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Microbes and Infection 7 (2005) 674681

www.elsevier.com/locate/micinf

Original article

Pathogenic significance of a-N-acetylgalactosaminidase activity found


in the hemagglutinin of influenza virus
Nobuto Yamamoto a,*, Masahiro Urade b
a

Division of Molecular Biology and Virology, Socrates Institute for Therapeutic Immunology, 1040 66th Avenue, Philadelphia, PA 19126-3305, USA
b
Department of Oral Surgery, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan
Received 9 December 2004; accepted 14 January 2005
Available online 22 March 2005

Abstract
Serum vitamin D3-binding protein (Gc protein) is the precursor for the principal macrophage activating factor (MAF). The precursor
activity of serum Gc protein was reduced in all influenza virus-infected patients. These patient sera contained a-N-acetylgalactosaminidase
(Nagalase) that deglycosylates Gc protein. Deglycosylated Gc protein cannot be converted to MAF, thus it loses the MAF precursor activity,
leading to immunosuppression. An influenza virus stock contained a large amount of Nagalase activity. A sucrose gradient centrifugation
analysis of the virus stock showed that the profile of Nagalase activity corresponds to that of hemagglutinating activity. When these gradient
fractions were treated with 0.01% trypsin for 30 min, the Nagalase activity of each fraction increased significantly, suggesting that the
Nagalase activity resides on an outer envelope protein of the influenza virion and is enhanced by the proteolytic process. After disruption of
influenza virions with sodium deoxycholate, fractionation of the envelope proteins with mannose-specific lectin affinity column along with
electrophoretic analysis of the Nagalase peak fraction revealed that Nagalase is the intrinsic component of the hemagglutinin (HA). Cloned
HA protein exhibited Nagalase activity only if treated with trypsin. Since both fusion capacity and Nagalase activity of HA protein are
expressed by proteolytic cleavage, Nagalase activity appears to be an enzymatic basis for the fusion process. Thus, Nagalase plays dual roles
in regulating both infectivity and immunosuppression.
2005 Elsevier SAS. All rights reserved.
Keywords: HA protein; Vitamin D-binding protein (Gc protein); Immunosuppression; a-N-acetylgalactosaminidase; Fusion; Proteolytic activation

1. Introduction
Early studies revealed that various oncogenic and nononcogenic viruses induce immunosuppression in their infected
hosts [14]. The recognition that viruses are able to compromise immunity dates back to the observation by Pirquet [5]
in 1908 that measles infection resulted in a reduced delayed
hypersensitivity response in patients who would normally
respond to tubercle bacillus antigens. This was followed a
decade later by a report in 1919 that influenza virus infection
could also suppress tuberculin reactivity [6]. In oncogenic

Abbreviations: Gc protein, vitamin D-binding protein; HA, hemagglutinin; MAF, macrophage activating factor; Nagalase, a-N-acetylgalactosaminidase.
* Corresponding author. Tel.: +1 215 424 4259; fax: +1 215 424 1850.
E-mail address: uradem@hyo-med.ac.jp (N. Yamamoto).
1286-4579/$ - see front matter 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.micinf.2005.01.015

and nononcogenic virus-infected hosts, both humoral and cellular immunity are shown to be depressed [14,7]. Many
investigators considered that virus-induced immunosuppression allows viral growth [8] and is also required for the establishment of persistent infection that leads to chronic disease
or tumor development and growth [4]. Most recent studies
have focused on understanding the molecular mechanisms
involved in virus-induced immunosuppression. Acquired
immunodeficiency syndrome (AIDS) is characterized by
opportunistic infections and by opportunistic neoplasms
(for example, Kaposis sarcoma) [9] as evidence of immunosuppression.
Influenza virus infection is common and a significant cause
of morbidity and mortality. The latter is usually due to secondary infections [1012]. The Spanish influenza pandemic of 1918 was exceptionally severe, high mortality of
more than 20 million people worldwide [13]. Influenza virus

N. Yamamoto, M. Urade / Microbes and Infection 7 (2005) 674681

infection can develop into fatal pneumonia in patients with


bacterial infection [14]. Impaired phagocytosis of bacteria by
alveolar macrophages of influenza virus-infected patients was
reported by Warshauer et al. [15].
Since macrophage activation for enhanced phagocytosis
and antigen presentation to B and T lymphocytes is the first
mandatory step in the immune developmental process, lack
of macrophage activation leads to immunosuppression and
secondary infection. Thus, we need to investigate the problem of macrophage activation in influenza virus-infected
patients. The inflammation-primed macrophage activation
cascade is the process for generation of the principal macrophage activating factor (MAF) [16,17]. Administration of
an inflamed membrane lipid metabolite, lysophosphatidylcholine (lyso-Pc), to mice activates macrophages for greatly
enhanced phagocytic and superoxide generating capacities
[16,18,19]. Activation of macrophages requires serum vitamin D3-binding protein (known as Gc protein) and participation of B and T lymphocytes [17,1923]. A trisaccharide composed of N-acetylgalactosamine with dibranched galactose
and sialic acid termini on Gc protein is hydrolyzed by the
inducible b-galactosidase (Bgli) of inflammation-primed (or
lyso-Pc-primed) B cells to yield the macrophage proactivating factor, which is in turn converted by the action of the
Neu-1 sialidase of T cells to a potent MAF, the protein with
N-acetylgalactosamine as the remaining sugar [17,20,21]
(Fig. 1a). Thus, Gc protein is the precursor for the principal
MAF. Since influenza virus infection can impair macrophage
function [15], the MAF precursor activity of the patient serum
Gc protein is first to be studied. In fact, the MAF precursor
activity of Gc protein of all influenza virus-infected patient
sera decreased. A decrease in the MAF precursor activity of
serum Gc protein is a consequence of deglycosylation of the
protein [24,25] (Fig. 1b).
In this communication, we report a virus-coded deglycosylating enzyme, a-N-acetylgalactosaminidase (Nagalase),
and discuss a mechanism of immunosuppression in influenza
virus-infected patients.

675

2. Materials and methods


2.1. Chemicals, reagents and culture media
Lysophosphatidylcholine (lyso-Pc) and p-nitrophenyl
N-acetyl-a-D-galactosaminide were purchased from Sigma
Chemical Co. (St. Louis, MO). Human Gc protein (Gc1:
major isomer) was purified by vitamin D affinity chromatography [26]. Gc1 protein carries a trisaccharide composed of
N-acetylgalactosamine with dibranched galactose and sialic
acid termini (see Fig. 1) [17,2022]. For manipulation in vitro
and cultivation of lymphocytes, monocytes and macrophages, 0.1% egg albumin-supplemented medium RPMI-1640
(EA medium) was used. Three cloned HA protein stocks
obtained from three independent influenza strains via baculovirus vector were kindly supplied by Connought Laboratories, Inc., Swiftwater, PA, USA.
2.2. Preparation of egg grown influenza virus
Ten-day-old embryonated eggs were inoculated with influenza virus strain A/Kitakyushu/159/93 (H3N2). Two days postinoculation of influenza virus, chorioallantoic fluid was harvested. The virus was purified by differential centrifugation.
Sucrose gradient-purified egg grown stocks of six epidemic
influenza strains were kindly supplied by Connought Laboratories, Inc.
2.3. Determination of the precursor activity of serum Gc
protein of influenza virus-infected patients
Blood samples of uninfected healthy humans were collected in tubes containing EDTA to prevent coagulation. The
blood samples were generally processed within 2 h from the
time of blood collection. Five milliliters of blood sample and
5 ml saline (0.9% NaCl) was mixed and gently laid on 15-ml
centrifuge tube containing 3 ml Lymphoprep (similar to
Ficoll), Polysciences, Inc., Warrington, PA. and centrifuged

Fig. 1. Schematic illustration of formation of MAF (a) and deglycosylation of Gc protein (b). *, Lyso-Pc-treated B cells.

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N. Yamamoto, M. Urade / Microbes and Infection 7 (2005) 674681

at 800 g for 15 min. Dense white cell band containing


monocytes/macrophages (macrophages for short) and lymphocytes (B and T cells) was collected using Pasteur pipettes.
The white cell mixtures were washed twice with 1 mM sodium
phosphate buffer containing 0.15 M NaCl (PBS), suspended
in EA medium and placed in 16-mm wells. Incubation for
45 min in a 5% CO2 incubator at 37 C allowed adherence of
macrophages to plastic. A mixture of lymphocytes and macrophages of uninfected humans was treated with 1 g lyso-Pc
per ml in EA medium for 30 min. Macrophages and lymphocytes were washed with PBS separately, admixed and cultured in a medium containing 0.1% serum of infected or uninfected humans as a source of Gc protein. After 3 h cultivation
the macrophages were assayed for superoxide generating
capacity. Loss or reduction of the precursor activity of patient
serum Gc protein results in a decrease in superoxide generation [2325].
2.4. Assay for generation of superoxide in the activated
macrophages
The adherent macrophages were washed twice with PBS.
The macrophages (approximately 3 105 macrophages per
well) were overlaid with 20 g cytochrome-c per ml and incubated for 10 min. Thirty minutes after addition of phorbol12-myristate acetate (0.5 g/ml), the superoxide generating
capacity of the macrophages was determined spectrophotometrically at 550 nm. The data are expressed as nanomoles
(nmol) of superoxide produced per min per 106 cells (macrophages). When a lyso-Pc-treated lymphocytes/macrophages
mixture of uninfected human was cultured with 0.1% patient
serum, the values of superoxide generation of the macrophages represent the precursor activity of the patient Gc protein
[2325].
2.5. Detection of Nagalase in influenza virus-infected
patient sera
Sera (1 ml) of infected patients and uninfected healthy
humans were precipitated with 70% saturated ammonium sulfate. The precipitates were dissolved in 50 mM citrate phosphate buffer (pH 6.0) and dialyzed against the same buffer at
4 C overnight. After dialysis, the volume of the samples were
made up to 1 ml and assayed for the enzyme [24,25,28]. The
enzyme activity was determined at 37 C in a reaction mixture of 1.0 ml containing 50 mM citrate phosphate buffer (pH
6.0) and 5 mol of p-nitrophenyl N-acetyl-a-D-galactosaminide as substrate. The reaction was initiated by addition
of 100 l of the dialyzed samples and stopped after 60 min by
adding 200 l of 10% TCA and centrifuged. The supernatant
was mixed with 300 l of a 0.5 M Na2CO3 solution. The
amount of released p-nitrophenol was determined spectrophotometrically at 420 nm with Beckman DU640 spectrophotometer and expressed as nmol/min per mg of serum for
the enzyme activity.

2.6. Sucrose gradient fractionation of influenza virions


An egg grown influenza A virus stock was concentrated
100-fold by ultracentrifugation. A 0.5 ml sample of the virus
stock was gently laid on the top of a preformed linear 550%
sucrose gradient and centrifuged at 100,000 g for 4 h.
Samples were collected for 18 fractions. Each fraction was
assayed for hemagglutinating capacity [29] and Nagalase
activity.
2.7. Fractionation of envelope glycoproteins using
mannose-specific lectin and electrophoresis
A purified egg grown influenza virus stock in 0.1 M Tris
HCl (pH 8.0) was treated with 6.6% sodium deoxycholate
for 30 min, diluted 10-fold and centrifuged at 100,000 g for
1 h. Supernatant was loaded to mannose-specific lectin (LeH)
Sepharose affinity column, eluted with 2% methyl-a-Dmannopyranoside [30]. This procedure selectively purified
two envelope glycoproteins, HA and neuraminidase (NA) proteins. Each fraction was assayed for Nagalase activity. The
major Nagalase peak fraction was electrophoresed for identification of the envelope protein by molecular weight.

3. Results
3.1. Precursor activity of serum Gc protein of influenza
virus-infected patients
It is well known, clinically, that most episodes of secondary bacterial pneumonia in humans occur 57 days after the
onset of acute influenza [12]. Therefore, we began assaying
for the MAF precursor activity of patient serum Gc protein in
the acute state, 3 days after recognition of influenza symptoms. As shown in Table 1, the MAF precursor activity of
patient serum Gc protein decreased significantly to approximately 50% of the control value as compared to 6.25 nmol of
Table 1
Precursor activity of Gc protein and Nagalase activity detected in influenza
patient blood stream on day 3 of acute state
Patient numbers

Healthy humans
Patient 1
Patient 2
Patient 3
Patient 4

Precursor activity a
superoxide produced
(nmol/min per 106 cells)
6.25 0.13
3.01 0.08
3.99 0.07
3.12 0.10
4.36 0.11

Nagalase specific
activity
(nmol/min per mg)
0.23 0.06b
1.96 0.08
1.75 0.09
1.68 0.12
1.53 0.13

a
Precursor activity was measured by superoxide generating capacity of
the patient Gc protein assayed on healthy human macrophages.
b
Very low levels of enzyme activity (average of five) were found in uninfected healthy human sera. This uninfected human enzyme is known to be
a-galactosidase [25,26]. a-Galactosidase is unable to deglycosylate Gc protein though it is able to hydrolyze p-nitrophenyl N-acetyl-a-Dgalactosaminide [24,25]. Because a-N-acetylgalactosaminidase and
a-galactosidase carry 46.9% amino acid sequence homology and share the
common chromogenic substrate [24,25,28].

N. Yamamoto, M. Urade / Microbes and Infection 7 (2005) 674681

superoxide produced per min per 106 macrophages with serum


Gc protein of uninfected healthy humans.
3.2. Detection of Nagalase activity in influenza
virus-infected patient sera
As in HIV-infected patients [24], the decreased MAF precursor activity of serum Gc protein of influenza patients led
us to suggest deglycosylation of Gc protein. Since Gc protein
is O-glycosylated [31,32], we anticipated the presence of
Nagalase in the influenza virus-infected patient blood stream
(see Fig. 1b). Accordingly, influenza virus-infected and uninfected human sera were assayed for Nagalase activity. On
day 3 of the influenza acute state, all four patient sera contained significant amounts of Nagalase (ranging from 1.53 to
1.96 nmol/min per mg) as shown in Table 1. Thus, the reduced
precursor activity of serum Gc protein is due to deglycosylation of Gc protein by the presence of Nagalase in patient blood
stream. In time course studies, the enzyme levels remained
constant at least for 9 days and gradually declined to approximately 50% (0.90 0.06 nmol/min per mg) of the acute state
level on days 19 and 30 as shown in Table 2. On day 60 all
four patient sera still showed 0.450.61 nmol/min per mg.
Additional eight patients, at various times in less than 2 weeks
post-infection, exhibited the same enzyme activity levels ranging from 1.78 to 1.95 nmol/min per mg as those of the patient
group in Table 2. On day 30 the enzyme activity was ranging
from 0.80 to 0.91 nmol/min per mg. On 60 days postinfection, the patients still carry low level
(0.50 0.05 nmol/min per mg) of Nagalase in their blood
stream, implying that the patients carry low levels of viral
infected cells without symptom.
3.3. Demonstration of Nagalase in an egg grown stock
of influenza A virus
An egg grown influenza virus stock had an infective titer
of 109.5 EID50 per ml. A large amount of Nagalase activity
was detected in the virus stock while uninfected chorioallantoic fluid showed no enzyme activity as shown in Table 3.
The Nagalase activity of these undisrupted virions suggests
that the enzyme resides on the outer structure of the virion.
Furthermore, this glycosidase enzyme should reside on an
envelope protein capable of interacting with O-glycans of the
host cell membrane. Thus, the Nagalase may be carried by
either HA protein or neuraminidase (NA) spike protein. If

677

Table 3
Nagalase activity and its proteolytic enhancement of an egg grown influenza
virus stock and purified HA protein

Virus stock (amount)a


Influenza A (0.25 ml)
Influenza A (0.25 ml)
Allantoic fluid (0.25 ml)
Protein fraction (amount)c
HA protein (12.5 g/ml)
HA protein (12.5 g/ml)

Treatment

Nagalase activity
(nmoles/min)

None
0.01% trypsin
None

80.2 3.2
99.4 2.7
UDb

None
0.01% trypsin

10.3 0.9
27.8 1.5

a
Egg grown influenza A virus stocks had an infective titer of 109.5 EID50
per ml. EID50, 50% egg infective dose.
b
Nagalase activity was undetectable in uninfected chorioallantoic fluid.
c
The Nagalase peak fraction of the mannose-specific lectin affinity fractionation was found to be HA protein fraction. The fractions 5 through 8 of
Fig. 3 was pooled and treated with 0.01% trypsin for 30 min.

the enzyme resides on HA protein, effect of trypsin on Nagalase activity should be tested because proteolytic cleavage of
HA protein is required for initiation of viral fusion and entry
to cells [33,34]. When the virus stock was treated with 0.01%
trypsin for 30 min, a 24% increase in Nagalase activity was
observed. This observation strongly suggests that Nagalase
is situated within HA protein of influenza virion and also that
the enzyme activity is expressed by proteolytic processing.
This small (24%) increase in the enzyme activity by trypsin
treatment appears to be the result of growth of influenza virus
in the chorioallantoic membrane cells of chick embryo where
proteolytic processing already occurred in the majority of the
HA proteins [35,36].
3.4. Sucrose gradient centrifugation analysis of Nagalase
and hemagglutinating activities of influenza virions
An egg grown virus stock was fractionated by sucrose gradient centrifugation and assayed for hemagglutinating and
Nagalase activities of each fraction. As shown in Fig. 2, the
gradient profile of the Nagalase activity is exactly the same
as that of hemagglutinating activity and the highest enzyme
activity corresponds to the peak fraction of hemagglutination. Top fractions (fraction numbers 16 through 18) of the
gradient also contained a very high level of Nagalase activity.
The result indicates that unassembled viral envelope proteins
are responsible for the high enzyme activity. However, the
unassembled HA protein does not appear to exhibit HA activity because of the monovalent nature of HA protein (Fig. 2).

Table 2
Time course study of Nagalase activity detected in blood stream of influenza virus-infected patients
Patient
no.
Patient 1
Patient 2
Patient 3
Patient 4
a

Days for symptoma:


(post-infection)

Post-infection. , Not determined.

1.96
1.75
1.68
1.53

1.95

1.63

Nagalase activity (nmoles/min/mg) in serum


9
19
30
1.85
1.72
1.61
1.50

0.94
0.87

0.93

0.86
0.88
0.91

60
0.58
0.55
0.45
0.61

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N. Yamamoto, M. Urade / Microbes and Infection 7 (2005) 674681

Fig. 2. Sucrose gradient centrifugation analyses of hemagglutinating and Nagalase activities of an egg grown influenza A virus stock. Each gradient fraction was
assayed for hemagglutination (HA activity) and Nagalase activity. Each fraction was treated with 0.01% trypsin for 30 min and assayed for Nagalase activity.

When each of these gradient fractions was treated with


0.01% trypsin for 30 min, Nagalase activity of each fraction
increased significantly as shown in Fig. 2. The result suggests that the proteolytic cleavage of HA to generate HA1
and HA2 subunits may be required for expression of Nagalase activity.
3.5. Demonstration of Nagalase activity in the envelope
HA protein: fractionation of deoxycholate-treated
influenza envelope glycoproteins using mannose-specific
lectin and electrophoretic demonstration of Nagalase in
the HA protein
A purified egg grown influenza virus stock was treated with
6.6% sodium deoxycholate and loaded onto mannose-specific
lectin affinity column, eluted and assayed for Nagalase activity. This lectin affinity chromatography selectively isolates
two envelope glycoproteins, HA and NA proteins [30]. Each
chromatographic fraction was assayed for Nagalase activity.
As shown in Fig. 3, the first protein peak fraction has a large
amount of Nagalase activity. This major peak fraction was
electrophoresed in SDS-PAGE and silver stained. Fig. 4 illustrates that the molecular weight of the Nagalase peak fraction coincides with the molecular weight (about 75 K) of the
HA protein. Therefore, we conclude that Nagalase is the
intrinsic component of the HA protein.
3.6. Deglycosylation of Gc protein with influenza HA
protein
When a purified Gc protein (1 ng/ml) was incubated with
the HA protein, the peak fraction of Nagalase activity
(2 mol), for 60 min and assayed for precursor activity, the
precursor activity of the Gc protein was reduced to roughly

Fig. 3. Mannose-specific lectin affinity fractionation of envelope proteins


after sodium deoxycholate treatment of an influenza virus stock and detection of Nagalase activity.

48% of that of the native Gc protein as shown in Table 4. This


result confirmed that the HA protein carries the Nagalase
capable to deglycosylate Gc protein.
3.7. Activation of Nagalase in HA protein by a proteolytic
process
Since the Nagalase peak fraction of the mannose affinity
fractionation was found to be HA protein, this peak fraction
(fraction numbers 5 through 8 of Fig. 3 was pooled) was
treated with 0.01% trypsin for 30 min. Nagalase activity
increased from 10.3 to 27.8 nmol/min as shown in Table 3.
This 2.7-fold increase over that of the untreated fraction confirmed the hypothesis that the Nagalase is expressed by pro-

N. Yamamoto, M. Urade / Microbes and Infection 7 (2005) 674681

679

Table 5
Nagalase activities in baculovirus-mediated cloned recombinant influenza
HA proteins
Clone

Influenza

No.
1
2
3
Control

Origin/type
A
A
B

Amount of
protein
for assay (g)
10
10
10
no protein

Nagalase (nmoles/min)a
without trypsin
0.082
0.095
0.078
0.093b

with trypsin
37.59
11.04
29.52

Nagalase activity is determined as p-nitrophenol production from substrate, p-nitrophenyl N-acetyl-a-D-galactosaminide.


b
The value is the amount of already hydrolyzed substrate (p-nitrophenol).
Thus, the values with untreated HA proteins had no Nagalase activity.

4. Discussion

Fig. 4. Electrophoretic identification of influenza HA protein having Nagalase activity after mannose-specific affinity fractionation of sodium
deoxycholate-treated influenza virus. SDS-polyacrylamide gel electrophoresis: Lane 1, proteins of influenza virion.
Lane 2, Nagalase peak fraction.
Table 4
Deglycosylation of Gc protein by influenza HA protein carrying Nagalase
activity as demonstrated by decrease in the precursor activity of Gc protein
after one hour incubation
Influenza
HA Nagalase
None
HA proteina

Gc protein
as substrate
Gc protein (1 ng)
Gc protein (1 ng)

Precursor activity
superoxide produced
(nmoles/min/106 macrophages)
4.48 0.18
2.14 0.15

Influenza HA protein having 2 mol of Nagalase activity as determined


by using the chromogenic substrate was incubated with 1 ng of Gc protein
for 1 h. The resultant reaction mixture was used for 3 h incubation with lysoPc-treated mixture of healthy human lymphocytes and macrophages to determine superoxide generating capacity of the macrophages as expressed as
precursor activity.

teolytic cleavage of the HA protein. In comparison with


trypsin treatment of egg grown viral stocks, a pronounced
increase (2.7-fold) in Nagalase activity of purified HA protein in probably due to better accessibility of trypsin to the
cleavage site of the protein. After breaking of the disulfide
bond of the HA protein with dithiothreitol, electrophoretic
fractionation revealed that HA1 subunit (2.5 g) carries Nagalase activity (2.57 nmol/min) whereas HA2 subunit has no
Nagalase activity.
3.8. Cloned HA protein required proteolytic process for
expression of Nagalase activity
Three HA proteins cloned from three independent epidemic strains showed no Nagalase activity. When these cloned
proteins were treated with 0.01% trypsin for 30 min, they
(influenza A as well as B) exhibited large amounts of Nagalase activity as shown in Table 5.

Influenza virus infection can be fatal, usually due to secondary complications [1012]. This is the major evidence for
immunosuppression in influenza virus-infected patients. The
proposed primary defender of the lung against bacterial pneumonia is the alveolar macrophages. We first characterized the
MAF precursor activity of the influenza patient Gc protein
and found that the MAF precursor activity of the serum Gc
protein was reduced in all the influenza virus-infected patients.
Decreased MAF precursor activity of the Gc protein was due
to the result of deglycosylation of the Gc protein [24,25]. Since
the Gc protein carries an O-glycan, the presence of Nagalase
in the patient blood stream was proposed (see Fig. 1b). In
fact, influenza virus-infected patients all carried Nagalase
activity in their blood stream. Thus, we speculated that the
virus-coded Nagalase is responsible for deglycosylation of
serum Gc protein causing immunosuppression. In the present
study, we concluded that the Nagalase resides on an envelope
glycoprotein, hemagglutinin HA, and that influenza virusinfected cells secrete viral Nagalase into the blood stream. In
our separate studies, we found that various enveloped viruses,
such as HIV, Sendai, rubella and measles viruses, carry Nagalase in their envelope glycoproteins (unpublished).
As we reported previously HIV-infected patients all carry
Nagalase in their blood stream [24]. This enzyme appears to
play a major role in immunosuppression in HIV-infected/
AIDS patients [24]. Impairment of immune function of the
hosts will enhance opportunities for survival and proliferation of neoplastic cells [8]. Thus, AIDS patients are at
extremely high risk for development of various forms of
malignant tumors [9]. As the disease of AIDS patients
progresses, the serum Nagalase activity level increases greatly
[24], resulting in severe immunosuppression. This should
explain why AIDS patients die with secondary infections
[37,38]. We also found Nagalase activity in the blood stream
of a variety of cancer patients [25,27]. All malignant cells but
not normal cells secrete this enzyme. Thus, the level of Nagalase activity in the blood stream is directly proportional to
tumor burden, i.e. the number of cancerous cells or the size
of tumor in the hosts [27,39,40]. The secretion of Nagalase
from cancerous cells to blood stream is responsible for deglycosylation of patient serum Gc protein [25,27], conse-

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N. Yamamoto, M. Urade / Microbes and Infection 7 (2005) 674681

quently loss of the MAF precursor activity and immunosuppression. Advanced cancer patient sera contain high Nagalase
activity, leading to severe immunosuppression [25,27]. Lack
of macrophage activation and immunosuppression explain
why cancer patients die from overwhelming infection, e.g.
pneumonia. Thus, the Nagalase activity in cancer bearing
hosts sera is an excellent diagnostic index for the pathogenicity of the disease and has been the most useful prognostic
index during cancer therapy [25,27,39,40].
The hemagglutinin of influenza virus is synthesized as a
single glycoprotein, HA (MW 75,000), which under certain
conditions of viral growth is post-translationally cleaved by
host cell proteases while in association with the host cell
plasma membrane into two smaller glycoproteins, HA1 (MW
49,000) and HA2 (MW 30,000), but still linked by disulfide
bond. This proteolytically cleaved subunit formation is
required for fusion capacity of the virus [3336]. We found
egg grown influenza virus stocks carry large amounts of Nagalase activity. Their most HA peptides are already cleaved to
HA1 and HA2 subunits because chorioallantoic membrane
cells of the embryonated egg carry a proteolytic enzyme
[35,36]. Treatment of the egg grown virus stock with trypsin
still increased Nagalase activity because the egg grown virus
stock contains some uncleaved HA proteins [35,36]. In HA
proteins cloned via baculovirus vector no Nagalase activity
was detected. When the cloned HA proteins were treated with
trypsin, they showed large amounts of Nagalase activities (see
Table 5). Since both fusion capacity and Nagalase activity
are expressed by proteolytic cleavage of HA protein to yield
HA1 and HA2, Nagalase activity seems to be an enzymatic
basis for fusion capacity. This concept is supported by our
recent observation that Nagalase resides on the fusion (F) protein of Sendai virus and gp160 of HIV and also that their
Nagalase activities are expressed by proteolytic cleavage to
generate F1 and F2 proteins for Sendai virus and gp120 and
gp41 for HIV, respectively (unpublished). Cloned gp160
(Intracel Corp., Cambridge, MA) showed no Nagalase activity. When cloned gp160 was treated with trypsin, Nagalase
activity was expressed. Thus, proteolytic cleavage of gp160 to
yield gp120 and gp41 is required for expression of Nagalase
activity. In fact cloned gp120 has Nagalase activity whereas
cloned gp41 has no Nagalase activity [41]. As the distal portion of the gp160 of HIV, gp120, carries Nagalase activity,
HA1 has Nagalase activity. Unlike liposomes, cell membranes are coated with glycans. Access of hydrophobic
N-terminal of HA2 to the cell membrane for fusion [42]
requires removal of O-glycans. Thus, it is tempting to speculate that Nagalase can promote HA2 to mediate the fusion of
the viral envelope with the cell membrane. Since fusion initiates viral infection [3336], Nagalase activity regulates
fusion capacity for infectivity.
Influenza virus-infected cells appear to release viral Nagalase (e.g. unassembled viral subunits) into the blood stream.
Since this enzyme can deglycosylate serum Gc protein resulting in loss of the MAF precursor activity, the deglycosylation
of Gc protein results in lack of macrophage activation and

thus appears to cause immunosuppression and secondary


infection in the influenza virus-infected patients. Therefore,
influenza HA-Nagalase plays dual roles in fusion and immunosuppression. Since fusion capacity regulates infectivity and
immunosuppression allows viral growth [8] and bacterial
pneumonia, Nagalase seems to contribute to virulence of the
virus. Recently we found that sucrose gradient-purified virus
stocks (egg grown) of six different influenza strains showed
varying levels of the enzyme activity ranging from 58.50 to
139.32 (nmol/mg per min). HA-Nagalase activity level varies from strain to strain. Thus, infections with influenza virus
strains carrying higher HA-Nagalase activity are likely to
cause severe immunosuppression.

Acknowledgements
This investigation was supported in part by US Public
Health Service Grant AI-32140. The authors are indebted to
Takako Namba and V.R. Naraparaju for their technical assistance.

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