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AIDS RESEARCH AND HUMAN RETROVIRUSES

Volume 22, Number 3, 2006, pp. 262271
Mary Ann Liebert, Inc.

Pathogenic Significance of
-N-Acetylgalactosaminidase

Activity Found in the Envelope Glycoprotein gp160 of
Human Immunodeficiency Virus Type 1
NOBUTO YAMAMOTO

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 lost or reduced in HIV-infected patients. These
patient sera contained
-N-acetylgalactosaminidase (Nagalase), which deglycosylates serum Gc protein. Deglycosylated Gc protein cannot be converted to MAF and thus loses MAF precursor activity, leading to immunosuppression. Nagalase in the blood stream of HIV-infected patients was complexed with patient immunoglobulin G, suggesting that this enzyme is immunogenic, seemingly a viral gene product. In fact, Nagalase
was inducible by treatment of cultures of HIV-infected patient peripheral blood mononuclear cells with a
provirus-inducing agent. This enzyme was immunoprecipitable with polyclonal anti-HIV but not with anticellular constitutive enzyme or with antitumor Nagalase. The kinetic parameters (km value of 1.27 mM and
pH optimum of 6.1), of the patient serum Nagalase were distinct from those of constitutive enzyme (km value
of 4.83 mM and pH optimum of 4.3). This glycosidase should reside on an envelope protein capable of interacting with cellular membranous O-glycans. Although cloned gp160 exhibited no Nagalase activity, treatment
of gp160 with trypsin expressed Nagalase activity, suggesting that proteolytic cleavage of gp160 to generate
gp120 and gp41 is required for Nagalase activity. Cloned gp120 exhibited Nagalase activity while cloned gp41
showed no Nagalase activity. Since proteolytic cleavage of protein gp160 is required for expression of both
fusion capacity and Nagalase activity, Nagalase seems to be an enzymatic basis for fusion in the infectious
process. Therefore, Nagalase appears to play dual roles in viral infectivity and immunosuppression.

INTRODUCTION

(AIDS) is characterized by opportunistic infections and by opportunistic

neoplasms (e.g., Kaposis sarcoma) as evidence of immunosuppression.1 Impaired phagocytosis and bacteriocidal process by
phagocytes of AIDS patients have been reported from a number
of laboratories.29 Since macrophage activation for phagocytosis
and antigen presentation to B and T lymphocytes represent the
first indispensable step in both humoral and cellular immunity
development,10,11 lack of macrophage activation leads to immunosuppression. We thus need to investigate the problem of
phagocyte activation in HIV-infected/AIDS patients.
The inflammation-derived macrophage activation cascade is
the process for generation of the principal macrophage-activating factor (MAF).12,13 Administration of an inflamed tissue
CQUIRED IMMUNODEFICIENCY SYNDROME

lipid metabolite, lysophosphatidylcholine (lyso-Pc), or other

lysophospholipids, to mice rapidly activates macrophages for
greatly enhanced phagocytic and superoxide-generating capacities.1214 The inflammation-primed macrophage activation process requires serum vitamin D3-binding protein (known as Gc
protein)1317 and participation of B and T lymphocytes.12,13,18
Gc protein carries a trisaccharide consisting of N-acetylgalactosamine with dibranched galactose and sialic acid termini.13
The inducible membranous -galactosidase (Bgli) of inflammation-primed (or lyso-Pc-treated) B lymphocytes modifies Gc
protein to yield the macrophage-proactivating factor. This is
further converted by action of the membranous Neu-1 sialidase
of T lymphocytes to the macrophage-activating factor,13,17,19
the protein with N-acetylgalactosamine as the remaining sugar
as shown in Fig. 1a. Thus, Gc protein is the precursor for the
principal MAF.13,15,19

Division of Molecular Virology, Socrates Institute for Therapeutic Immunology, Philadelphia, Pennsylvania 19126-3305.

262

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-N-ACETYLGALACTOSAMINIDASE OF HIV

263

FIG. 1. Schematic illustration of formation of macrophage-activating factor (a) and deglycosylation of Gc protein (b). *Inflammation-primed B cells: B cells can be treated with an inflamed membranous lipid metabolite, e.g., lysophosphatidylcholine.

When peripheral blood mononuclear cells (PBMCs) containing monocytes/macrophages and lymphocytes of HIV-infected patients were treated with lyso-Pc and cultured in
medium containing purified Gc protein (1 ng/ml) or healthy human serum (0.1%) as a source of Gc protein, the lyso-Pc-primed
lymphocytes of all patients were fully capable of converting Gc
protein to MAF, resulting in activation of their macrophages.10
Thus, a possible dysfunction of HIV-infected patient
PBMCs20,21 does not affect macrophage activation, because
only a small proportion of PBMCs expressing CD4 is infected.22,23 However, cultivation of lyso-Pc-treated patient
PBMCs in medium containing the patients own serum (0.1%)
resulted in the lack or reduced levels of macrophage activation,10 as a consequence of the lost or decreased MAF precursor activity of the patient serum Gc protein.10 Loss of MAF
precursor activity is due to deglycosylation of serum Gc protein by
-N-acetylgalactosaminidase (Nagalase) found in the
blood stream of HIV-infected patients10 (Fig. 1b). The deglycosylated Gc protein cannot be converted to MAF, thus leading to immunosuppression.10 As the disease advances patient
serum Nagalase activity increases while the MAF precursor activity of Gc protein decreases greatly.10 Lack of macrophage
activation and immunosuppression may explain why AIDS patients die from overwhelming infection.24,25
Nagalase activity was detected in the blood stream of all
HIV-infected patients but not in the blood of healthy humans.10
Nagalase appears to be secreted from HIV-infected cells.10
Thus, serum Nagalase seems to be of viral origin. In this article, we report that Nagalase is the intrinsic component of an
envelope protein promoting fusion for the initiation of infection. Because Nagalase in the blood stream causes immuno-

suppression,10 Nagalase has pathogenic significance in the regulation of both HIV infectivity and immunosuppression.

MATERIALS AND METHODS

Reagents, media, and cells
p-Nitrophenyl N-acetyl-
-D-galactosaminide was obtained
from Sigma (St. Louis, MO). Mitomycin C, a provirus-inducing
agent,26 was obtained from ICN Pharmaceutical (Costa Mesa,
CA). Macrophage-SSM serum-free medium was purchased from
Invitrogen (GIBCO-BRL Life Science Technology, Grand Island,
NY). Protein GSepharose was obtained from Pharmacia Biotech
(Piscataway, NJ). Polyclonal anti-HIV-1 antibodies were purchased from Biodesign (Kennebunk, ME). Polyclonal anti-cellular constitutive enzyme and anti-tumor Nagalase antibodies27 were
prepared in our institute. Antibodies were diluted with 10 mM
phosphate buffer. Cloned envelope glycoproteins gp160, gp120,
and gp41 were purchased from Intracel (Cambridge, MA) and
Trinity Biotech (Carlsbad, CA). These proteins were all baculovirus vector-based cloned products. For manipulation in vitro
and cultivation of lymphocytes and macrophages, 0.1% egg albumin-supplemented medium RPMI 1640 (EA medium) was
used. MT-4 cells were used for plaquing infectious centers to determine the number of HIV-infected cells.

Determination of precursor activity of serum

Gc protein of HIV-infected patients
Blood samples of two uninfected healthy humans were collected in tubes containing EDTA to prevent coagulation. The

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264
blood samples were generally processed within 2 hr from the
time of blood collection. A 5-ml blood sample and 5 ml of
saline (0.9% NaCl) were mixed and gently placed in a 15-ml
centrifuge tube containing 3 ml of Lymphoprep (similar to Ficoll; Polysciences, Warrington, PA) and centrifuged at 800
g
for 15 min. The dense white cell band, PBMCs containing
monocytes/macrophages (macrophages for short) and lymphocytes (B and T cells), was collected with a Pasteur pipette. The
white cell mixture was 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 37C allowed adherence of macrophages. The mixture of lymphocytes and adherent macrophages
of uninfected humans was treated with 1 g of lyso-Pc/ml in
EA medium for 30 min. Because of adherence of macrophages
to plastic substrata, lymphocytes and macrophages were separately washed with PBS, admixed, and cultured in EA medium
containing 0.1% serum from an infected or uninfected human
as a source of Gc protein. After 3 hr of cultivation the macrophages were assayed for superoxide-generating capacity. The
values of superoxide generation of the macrophages represent
the precursor activity of the patient Gc protein.10,28,29 Lost or
reduced precursor activity of patient serum Gc protein is exhibited as a decrease in superoxide generation as compared with
uninfected human Gc protein. Thus, this procedure measures
the ability of an individual patient to activate macrophages.

Assay for generation of superoxide in macrophages

Adherent macrophages were washed twice with PBS. The
macrophages (approximately 3
105 macrophages per well)
were overlaid with 20 g of cytochrome c/ml and incubated
for 10 min. Thirty minutes after addition of phorbol-12-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 minute per 106 cells.10,28,29

YAMAMOTO
man DU640 spectrophotometer and expressed as nanomoles per
minute per milligram of serum protein for the enzyme activity.
Protein concentrations were estimated by the Bradford
method.30

Procedures for immunologic characterization of

HIV-infected patient serum Nagalase
Six-fold diluted patient serum (300 l) was precipitated with
70% ammonium sulfate and dialyzed against 50 mM citrate
phosphate buffer for assay of the total Nagalase activity in individual patient serum samples. To immunoprecipitate the enzyme, 50 l of patient serum and 50 l of 10 mM phosphate
buffer with or without 100-fold diluted polyclonal anti-HIV-1
IgG were mixed in a microcentrifuge tube and incubated for 60
min at 37C. Protein GSepharose (40 mg) in citrate phosphate
buffer (400 l) was added to anti-HIV-treated or untreated
serum (100 l) of individual patients and incubated with tumbling motion for 60 min. The protein GSepharose was washed
with 50 mM citrate phosphate buffer (pH 6.3), and the enzyme
was eluted from the protein GSepharose with 300 l of citrate phosphate-buffered saline containing 0.15 M NaCl (pH 6.5)
and assayed for Nagalase activity. Substrate solution (250 l)
containing 5 mol of p-nitrophenyl N-acetyl-
-D-galactosaminide in 50 mM citrate buffer (pH 6.0) was added to
250 l of the dialysates to determine total serum Nagalase activity or to samples eluted from protein GSepharose and incubated for 60 min at 37C. The reaction was stopped 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 and expressed as nanomoles per minute per
milligram of serum. All eluted enzyme activities were calculated for specific activity against the 70% ammonium sulfateprecipitable protein concentration in the volume of the original
patient serum used. Supernatant after protein GSepharose precipitation was also precipitated with 70% ammonium sulfate
and assayed for Nagalase activity.

Detection of Nagalase in HIV-infected patient sera

All blood serum samples represent portions of diagnostic
specimens from three different institutions. Serum samples from
HIV-infected patients under chemotherapy were not used for
this study, because their serum Nagalase levels are not indicative of the state of disease: chemotherapeutic drugs are metabolic inhibitors, which suppress the production of Nagalase
from HIV-infected cells.
Sera (1 ml) of infected 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 4C overnight. After dialysis, the volume of the samples were made up to 1 ml
and assayed for the enzyme.10,28 Enzyme activity was determined at 37C 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-
-D-galactosaminide as substrate. The reaction was initiated by addition of 100 l of each dialyzed sample 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 a Beck-

Treatment of patient PBMCs with a provirus-inducing

agent, mitomycin C, and detection of induced
Nagalase in culture media
The desired number of patient PBMCs (approximately 2

106 cells) was placed in 35-mm wells and cultured for 24 hr in
5% FCS-supplemented medium Macrophage-SSM. After 24 hr
of cultivation, PBMCs were treated with 5 g of mitomycin
per milliliter as provirus inducer for 30 min.26 Lymphocytes
and macrophages were separately washed with PBS three times
and admixed in 5% FCS-supplemented medium MacrophageSSM. After 72 hr of cultivation, culture media of mitomycintreated or untreated PBMCs were harvested and assayed for Nagalase activity for indication of viral induction.

Determination of numbers of HIV-infected PBMCs

To determine the number of HIV-infected PBMCs, an assay
method for plaques as infectious centers was employed. Sixwell tissue culture plates were treated with poly-L-lysine (50
g/ml) for 1 hr at room temperature. The wells were then
washed three times with distilled water and left to dry in air.

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-N-ACETYLGALACTOSAMINIDASE OF HIV
MT-4 cells were washed in serum free EA medium twice and
resuspended at a final cell concentration of 12
106/ml. Cells
were then allowed to adsorb to the plates (1 ml/well) for 30 min
at room temperature. Unbound cells were subsequently removed by aspiration. To determine the number of HIV-infected
PBMCs, 0.1 ml of mitomycin-treated patient PBMCs was added
to 1.5 ml of assay medium (RPMI-1640 with 20% FCS) containing 0.8% molten agarose (Sea Plaque agarose; FMC Bioproducts, Rockland, ME), prewarmed to 38.5C, and added to
each well. The plates were kept at 37C for a few minutes and
then centrifuged31 in plate carriers at 800
g for 20 min in a
centrifuge precooled to 10C. After the agarose medium had
set, the plates were incubated at 37C for 3 days, at which time
a second agarose overlay was added. Neutral red stain was
added in the second agarose overlay at a concentration of
0.00033% and incubated in the dark for 20 hr, and the plaques
were counted with an inverted microscope.

Enzymatic characterization of Nagalase from HIVinfected patient sera and cellular constitutive enzyme
from normal lung tissue homogenates
The constitutive enzyme and HIV-infected patient serum enzyme were purified by the method of Dean and Sweeley.32 HIVinfected patient serum and healthy lung tissue homogenates
were precipitated with 70% saturated ammonium sulfate and
dialyzed in 10 mM phosphate buffer (pH 6.5). The dialysates
were loaded onto DEAE-cellulose columns and eluted in a stepwise fashion with phosphate buffer containing 50 mM NaCl,
150 mM NaCl, and 175 mM NaCl. Phosphate buffer containing 50 and 150 mM NaCl elutes
-galactosidase and other glycosidases, respectively.32 Phosphate buffer containing 175 mM
NaCl elutes Nagalase exclusively. The enzyme fractions were
pooled, dialyzed, loaded onto hydroxyapatite columns, and
eluted with 30 mM phosphate buffer (pH 5.5). The eluted fractions were again loaded onto DEAE-cellulose columns and
eluted with 15-fold diluted ampholytes displacement chromatogram (pH 46). This fraction was loaded on Sephadex G150 to remove ampholytes. The purified lung Nagalase and HIV
patient serum Nagalase were characterized for their enzyme kinetic parameters. The pH optima and km values of these enzymes provide us with clues to determine whether the origin of
the HIV patient serum enzyme is viral or inducible cellular
isozyme.

RESULTS
Illustration of inverse correlation between MAF
precursor activity of serum Gc protein and serum
Nagalase activity of HIV-infected patients
As shown in Table 1, various reduced levels of the MAF
precursor activity of serum Gc protein were found in HIV-infected patients. Since a decrease in the MAF precursor activity
of serum Gc protein is due to deglycosylation of the O-glycan
of Gc protein10 (Fig. 1b), serum deglycosylating enzyme, Nagalase, was measured in those patients. As the disease progresses serum Nagalase increases.10 As the MAF precursor activity decreased, serum Nagalase activity increased (Table 1).
This inverse correlation between MAF precursor activity and

265
TABLE 1.
AND

BLOOD

PRECURSOR ACTIVITY OF SERUM Gc PROTEIN

NAGALASE ACTIVITY DETECTED IN THE
STEAM OF HIV-INFECTED/AIDS PATIENTS

Patient
no.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Healthy humans

Precursor activity:
superoxide produced
(nmol/min/106 cells)

Nagalase
specific activity
(nmol/min/mg)

2.25
2.84
0.74
0.54
3.42
0.69
4.43
5.05
1.51
0.49
0.29
5.01
3.10
0.05
2.91
2.48
1.18
0.49
3.62
1.64
1.34
0.72
1.91
2.53
6.25a

3.60
3.48
7.75
10.02
2.51
9.03
1.43
0.51
7.38
8.42
7.14
0.81
2.63
8.66
3.15
3.55
6.87
9.47
2.28
6.26
6.88
6.65
5.75
4.71
0.23b

aPrecursor

activity was measured by superoxide-generating

capacity of healthy human macrophages when treated with
0.1% of patient serum as a source of Gc protein.
bExtremely low levels of Nagalase activity was an average
of 5 uninfected healthy human sera. This uninfected human
enzyme is known to be
-galactosidase.10,28 This enzyme is unable to deglycosylate Gc protein although it is able to hydrolyze p-nitrophenyl N-acetyl-
-D-galactosaminide.10,28
-NAcetylgalactosaminidase and
-galactosidase are evolutionarily
conserved, carry 46.9% amino acid sequence homology, and
share a common chromogenic substrate for their catabolic capacities.10,28

serum Nagalase activity of 24 HIV-infected patients is illustrated in Fig. 2.

Origin of Nagalase found in HIV-infected patient

blood stream
If Nagalase in the patient blood stream is a viral protein, antiHIV-1 antibodies should bind to Nagalase and the antibodyNagalase complex should be precipitable by immobilized
protein A or protein G. However, the HIV-1 envelope protein
gp120 nonspecifically binds to immunoglobulin M,33 which can
be precipitated with protein A but not with protein G. Thus, the
IgGNagalase immune complexes should be precipitated with
protein GSepharose.
Four patients were chosen for characterization of their serum
Nagalase. Polyclonal anti-HIV-1 IgG was used for immuno-

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266

FIG. 2.

YAMAMOTO

Inverse correlation between the precursor activity of serum Gc protein and Nagalase activity in patient blood stream.

precipitation of the enzyme. With anti-HIV-treated patient sera,

nearly total Nagalase activity of all four patients was precipitated with protein GSepharose as shown in Table 2. However,
protein GSepharose precipitated Nagalase activities from patient sera that have not been treated with anti-HIV (Table 2).
Thus, protein G precipitation of serum Nagalase activity did not
require treatment of patient serum with anti-HIV, indicating that
the Nagalase in the patient blood stream was already complexed
with the patients own immunoglobulin G. This observation indicates that the enzyme is immunogenic to humans, implying
that the enzyme is encoded by the virus.

g/ml), and cultured in 5% FCS supplemented medium Macrophage-SSM for 72 hr, significant amounts of Nagalase were
induced in the culture media. In a few cases (e.g., patients 10
and 18), no significant induction of Nagalase was shown, indicating that these cultured PBMCs have small numbers of infected cells. In fact, the numbers of HIV-infected PBMCs, as
determined by plaque (infectious center) assay, showed that patients 10 and 18 had 10-fold less infected cells (0.7
104 and
1.0
104) than the others (0.73.2
103) as shown in Table
3. Nevertheless, we conclude that the Nagalase is inducible by
the provirus-inducing agent.

Secretion of Nagalase from cultured HIV-infected

patient PBMCs and induction of Nagalase by a
provirus-inducing agent

Serological characterization of Nagalase secreted from

HIV-infected PBMCs

All HIV-infected patients but not healthy humans carry Nagalase in their blood stream,10 suggesting that the HIV-infected
cells secrete Nagalase. To distinctly demonstrate secretion and
induction of Nagalase from HIV-infected patient CD4 cells,
we isolated PBMCs from 19 patients carrying rather low serum
Nagalase levels. After these PBMCs were cultured in 5% FCS
supplemented medium Macrophage-SSM for 72 hr, small
amounts of Nagalase activity were detected in their cultured
media as shown in Table 3. When these patient PBMCs were
treated with a provirus-inducing agent,26 mitomycin C (5

Culture media (100 l) of HIV-infected patient PBMCs with

or without mitomycin pretreatment were mixed with 100 l of
100-fold diluted polyclonal anti-HIV antibody and incubated
for 60 min. The total Nagalase activity of all four patients was
precipitated with protein GSepharose as shown in Table 4.
This result confirmed the previous observation that the total Nagalase activity secreted from mitomycin-treated AIDS patient
PBMCs is immunoprecipitable with anti-HIV-1 IgG.34 When
these culture media were incubated with anti-constitutive enzyme and with anti-tumor Nagalase, the Nagalase activity was
not precipitated by protein GSepharose.

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-N-ACETYLGALACTOSAMINIDASE OF HIV
TABLE 2.

DETECTION

OF IMMUNOGLOBULIN

267

G-BOUND NAGALASE ACTIVITY

IN

SERUM

OF

HIV-INFECTED PATIENTS

Nagalase (nmol/min/mg)
Patient
no.
1
2
3
4

Anti-HIV
pretreated

Protein G
precipitation













Controla

Precipitateb

Supernatantc

3.09
3.13

0.26
0.06

2.23
1.91

0.17
0.35

2.71
2.58

0.15
0.03

2.66
2.41

0.11
0.38

3.14
2.61
2.87
2.81

aSix-fold diluted patient serum (300 l) without protein GSepharose precipitation was precipitated with 70% ammonium sulfate, dialyzed against 50 mM citrate phosphate buffer, and assayed for enzyme activity as the total enzyme activity in serum as
described previously.10
bNagalaseantibody complex was eluted from protein GSepharose with 0.1 M citrate and assayed for Nagalase activity.
cSupernatant after protein GSepharose precipitation was precipitated with 70% ammonium sulfate and assayed for the
enzyme.

Enzyme kinetic parameters of Nagalase in blood

stream of HIV-infected patients
To compare enzyme kinetic parameters of the serum Nagalase of HIV-infected patients with those of the constitutive
enzyme, these enzymes were purified from HIV-infected pa-

tient sera and healthy human lung tissue. As shown in Table 5,

the km value for hydrolysis of p-nitrophenyl N-acetyl-
-D-galactosaminide by the HIV-infected patient serum enzyme is 1.27

103 M while the km value of the constitutive enzyme is 4.83

103 M. The optimal pH for the HIV patient serum enzyme is
6.1 while the optimal pH for the constitutive lung enzyme is

TABLE 3. INDUCTION OF NAGALASE ACTIVITY FROM CULTURED HIV-INFECTED PATIENT

PBMCS BY TREATMENT WITH PROVIRUS-INDUCING AGENT MITOMYCIN C
Nagalase (nmol/mg/min)
Medium of 72-hr culture after mitocyin
Patient no.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Healthy humansa
aAverage

Serum enzyme
activity

No treatment

Treatment

1.91
3.70
1.91
2.04
2.01
1.55
1.63
2.87
3.07
1.05
3.63
2.96
3.91
2.16
3.80
3.20
1.85
1.11
2.43
0.27

0.67
0.98
0.44
1.12
0.71
0.48
0.95
1.09
0.51
0.54
0.54
0.56
0.53
0.78
0.87
0.84
0.65
0.55
0.54
0.03

0.85
2.51
1.12
3.18
2.09
1.82
1.43
2.59
2.55
0.55
2.92
1.82
2.75
2.15
2.56
2.19
1.27
0.65
1.96
0.02

of three. , not determined.

Rate of
infected
PBMCs
0.7
2.6
0.9
3.2
2.1
1.8
2.0
0.7
2.5
1.1
2.5
2.7
1.2
1.0
1.8

103

103

103

103

103

103

103

104

103

103

103

103

103

104

103

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268

YAMAMOTO
TABLE 4. SEROLOGICAL SPECIFICITY OF NAGALASE RELEASED FROM HIV-INFECTED PATIENT PBMCS WAS EXAMINED
IMMUNOPRECIPITATION WITH ANTI-HIV, ANTI-CELLULAR CONSTITUTIVE ENZYME, OR ANTI-TUMOR NAGALASE

BY

Nagalase (nmol/min/mg) after protein G precipitationa

Patient
no.
1
2
3
4

Provirusinducing
agent

No antibodies

HIVb

Constitutivec

Tumor nagalased

None
Mitomycin
None
Mitomycin
None
Mitomycin
None
Mitomycin

1.12
3.18
0.87
2.56
2.31
6.25
3.31
7.58

0.04
0.09
0.02
0.10
0.03
0.07
0.07
0.18

1.12
3.22
0.89
2.59
2.29
6.25
3.29
7.71

1.24
3.17
0.84
2.49
2.33
6.09
3.36
7.15

Antibodies against

aCulture

medium of patient PBMCs was mixed with anti-HIV, anti-cellular constitutive enzyme, or anti-cancer Nagalase and
incubated for 1 hr. After protein GSepharose precipitation, supernatants were precipitated with 70% ammonium sulfate, dialyzed against 50 mM citrate phosphate buffer, and assayed for enzyme activity as the total enzyme activity in plasma as described
previously.50 Supernatant after protein GSepharose precipitation was assayed for the enzyme.
bAnti-HIV.
cAnti-cellular constitutive enzyme.
dAnti-cancer Nagalase.

4.3. These distinct enzymatic parameters support the idea that

the HIV patient serum Nagalase is a viral enzyme, not of cellular origin.

Searching for Nagalase activity in an HIV envelope

protein, using baculovirus vector-based cloned
envelope proteins
If HIV encodes this glycosidase, we anticipate that the enzyme activity should reside on an envelope glycoprotein capable of interacting with O-glycans of the host cell membranes.
Therefore, cloned HIV-1 envelope proteins were examined for
Nagalase activity. The enzyme activity was determined at 37C
in a reaction mixture of 500 l containing 50 mM sodium citrate phosphate buffer (pH 6.0), 5 mol of the chromogenic substrate, and a cloned envelope protein. The amount of released
p-nitrophenol was determined and expressed as micromoles per
hour. After a 60 min incubation of 5 g of cloned gp160 with
the substrate, Nagalase activity was undetectable, less than 0.03
mol/hr. However, when cloned gp160 (5 g) was treated with
a low concentration (0.01%) of trypsin for 30 min, Nagalase
activity of 0.42  0.02 mol/hr (7.0 nmol/min) was found.
Thus, trypsin treatment of cloned gp160 is required for ex-

TABLE 5. ENZYMATIC PARAMETERS OF

-N-ACETYLGALACTOSAMINIDASE ACTIVITY
IN SERUM OF HIV-INFECTED PATIENTS

-N-Acetylgalactosaminidase
Origin
HIV patient serum
Constitutive enzyme

km value (
103 M)

Optimal pH

1.27
4.83

6.1
4.3

pression of Nagalase activity. Kinetic analyses of the enzyme

activity of cloned gp160 (10 g) were performed after incubation for 60, 90, or 180 min with the substrate. As shown in Fig.
3, cloned gp160 (10 g) exhibited no activity. When cloned
gp160 was treated with trypsin for 30 min, the enzyme activity greatly increased to 0.91 mol/hr (15.16 nmol/min). These
observations confirmed that proteolytic cleavage of gp160 to
generate gp120 and gp41 is required for expression of Nagalase
activity. To demonstrate whether gp120 or gp41 carries Nagalase activity, 5-g samples of cloned gp120 and cloned gp41
were examined. As shown in Fig. 3, cloned gp120 (5 g) exhibited Nagalase activity of 0.26 mol/hr (4.33 nm/min)
whereas cloned gp41 (5 g) showed insignificant activity, less
than 0.03 mol/hr. Thus, the result suggests that Nagalase is
the intrinsic component of the envelope glycoprotein gp120.

DISCUSSION
HIV infection leads to an immune response in patients who
nonetheless develop inadequate immunity to the disease. Consequently, a persistent infection is established. HIV-infected
cells can secrete Nagalase into the blood stream. In fact, spontaneously induced Nagalase from the culture of HIV-infected
patient PBMCs was immunoprecipitable by anti-HIV-1 IgG but
not by polyclonal anti-constitutive enzyme or by anti-tumor Nagalase antibodies. Furthermore, enzyme kinetic parameters of
serum Nagalase indicate that HIV-patient serum Nagalase is not
of cellular origin. Thus, the serum Nagalase activity seems to
be the enzyme activity of gp120 and the whole HIV virion.
Since HIV patient Nagalase appears to be an intrinsic component of the envelope glycoprotein gp120, which is immunogenic to humans, serum Nagalase was complexed with each patients own immunoglobulin G. The immunoglobulin G-bound
Nagalase retained its enzymatic activity and was able to de-

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269

FIG. 3. Kinetics of Nagalase activity in cloned HIV envelope proteins. Enzyme activity was determined at 37C in a reaction
mixture of 500 l containing 50 mM sodium citrate phosphate buffer (pH 6.0), 5 mol of p-nitrophenyl N-acetyl-
-D-galactosaminide as a substrate, and the indicated amount of cloned envelope proteins. To demonstrate whether these cloned proteins
carry Nagalase activity, 10 g for cloned gp160 and 5 g for cloned gp120 and cloned gp41 rather than their equimolar concentrations were used.
glycosylate Gc protein. The deglycosylated Gc protein could
not be converted to MAF, leading to immunosuppression.10
Since various viruses induce immunosuppression in their
hosts,3537 Nagalase could be a generalized function required
for growth of a variety of viruses. In fact, in our separate studies, we found that various enveloped viruses such as influenza,38
Sendai, measles, and rubella viruses (unpublished data) carry
Nagalase in their envelope glycoproteins.
As we reported previously, Nagalase in the blood stream
seems to play a major role in immunosuppression in HIV-infected/AIDS patients.10 Impairment of the immune function of
the hosts will enhance opportunities for survival and proliferation of neoplastic cells.39 Thus, AIDS patients are at extremely
high risk for development of various forms of malignant tumors.1 As the disease advances, the serum Nagalase activity
level increases greatly,10 resulting in no macrophage activation
and severe immunosuppression. This should explain why AIDS
patients die with secondary infections.24,25 We also found Nagalase activity in the blood stream of a variety of cancer patients.28,29 All malignant cells but not normal cells secrete this
enzyme.28,29 Thus, the serum tumor Nagalase is detectable in
patients with malignant tumors exclusively. Enzymatic parameters of the tumor Nagalase are distinct from those of the constitutive enzyme.40 The level of Nagalase activity in the blood
stream is directly proportional to tumor burden, i.e., tumor size
or the number of cancerous cells in the host.28,29,41 The secre-

tion of Nagalase from cancerous cells into the blood stream is

responsible for deglycosylation of patient serum Gc protein,28,29
consequently causing the loss of MAF precursor activity and
leading to immunosuppression. Advanced cancer patient sera
contain high Nagalase activity, resulting in severe immunosuppression.28,29 Lack of macrophage activation and immunosuppression explain why cancer patients die from overwhelming infection, e.g., pneumonia. Thus, the Nagalase activity in
the sera of cancer-bearing hosts is an excellent diagnostic index for the pathogenicity of the disease and has been the most
useful prognostic index during cancer therapy.28,29,41,42
The envelope glycoprotein gp160 of HIV have been shown
to play a critical role in virus binding to the CD4 receptor molecule1 and in fusion of the viral envelope with the host cell
membrane for the infectious process.43 The envelope protein
gp160 is synthesized as a polyprotein precursor that is proteolytically cleaved by host cell protease to yield two smaller glycoproteins, the external receptor-binding glycoprotein gp120
and the transmembrane glycoprotein gp41.44,45 The proteolytic
cleavage of the envelope protein gp160 is required for expression of its fusion capacity for the initiation of infection.4446
Similar phenomena are also known for the envelope proteins
of other enveloped virus families such as influenza and Sendai
viruses.46,47 The present study demonstrated that the proteolytic
cleavage of the protein gp160 is required for expression of Nagalase activity. Nagalase also resides on the HA protein of in-

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fluenza virus38 and the fusion (F) protein of Sendai virus and
their Nagalase activities are also expressed by proteolytic cleavage to generate HA1 and HA2 of influenza virus38 and F1 and
F2 of Sendai virus (our unpublished data). Since both Nagalase
activity and the fusion capacity of the protein gp160 are expressed by proteolytic cleavage, Nagalase activity is seemingly
an enzymatic basis for fusion. As the distal portion of the HA
protein of influenza virus, HA1, carries Nagalase activity,38
gp120 has Nagalase activity. Thus, it is tempting to speculate
that gp120Nagalase can act on the host cell membrane. This
event may promote the hydrophobic amino terminus of gp41
to mediate the fusion of the viral envelope with the cell membrane. Thus, Nagalase seems to trigger fusion for the infectious
process. Therefore, Nagalase plays dual roles in infectivity and
immunosuppression.
This study used in vitro experiments with HIV-infected patient specimens to develop a molecular understanding of how
viral infection can also suppress normal immune functions in
vivo. Since similar phenomena with Nagalase were found in
various enveloped viruses and cancers, pathogenic significance
of the Nagalase is greatly broadened.

10.

11.

12.

13.

14.

15.

ACKNOWLEDGMENTS

16.

This investigation was supported in part by U.S. Public

Health Service grant AI-32140. The author is indebted to Masumi Ueda, Yoshihiko Koga, V.R. Naraparaju, and Naofumi
Ushijima for technical assistance.

17.

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Address reprint requests to:

Nobuto Yamamoto
Socrates Institute for Therapeutic Immunology
1040W 66th Avenue
Philadelphia, PA 19126-3305
E-mail: nobutoyama@peoplepc.com