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Original article
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
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Fig. 1. Schematic illustration of formation of MAF (a) and deglycosylation of Gc protein (b). *, Lyso-Pc-treated B cells.
676
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].
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Table 3
Nagalase activity and its proteolytic enhancement of an egg grown influenza
virus stock and purified HA protein
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
1.96
1.75
1.68
1.53
1.95
1.63
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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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.
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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
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 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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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
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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