatin granules, x 18,400. Bar = 1.0 tm.

Immunogold
Antibody
Application

Labeling
Against
of MAb[I] against

with

Mouse
in the

Monoclonal
Nuclei
by the strepcases two followed

H Antigen

HPA and GSAI-B4 reacted with secretory granules from blood group A, B, and AB secretor individuals in a blood group-specific manner (Figure 5 and Table 1). UEA-I staining ABO these in secretory blood lectins granules was observed individuals (Table in nonsecretor independent of the 1). No reaction with group of the was observed

H antigen,

tavidin-biotin bridge technique, in sections from all the resulted in negative labeling in all regions of the nuclei. Secretory granules from three blood group A secretors and

individuals.

blood group 0 secretors were intensely stained with MAb[I] against H antigen. Markedly reduced or negative labeling in the secretory granules was seen in control sections.

Discussion
In previous
procedure

studies,
with

we have or the
The

used
antibodies

immunocytochemical
and the streptavidin-bio-

staining to inves-

monoclonal

Cytochemical
Lectins
In three
with

Labeling the Nuclei

A and two group
complex solution

with

Blood

Group-specific
sections the other complex examined incubated labelcases were of 5). revealed (Figure

tin bridge tigate

salivary

technique distribution
(30,32).

lectin-gold group
usefulness

staining

method

the have

of blood established level.

these

in
group
HPA-gold

glands

ABH antigens in human and advantages ofthese techthese study,

in tissue

AB individuals,
showed probable

niques the
both

been

for demonstrating In the present

antigens

antigens we employed

at

positive

electron
techniques

microscopic
to localize

ing in heterochromatin unlabeled sections negative with incubated labeling HPA-gold of the

regions. in GSAI-B4nuclei

Sections in the

from

sections

of hu-

complex

nuclei.

Examination

or UEA-I-gold in all cases

man uterine cervical glands, resulting in the discovery of the existence of glycoconjugates expressing blood group antigens in heterochromatin regions of nuclei. This is the first report implicating

BLOOD

GROUP

A AND

B ANTIGENS

IN

NUCLEAR

HETEROCHROMATIN

475

Figure 4. Secretory cellsfrom a blood group B secretor (the same individual as in Figure 3)incubated with (a) MAb(B]and(b) MAb[O] against B antigen. (a) Moderate (+ +) to (b) weak(+)labeling is seen withthese antibodies. MAb[O] reacts more preferentially with secretory granules than with the nuclear heterochromatin region. Original magnifications: a x 25,000; b x 21,500. Bars = 1.0 sm.
a.

r.#{149}.

2-

.;-;;
S

;p!1
--I.

.t

-S

.-_

4b
f.

4
-

_
-,

. tt

:
t_

. i:

4 - -

I . - , _%-

. ., -

#{149}-$,,(,

st,;.*

.;.,

-.

..

--

.;Sfr

-_)

Figure 5. Secretory cells from a blood group B secretor (the same individual as in Figure 3) incubated with GSAI-B4. GSAI-B4 reacts strongly with secretory granules. No significant labeling is observed in nuclei and in the other cytoplasmic organdies. Original magnification x 9500. Bars = 1.0 tm.

.-,:
I

-..--::-,,
.

-.#{149}--4

476

NAKAJIMA,

ITO,

NISHI,

OKAMURA,

KAWAHARA,

HIROTA

the possible involvement of blood lation of nuclear function. The reactivity chromatin of the donor, results obtained against of nuclei of MAb regions blood

group

substances study

in the moduthat with blood the

ity is affected

by the

internal

carrier

carbohydrate

chain

(2,3,13,24).

In fact, demonstrate variant We have

tins

several structures

different of the localization

variant

kinds antigens

of monoclonal have been structures ofblood

of antigens

antibodies developed group-specific

in tissue

against and used 1ccsections

in the present group

of MAb

A or B antigen on the ABO

against A antigen

heterogroup

for histochemical
to discriminate

of these
structures

(S ,9, 10,2 3 ,28,3 5).

was dependent

also suggested

the usefulness observations).

with secretory

i.e. , the reactivity

was found

only in group A and group B antigen was mainly shown MAb obtained against with A or B antigen these MAb

AB donors and that of MAb against in group B donors. Although we used from were three essentially different sources, the results differsources used were subthe same. Slight

(Ito et a]. , unpublished

ity of MAb and lectins

Thus,
granules

differences
and nuclear

in reactivhetero-

chromatin due
binding

may
ability

be ascribed of the
among MAb

in part core
used

to the difference chain.

this study

in antigenicity The
also

to variations of different

saccharide
in

difference
suggests

in
the

ences in recognition may be due to the for producing these

ability among these MAb from difference in the immunizing MAb, i.e., MAb[I] was obtained

different substances

existence

variant against
variants be due

species B antigen
(33; and inability

of A and may
Ito

B antigens a restricted
to detect

even

in

by immuni-

zation ofthe synthetic trisaccharide, and MAb[B] and MAb[O] produced by immunization of erythrocytes or blood group stances group

nuclear heterochromatin. Commercial MAb

ificity vation). for B antigen This may

have

specobserB anti-

et al.,

unpublished

(27). The specificity ofthe MAb for the corresponding blood antigens was confirmed by a series of control experiments.

to the

of MAb

These results are therefore taken as evidence that binding sites recognized with MAb in heterochromatin regions could consist of the
same

gen in cells from blood neighboring substances be considered Although to be actual hydrate-protein eties
tion

group AB individuals. Steric hindrance by such as A antigenic determinants may also unexpected have been results. suggested

epitopes of blood group antigens and on the erythrocyte membrane. It is well known that blood group or glycolipids residues
of blood

as found substances

in body

secretions H are the ter-

for an explanation of such several types of glycoconjugates nuclear components, neither linkages nor the structures

A, B, and

the nature of the carboof the saccharide moiwe have little informaand biosynthesis protein-sacchathe questions

glycoproteins minal
terminants

and

in these

glycoconjugates side chains

have

been

elucidated
actual

(15).

At present,

sugar

of the oligosaccharide

are the de-

as to the

the lipid tion

fraction

group specificity (43). Since is extracted by ethanol treatment specimens, the glycoconjugates that

it is expected that during dehydraobserved in this of

their ride

transport, linkage of

site and mechanism except for the recently O-GlcNAc (15,16,22). in this (N-linked)
mucin

of their established Concerning study, these

nuclear also

of the tissue

study may represent glycoprotein species. The present results further demonstrated

glycoconjugates demonstrated remain to be answered. Classical

(threonine)-linked known

the reactivity

asparagine-linked
(0-linked)

oligosaccharide
type oligosaccharides

or serine
are

MAb and lectins with nuclei was quite different secretory granules in the same mucous cells (33): ing intensity with MAb was dependent on the donors in secretory granules group B individuals positive was observed in nucleus but

from that with i.e., (a) the labelsecretor status of

to be added

to proteins

in luminal

components

of endoplas-

but not in nuclei; (b) in some blood labeling with MAb against B antigen not in secretory granules in the same

mic reticulum and the Golgi apparatus (18). Thus, the existence of glycoproteins in nucleus would not be consistent with current models ofglycoprotein biosynthesis or transport (15,16). Antigenic determinants of the ABH antigens are also known to be carried
by both

cells; (c) positive labeling was observed with MAb against H antigen in secretory granules from group A and 0 donors but not in nuclei from any donor; (d) blood group antigens could be detected with with group-specific any lectin, except lectins in secretory labeling granules with but HPA-gold not in nuclei complexes for weak

N- and

0-linked (2,13).

oligosaccharides In this context,

ofmembrane we must

and postulate

secrethat

tory

glycoproteins

the mechanism of biosynthesis and intracellular clear glycoconjugates with blood group specificity and found ferases topologically different from that of secretory in the same cells. However, involved in the synthesis

transport of nuis biochemically glycoproteins

in nuclei. Such differences may be due to the difference in the molecular properties of glycoconjugate found in nuclei and secretory
granules.

it is conceivable that glycosyltransof ABH antigenic determinants are also responsible for since the appearance dependent on the ABO data and and are pores

with rived

blood from

Thus, we could exclude the possibility that glycoconjugates group specificity in nuclear heterochromatin are desecretory granules. study, we did not investigate specimens from in-

ofsecretory and membrane glycoconjugates the synthesis of nuclear ABH determinants, of these determinants in nuclei blood group of individuals. have Most biochemical, indicated that regions cytochemical glycoproteins known events is strictly

In the present

dividuals other than those in the proliferative stage of the menstrual cycle. It may be important to know the pattern of expression

and immunocytochemical are localized in nuclear of transcriptional roles ofthese the

pores

of blood
at other

group-related
stages in the

antigens
menstrual

in nuclei
cycle. Such

and granules
findings

from cells
may provide

euchromatin post-transcriptional presumed and

to be the sites (19). The exchanges

glycoproteins (15,16,19,20) nuclear

a new means of approaching changes in the expression

certain problems such of carbohydrate antigens

infertility associated

as malignant in carcinoma

to be involved

in transcriptional through information regions,

processes

nucleo-cytoplasmic

ofthe
milieu that ent

uterine

cervix incompatibility biochemical group

(4) and
and

with semen-cervical studies have shown differspecific-

(44). immunochemical determinants chains and that antigenic are built on many

(8,11,15,16). In contrast, proteins in heterochromatin

scriptionally inactive,

as to the presence which are thought

cells, and the

of glycoto be tranmeaning

Recent blood types

is restricted

to certain

of core saccharide

the antigenic

of their presence In a previous

in these regions is still unknown study, we could not demonstrate

(25,26,37,40). the blood group

BLOOD

GROUP

A AND

B ANTIGENS

IN

NUCLEAR

HETEROCHROMATIN

477

antigens

in nuclei

ofhuman abundant granules reaction

labial

and

submandibular

gland

mu-

the nuclear
tein and

cous cells even though ent in their secretory nary studies, positive in nuclear was observed

amounts (31,32). with MAb

ofthese However, against regions

antigens in our ofdifferent

are presprelimitypes

pore complex inhibits nucleocytoplasmic RNA in vivo. ) Cell Biol 107:1289, 1988

transport

of pro-

A or B antigen

heterochromatin

9. Feizi T Demonstration by monoclonal antibodies that carbohydrate structures of glycoproteins and glycolipids are onco-developmental antigens. Nature 314:53, 1985
10. Feizi T, Childs RA: proteins. Biochem

of cells of human and monkey stomach and rat duodenal mucosa. In addition to these observations, definite positive labeling with MAb against blood group A antigen has been demonstrated gland, labeling in nuclear heterochromatin authors stated their fore,
glands.

Carbohydrates 245:1, 1987

as antigenic

determinants

of glyco-

11.

regions of rat salivary findings as nonspecific of blood

ubiquitous

although the (29). Thereheterocervical indicate blood not

14. 12.

Finlay DR. Newmeyer DD, Price TM, Forbes DJ: Inhibition nuclear transport by a lectin that binds to nuclear pores. 104:189, 1987 Goldstein Goldstein in biology

ofin

vitro

) Cell Biol

the presence
Their rather

group to mucous

determinants cells ofhuman

in nuclear

in nuclear uterine may these

U Poretz RD: Isolation,

specificity

and carbohydrate-binding

physicochemical characterization, oflectins. In Liener I, Sharon N,

chromatin from group yet been various

is not confined human and

,
and

presence

heterochromatin 13.

eds. The lectins, properties, functions, and applications medicine. New York, Academic Press, 1986, 33 ABH and Ii antigens ofhuman erythrocytes:

other

mammalian of glycoconjugates

tissues with

the physiological determinants. Although

importance the functional with these

Hakomori chemistry,

Hematol roles of blood array structures. group antigens have a diverse

galactosyltransferase,

5: Blood group polymorphism, 18:39, 1981

and

their

developmental
electron Gynecol Kelly

change.

Semin
on

determined,

of macromolecules

is known
15.

Hamada Y: Transmission and scanning the human uterine cervix. Adv Obstet Hart GW, Haltiwanger RS, Holt GD,

microscopic 25:349,

study 1973

to be decorated testinal hydrolases, ma] growth GaINAc

in determining

These include mucins, inand the receptor for epider(10). Recently, suggested
affinity,

nucleus
16. Hart GW,

and cytoplasm.
Holt GD,

Annu linkages

Rev Biochem
RS: Nuclear

WG: Glycosylation 58:841, 1989 and cytoplasmic

in the

factor,

as well as glycosphingolipids of A antigen

epidermal growth

the
pro-

Haltiwanger

glycosy-

residue(s)

has been

to be involved
17.

lation:
Sci

novel saccharide
1988

in unexpected

places.

Trends Biochem
Ultrastructural

factor-receptor

13:380,

tein-tyrosine kinase activity, and turnover in A431 cells (6). The finding ofnuclear glycoconjugates with blood group specificity may provide an important clue to the role of the antigens as well as to the mechanism
tein, since blood

Hinglais N, Bretton R, Rouchon M, Oriol R, BarietyJ: localization ofblood group A antigen in normal human truct Res 74:34, 1981 Hirschberg endoplasmic
1987

kidneys.)

Ultras-

of biosynthesis
group antigens

and in terms Further

the

transport
the

of nuclear
best

glycoprohu-

18.

CB, Snider reticulum

MD: Topography of glycosylation in the rough and Golgi apparatus. Annu Rev Biochem 56:63,

represent

understood

man genetic
our

antigenic
laboratory

determinants (43).
substantiate to

ofstructure, studies
present study.

biosynthesis, in progress

and in

transmission

are now

19.

Hubert), proteins

Dcv

Seve AP, Monsigny M: Are nuclear lectins and nuclear involved in the modulation of nuclear function? Cell 27:69, 1989 R: 0-Glycosylation for mechanisms of eukaryotic of transcriptional transcription regulation.

glycoDiffer

Literature
1. Bennett

Cited
G, Hemming and R, Lavoie [3H]galactose. PA: Presence Eur) Cell of glycoproteins Biol 42:246, 1986 in the

20. )ackson SP, Tjian tors: implications 55:125, 1988 21.

facCell

cell nucleus
of [3H]fucose

as shown by radioautographic

studies

after administration
group antigens; their distribution. 22.

Kan IWK, da Silva PP: Preferential euchromatin regions of cross-fractured label. ) Cell Biol 102:576, 1986 Kelly WG, Hart GW: Glycosylation

association nuclei

ofglycoproteins to the is revealed by fracture-

2. Clausen H, Hakomori 5: ABH and related histo-blood immunochemical differences in carrier isotypes and Vox Sang 56:1, 1989 3. Clausen antigens: regulation 4. H, Levery SB, Dabelsteen E, Hakomori a new series of blood group A-associated and tissue distribution). Transplant

of chromosomal

proteins:

local-

ization
57:243, 23.

ofO-linked
1989 Lambert

N-acetylglucosamine
F, Samuelsson

in Drosophila
B, Breimer

chromatin.
RC,

Cell

5: Blood group ABH structures (genetic Proc 19:4408, 1987

Le Pendu),

ME, Seitz

Urdaniz

MP, Suesa N, Ratcliffe M, Francois A, Poschmann A, Vinas ), Oriol R: Monoclonal antibodies specific for type 3 and type 4 chain-based
blood group Glycoconjugates 24. Lloyd KO: determinants: ) 3:255, Blood group relationship 1986 antigens as markers to the Ai and A2 subgroups.

Coon )S, Weinstein RS: Blood group-related antigens as markers of malignant potential and heterogeneity in human carcinomas. Hum Pathol 17:1089, 1986

for normal

differentiation

5. Dabelsteen

E, Graem

N, Clausen

H, Hakomori
normal

S: Structural
colon and

variations
carcinomas. 25.

and malignant
Londono

change

in human

tissues. Am)

Clin Pathol

87:129, 1987
of neuraminic

of blood group A antigens Cancer Res 48:181, 1988

in human

I, Bendayan

M: High-resolution

cytochemistry

6. Defize LHK, Arndt-)ovin D), )ovin TM, Boonstra), Meisenhelder), Hunter T, de Hay HT, de Laat SW: A431 cell variants lacking the blood group A antigen display increased high affinity epidermal growth factor-receptor number, protein-tyrosine kinase activity, and receptor turnover.

and hexuronic acid-containing macromolecules applying the zyme-gold approach. J Histochem Cytochem 36:1005, 1988 26. Londono I, Bendayan M: Ultrastructural localization of mannoside residues on tissue sections: comparative evaluation ofthe enzyme-gold and the lectin-gold approaches. Eur ) Cell Biol 45:88, 1987
27.

en-

Cell

Biol

107:939,

1988

Lowe AD, Lennox

pernatants with

ES, Voak V: A new monoclonal

the performance of hyperimmune

anti-A.
human

Culture
reagents.

su-

7. Dc Harven heterogeneous Cytol 19:639,

E, Hanna
deletion
1987

SI-lW, Bootsma
of the ABH

G, Connolly)G:
antigen from

Phenotypically
28.

der urothelium.
8. Featherstone

A scanning
C, Darby MK,

electron
Gerace

microscope
L: A monoclonal

the transformed bladstudy. ) Submicrosc antibody against

Vox Sang 46:29,

Mollicone

1984
ABH

R, Le Pendu), Bara), Oriol R: Heterogeneity ofthe antigenic determinants expressed in human pyloric and duodenal sac. Glycoconjugates ) 3:187, 1986

muco-

478

NAKAJIMA,

ITO, NISHI,

OKAMURA,

KAWAHARA,

HIROTA

29. Moreira)E, Tabak LA, Bedi GS, CuIp D), Hand AR: Light and dcctron microscopic immunolocalization of rat submandibular gland mucin glycoprotein and glutamine/glutamic acid-rich proteins. ) Histochem Cytochem 37:515, 1989
30. Nakajima Matsuda

mobility 37.

group

proteins.

Proc

NatI

Acad

Sci USA

78:6704,

1981

Roth): Application of lectin-gold complexes for electron localization ofglycoconjugates on thin sections.) Histochem 31:987, 1983 RothJ, Greenwell P, Watkins WM: Immunolocalization

microscopic Cytochem

demonstration of blood group antigen A in the human Hassalls corpuscles using Dolichos biflorus anti A lectin-colloidal gold method. ) Nara Med Ass 38:14, 1987
31. Nakajima M, Ito N, Nishi K, Okamura Y, Hirota T: Cytochemical lo-

M, Ito N, Nishi K, Ishitani Y, Hirota P Ultrastructural

A, Okamura

Y, Mizumoto),

38.

ofbbood

group

A gene specified

cii, 3 N-acetylgalactosaminyl-transferase
trans-tubular network goblet cells. Eur) Paulson)C,

and blood

group A substance in the tus and mucus of intestinal 39. Roth), l#{224}atjes ), Weinstein), D

of the Golgi apparaCell Biol 46:105, 1988 P, Watkins WM:

calization lectin-gold
32.

of blood group substances complexes. ) Histochem

in human Cytochem
salivary

salivary 36:337,
glands

glands
1988

using label-

Greenwell

Differential apparatus
261:14307, 40.

subcompartmentation ofterminal glycosylation in the Golgi of intestinal absorptive and goblet cells. ) Biol Chem
1986 M: In de-

Nakajima M, Ito N, Nishi K, Okamura ing of blood-group antigens in human

Y, Hirota I Immunogold
using

monoclo-

nal antibodies
87:539, 33. 1987

and the streptavidin-biotin

technique.

Histochemistry
and variain human lectins and

Seve AP, Hubert), situ distribution

tected
antigens granules using 1990 41.

Bouvier D, Masson G, Geraud G, Bouteille in different cell types of nuclear glycoconjugates by two lectins. J Submicrosc Cytol 16:631, 1984 HL: A new cytochemistry. The histological method Eur distribution
111:785,

Okamura Y: Heterogeneity of blood group ABH lion in the expression of these antigens of secretory cervical glands. An electron microscopic observation monoclonal antibodies. Histochemistry 94:489, Okamura in human 2. Berlin,

Slot )W, Geuze multiple-labeling Szulman AE:

of preparing

) Cell Biol
of blood 1960

gold probes 38:87, 1985 group substances

for

42.

34.

Y: Mucosubstance and lectin histochemistry ofcervical glands uteri. In Mayer WR, ed. Advances in forensic haemogenetics Heidelberg, Springer-Verlag, 1988, 521

A and B in man. ) Exp Med

43.

Watkins WM: Genetics and biochemistry Proc R Soc Lond [B] 202:31, 1978
Yarewicz position, EC, Moghissi and KS: Purification

ofsome
ofhuman

human
midcyle

blood groups.
cervical mucin 1981

35. Philipsen EK, Clausen carbohydrate antigens Scand 96:1109, 1988 36. Reeves R, Chang

H, Dabelsteen E, Graem in human fetal pancreas. SC: Carbohydrate

N: Blood group related Acta Pathol Microbiol modifications ofthe high

44.

and characterization

of its oligosaccharides

with respect
Chem 256:11895,

to size, com-

microheterogeneity.

Biol

D, Chung