From the Institute of Biochemistry, University of Giessen, Friedrichstrasse 24, D-35392 Giessen, Germany, the § Institute of Medical Virology, University of Giessen, Frankfurter Str. 107, D-35392 Giessen, Germany, and the ¶ Institute of Medical Physics and Biophysics, University of Münster, Robert-Koch-Strasse 31, D-48149 Münster, Germany
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ABSTRACT |
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The surface antigen of hepatitis B virus
comprises a nested set of small (S), middle (M), and large (L)
proteins, all of which are partially glycosylated in their S domains.
The pre-S2 domain, present only in M and L proteins, is further
N-glycosylated at Asn-4 exclusively in the M protein. Since
the pre-S2 N-glycan appears to play a crucial role in the
secretion of viral particles, the M protein may be considered as a
potential target for antiviral therapy. For characterization of the
pre-S2 glycosylation, pre-S2 (glyco)peptides were released from native,
patient-derived hepatitis B virus subviral particles by tryptic
digestion, separated from remaining particles, purified by
reversed-phase high performance liquid chromatography, and identified
by amino acid and N-terminal sequence analysis as well as
matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry (MALDI-TOF-MS). Pre-S2 N-glycans were
characterized by anion exchange chromatography, methylation analysis,
and on target sequential exoglycosidase digestions in combination with
MALDI-TOF-MS, demonstrating the presence of partially sialylated
diantennary complex-type oligosaccharides. In addition, the pre-S2
domain of M protein, but not that of L protein, was found to be
partially O-glycosylated by a Gal( Hepatitis B virus
(HBV),1 belonging
to the virus family hepadnaviridae, is an important etiological agent
of acute and chronic liver disease (1, 2). Chronic HBV infection may lead to liver cirrhosis and hepatocellular carcinoma, which result in
about 1 million deaths per year worldwide. The virus replicates in the
liver and is secreted in large amounts of up to 1010
particles/ml into the blood (3). In addition to 42-nm DNA containing
virions, infected hepatocytes produce subviral, noninfectious 22-nm
spherical or filamentous particles in vast excess. The envelopes of
virions and subviral particles contain varying amounts of three related
HBV-encoded (glyco)protein species termed large (L), middle (M), and
small (S) proteins, which are together referred to as HBV surface
antigen (HBsAg). S protein is the major component of virions and both
spherical and filamentous HBsAg particles, while filaments and virions
contain more M and, in particular, more L proteins than spheres (4,
5).
All envelope proteins are produced from a single open reading frame
(see Fig. 1A) by the use of three different translation start sites, dividing this open reading frame into three domains: the
amino-terminal pre-S1 domain, which occurs exclusively in the L
protein; the pre-S2 domain, which is present in both M and L proteins
and forms the amino-terminal end of the M protein; and the S domain,
which is common to S, M, and L proteins. All proteins possess a
potential N-glycosylation site at Asn-146 of the S domain,
which, however, is only partially utilized. Hence, the proteins exist
in two isomeric forms, being either glycosylated or nonglycosylated in
this position, and migrate as doublets in SDS gel electrophoresis (Fig.
1B). The second potential N-glycosylation site,
present at Asn-42
of the pre-S2 domain, is solely used in the M but not in the L protein
(4). In addition to N-glycosylation, pre-S2 domains of HBV M
protein expressed in mammalian cell culture (6-8) or of M protein of
the related woodchuck hepatitis virus (WHV) (9) have been reported to
be O-glycosylated in as yet unidentified positions.
HBV envelope (glyco)proteins are functionally important during the
viral life cycle. L protein mediates binding to human hepatocytes of
the virus via its pre-S1 domain (10, 11). Furthermore, L protein has
been shown to be important for virion envelopment and secretion (12).
In order to fulfill these functions, the pre-S domain of the L protein
has a dual topology. During and directly after translation, it is
located at the cytosolic side of the endoplasmic reticulum and, hence,
within the virion after budding. Therefore, L is not
N-glycosylated in its pre-S region. Later, L molecules
change, in part, their topology by an unknown mechanism, resulting in a
surface exposure of pre-S1 and pre-S2 at HBsAg particles and virions
(13). In contrast, the pre-S2 domain of M protein is translocated
cotranslationally to the endoplasmic reticulum lumen and is, thus,
N-glycosylated. The role of M protein was not clear for a
long time. Recent studies provided evidence, however, that M protein
glycosylation at Asn-4 of the pre-S2 domain as well as
subsequent trimming of this N-glycan play an important role
in the secretion of HBV virions (8, 14-19). Prevention of M protein
N-glycosylation by either disruption of the
Asn-4-X-Ser-6 sequon (16) or by
tunicamycin treatment of HBV-expressing cells (15) suppressed secretion
of HBV particles. Likewise, treatment of cell cultures with inhibitors
of the oligosaccharide-trimming enzymes The pre-S1 and pre-S2 domains of HBsAg display numerous T- and B-cell
epitopes (23, 24), which are capable of inducing neutralizing
antibodies and immune protection (25, 26). Hence, L and M proteins are
also attractive candidates for the development of improved vaccines
against HBV, which might (a) override nonresponsiveness to
the standard HBV vaccine consisting only of S protein (27), (b) allow immunotherapeutic treatment of chronic HBV
infections (28), and (c) prevent selection of escape mutants
with mutations in the S protein (29). Several candidate hepatitis B
vaccines containing pre-S2 sequences have been developed that are
produced in transfected cell cultures (27, 28, 30-33). The usefulness of these vaccines is not completely proven yet, particularly because the anti-pre-S response induced by these vaccines in human recipients is rather weak.3 One reason
for this observation may reside in modifications of the pre-S domain.
Since naturally generated HBsAg is the target of neutralizing
antibodies in vivo, knowledge on post-translational modifications of the pre-S2 sequence is important for the design and
evaluation of future hepatitis B vaccines. Current data on N- and O-glycosylation or phosphorylation (8, 34)
of the pre-S2 region were obtained from transfected rodent cell
cultures. In order to initiate a detailed structure analysis of the
in vivo pre-S2 glycosylation, we isolated HBsAg spheres from
the plasma of two chronically infected HBV carriers, released the pre-S
peptides from the HBsAg particles by trypsin digestion, and determined the primary structure of tryptic glycopeptides.
Isolation of HBsAg--
Subviral spherical and filamentous
particles were purified from the sera of two chronically HBV-infected
donors, genotype D (HBsAg subtype ayw2), with a virus titer of about
6 × 109/ml and a HBsAg concentration of 100 µg/ml.
The genotype and the HBsAg subtype were determined by sequencing of the
viral genomes as described previously (35). 18 ml of serum were
ultracentrifuged in a discontinuous sucrose density gradient (15, 25, 35, 45, and 60% (w/w)) in TNE buffer (20 mM Tris-HCl, pH
7.4, 140 mM NaCl, 1 mM EDTA) for 15 h at
25,000 rpm at 10 °C in a TST 28.38 rotor (Beckman, München,
Germany). Fractions containing 20-40% sucrose were analyzed on a 12%
gel by Laemmli SDS-PAGE under reducing conditions and silver-stained.
Fractions with the typical HBsAg protein pattern (Fig. 1B)
were pooled, adjusted to a density of 1.31 g/ml with solid KBr, and
layered for further purification between a KBr density gradient ranging
from 1.16 to 1.34 g/ml. Centrifugation for 36 h and analysis of
fractions with densities of 1.20-1.25 g/ml were performed as described
above. Pooled fractions were desalted and concentrated by
ultrafiltration (Centriplus-100 filter units; Millipore, Eschborn,
Germany) washing three times with TNE buffer. The concentration of the
purified HBsAg was estimated by A280, assuming a
value of 4.3 for 1 mg/ml (36), and by amino acid analysis (see below).
Purified HBsAg was stored at Isolation of Tryptic L and M Protein-derived
Peptides--
Digestion of purified native HBsAg particles (4 mg) was
carried out with trypsin (Sequencing Grade, Roche Molecular
Biochemicals, Mannheim, Germany) in 1.2 ml of TNE buffer for 1 h
at 37 °C using an enzyme:substrate ratio of 1:40 (w/w). Peptides and
particles were separated by ultrafiltration using one Microcon-100
filter unit (Millipore). Tryptic peptides were isolated by
reversed-phase HPLC (rHPLC) on a C18- column (5 µm, 30 nm, 4.6 × 250 mm; Vydac, Hesperia, CA) using 0.1% (v/v) aqueous
trifluoroacetic acid with an acetonitrile gradient (0-60% in 60 min)
and a flow rate of 1 ml/min at 30 °C. Peptides were monitored by
absorption at 220 nm, and fractions were collected semiautomatically.
Peptide-containing fractions were stored at Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass
Spectrometry (MALDI-TOF-MS) of (Glyco)peptides and Released
Oligosaccharides--
Molecular masses of rHPLC-purified peptides were
determined by MALDI-TOF-MS on a Vision 2000 mass spectrometer (Finnigan
MAT, Bremen, Germany). 1 µl of peptide solution (1-5 pmol) was mixed with 1 µl of matrix solution (10 mg of 2,5-dihydroxybenzoic acid/ml of 0.1% (v/v) trifluoroacetic acid, 30% (v/v) acetonitrile) and allowed to air-dry. Ions were generated by irradiation with a pulsed
nitrogen laser (emission wavelength 337 nm; laser power density about
106 watts/cm2), and positive ions were
accelerated and detected in the reflectron and linear mode. For
analysis of released oligosaccharides, 50 pmol of glycans in aequeous
solution were used and analyzed as described above. For calibration of
the peptide mass spectra, human angiotensin and bovine insulin (both
from Sigma, Deisenhofen, Germany) were used as external standards. For
calibration of the mass spectra of released oligosaccharides, the
diantennary oligosaccharide standard NA2
(Gal Amino Acid Analysis--
20-50 ng of protein or peptide were
lyophilized and hydrolyzed in the gas phase over 6 N HCl,
with 0.02% mercaptoethanol, for 24 h at 110 °C. Free amino
acids were dissolved in 20 µl of 0.5 M borate buffer, pH
7.7, derivatized with Fmoc (N-(9-fluorenyl)methoxycarbonyl), and analyzed by rHPLC on a Merck-Hitachi (Darmstadt, Germany) system
composed of an AS-4000 autosampler, a L-6200A pump, a F-1050 fluorescence detector, and a D-6000 interface.
Peptide and Protein Sequencing--
HPLC-purified peptides
(50-100 pmol) or HBV subviral particles after tryptic digestion (about
500 pmol) were amino-terminally sequenced by automated Edman
degradation on an Applied Biosystems (Foster City, CA) pulsed liquid
phase sequencer, model 477A or 471A, under standard conditions.
Phenylthiohydantoin-derivatives of amino acids were identified by an
on-line analyzer, model 120A or 140B (Applied Biosystems), with a
repetitive yield of 92-95%.
N-terminally blocked tryptic peptides were further digested with
chymotrypsin (sequencing grade; Roche Molecular Biochemicals). Resulting products were fractionated by rHPLC and sequenced by Edman
degradation as above.
Liberation of N-Linked Oligosaccharides from
Glycopeptides--
HPLC fractions containing the
N-glycosylated pre-S2 tryptic peptides (20 µg, about 10 nmol) were pooled, lyophilized, resuspended in 150 µl of 20 mM sodium phosphate buffer, pH 7.5, and digested with 100 milliunits of
peptide-N4-(N-acetyl- High pH Anion Exchange Chromatography (HPAEC) and Gel
Filtration--
Separation of released oligosaccharides was carried
out at room temperature on a Dionex (Sunnyvale, CA) BioLC system using a CarboPac PA-100 column (4.6 × 250 mm) in series with a CarboPac PA guard column as described in detail earlier (38). A sodium acetate
(210 mM) gradient (0-100% in 48 min) in 100 mM NaOH was used at a flow rate of 1 ml/min. Fractions (1 ml) were collected and immediately neutralized with 25 µl of 1 M acetic acid. Oligosaccharide-containing fractions were
pooled, lyophilized, and resuspended in water. Desalting was performed
by Bio-Gel P2 (Bio-Rad) chromatography as reported earlier (39).
Carbohydrate Constituent and Methylation Analysis--
The
carbohydrate constituents were analyzed as detailed elsewhere (40). In
short, hydrolysis of liberated oligosaccharides was performed with 4 M trifluoroacetic acid at 100 °C for 4 h; for
hydrolysis of glycoproteins, 0.5 N sulfuric acid in 80%
acetic acid (v/v) was used at 80 °C for 6 h. Following
reduction with sodium borohydride and peracetylation, alditol acetates
were analyzed by capillary gas-liquid chromatography/mass spectrometry
using the instrumentation and microtechniques described earlier (41). For linkage analyses, oligosaccharide alditols were permethylated and
hydrolyzed (42). Partially methylated alditol acetates obtained after
reduction and acetylation were analyzed as above.
On Target Sequential Enzymatic Digestion of Carbohydrates with
Glycosidases in Combination with MALDI-TOF
Analysis--
rHPLC-purified O-glycosylated peptides or
released complex diantennary glycans of the pre-S2 domain were
sequentially digested directly on the MALDI target (43).
Western Blotting, Lectin Analysis, and Immunostaining of HBsAg
Proteins--
HBsAg filaments (0.5-20 µg, untreated and after
digestion with A. ureafaciens sialidase) were separated by
12% denaturating Laemmli SDS-PAGE and blotted onto a polyvinylidene
difluoride membrane (Millipore).
For lectin analysis, the membrane was blocked with 1% (w/v) BSA (1×
crystallized, Sigma) in Tris-buffered saline overnight and incubated
with digoxigenin-labeled peanut agglutinin (PNA, Roche Molecular
Biochemicals) diluted 1:100 in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM MgCl2, 1 mM MnCl2, 1 mM CaCl2
with 1% BSA for 2 h at 37 °C. Thereafter, anti-digoxigenin
antibodies conjugated with horseradish peroxidase (Roche Molecular
Biochemicals) were used at a concentration of 1:200 and developed with
diaminobenzidine-H2O2 substrate (Sigma).
For immunostaining of the blotted proteins, the membrane was blocked in
5% low fat milk powder in phosphate-buffered saline, pH 7.5, and
incubated either with monoclonal anti-mouse antibodies Q19/10 (specific
for the N-glycosylated N terminus of M protein) or MA18/7
(specific for the peptide sequence Asp-Pro-Ala-Phe (pre-S1 20-23) in
the pre-S1 domain of L protein) to a concentration of 0.5 ng/µl in
phosphate-buffered saline with 1% low fat milk powder. Thereafter,
anti-mouse antibodies conjugated with alkaline phosphatase (250 units/ml; Roche Molecular Biochemicals) were used at a dilution of
1:1000, and the membrane was developed with
5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium substrate
(Sigma FastTM, Sigma).
Carboxypeptidase Digestion of Peptides--
125 pmol of
lyophilized C-terminal tryptic (glyco)peptides of the pre-S2 domain
(19-48) were resuspended in 8 µl of 20 mM
ammonium citrate buffer, pH 6.0 according to Ref. 44. Aliquots (1 µl)
of carboxypeptidases P and Y from Penicillium janthinellum
(sequencing grade; Roche Molecular Biochemicals) and yeast (excision
grade; Calbiochem), each diluted to 50 ng/µl with digestion buffer,
were added. Digestion was carried out at 37 °C for different times
(0 min to 2 × 24 h). 15 pmol (1.2 µl) of the digest were
mixed with 1 µl of 2,5-dihydroxybenzoic acid on the target, and
MALDI-TOF analysis was performed as described above.
Quadrupole Time-of-flight (QTOF) Electrospray Ionization (ESI)
Mass Spectrometry of Glycopeptides--
Positive ion ESI-MS and
ESI-MS/MS was performed on a hybrid QTOF electrospray mass spectrometer
(Micromass, Manchester, UK) in the nanospray mode. The ions were
produced in an atmospheric pressure ionization/ESI ion source, using
argon as a collision gas, and were transported to the mass spectrometer
through a hexapole lens for optimal transmission. The nanospray
capillaries were produced in the laboratory in Münster, using a
Kopf vertical pipette puller/model 720 (David Kopf Instruments,
Tojunga, CA). The capillaries were not gold-coated, but an internal
wire electrode was used.
Nanoelectrospray low energy collision-induced dissociation was
performed as described recently (45) with the peptide
19-48 carrying one Gal-GalNAc disaccharide unit. The
sample was dissolved in water to a concentration of 5 pmol/µl and
introduced to the nanospray needle. The collision-induced dissociation
conditions were optimized for the maximal signal intensity of
glycosylated fragments (minimal deglycosylation). For MS/MS sequencing,
the triply charged precursor ion was selected in the quadrupole,
fragmented in the hexapole collision cell, refocused in the radio
frequency-only-hexapole, and extracted orthogonally into the TOF analyzer.
Isolation of the pre-S2 Glycopeptides--
HBV subviral particles
were purified separately from the sera of two well characterized HBV
carriers infected with two different but typical strains of genotype D
virus. In both cases, identical results were obtained. Therefore, only
one set of data is presented. To exclude contamination by glycopeptides
containing Asn-146, native HBsAg particles were incubated with trypsin.
Since the S domain is known to be highly resistant to trypsin, whereas
pre-S cleavage sites are very sensitive (46), only glycopeptides of the
pre-S region were released (Fig. 1).
After removal of the digested HBsAg particles by ultrafiltration,
released peptides were isolated by rHPLC (Fig.
2). Peptides eluting in the interval of
32-50 min were identified by amino acid analysis, MALDI-TOF-MS, and
peptide sequencing (Table I). Compounds
eluting prior to peptide 1 could be neither registered by MALDI-TOF-MS
nor characterized by amino acid and sequence analysis and are,
therefore, assumed to represent nonpeptide contaminants. In parallel,
residual trypsin-treated HBsAg particles (cf. Fig.
1B, lane 3) were also analyzed by
Edman sequencing, demonstrating (a) complete removal of
pre-S tryptic peptides, (b) Arg-48 to be the
most C-terminal cleavage site accessible for trypsin in native
particles, and (c) a molar ratio of about 20% of
pre-S2-containing proteins (M and L proteins) versus 80% of
S proteins in spherical particles.
The N-terminal pre-S2 tryptic peptide (1-16;
cf. Fig. 1C) was found by MALDI-TOF-MS analysis
to be N-terminally acetylated to an extent of about 90% and was,
therefore, not directly accessible to Edman sequencing. Following
further digestion of this glycopeptide with chymotrypsin and
subfractionation of the resulting peptides by rHPLC, two internal
fragments (9-12 and 13-16) were identified by
their molecular masses, and a third (4-8) could be
assigned by Edman degradation (data not shown). The original glycopeptide eluted from the rHPLC column as several successive peaks
(marked as Ia in Fig. 2) due to its heterogeneity in
glycosylation and acetylation and/or partial oxidation of the
N-terminal Met yielding methionine sulfoxide or methionine sulfone
derivatives. Oxidation of Met-1 is indicated by the observation that
the original pre-S2 glycopeptides (1-16) displayed, in
part, additional mass increments corresponding to one or two oxygen
atoms, which were not registered in the case of chymotryptic
subfragments (4-8, 9-12, and
13-16). Furthermore, removal of the N-terminal Met by BrCN
cleavage was not possible, in agreement with literature data (47),
which confirm that oxidized methionine residues are not cleaved by this
treatment. It is not clear, however, whether the oxidation already
occurred in vivo or reflects an artifact produced during
sample handling in the laboratory. In accordance with literature data,
we could further demonstrate that the M protein is
N-glycosylated to 100% in the pre-S2 domain, whereas the L
protein is not glycosylated in this position, as confirmed by the
analysis of the peptide ( Structural Analysis of Pre-S2 N-Glycans--
For carbohydrate
structure analysis, pre-S2 N-glycans were preparatively
released from peptides 1-6 (cf. Fig. 2) by treatment with
peptide-N4-(N-acetyl-
This structure was verified by sequential exoglycosidase digestion in
combination with MALDI-TOF-MS (Fig. 3)
and by methylation analysis. MALDI-TOF-MS spectra of the released
native oligosaccharide fraction displayed three major signals
indicative of a complex-type diantennary N-glycan carrying
zero, one, or two sialic acid residues (Fig. 3A). Sialylated
oligosaccharides and glycopeptides are known to lose sialic acid very
easily under MALDI conditions. Even with 6-aza-2-thiothymine as matrix,
usually less than 50% of sialylated molecules pass through the
reflector intact (48). Therefore, it was not possible to quantify the
degree of sialylation from these experiments. After treatment with
sialidase from A. ureafaciens, peaks reflecting sialylated
components shifted to the first one (Fig. 3B). Subsequent
treatment with
In order to determine the degree of sialylation, the released
oligosaccharides were separated by HPAEC (Fig.
4A), which revealed that 50%
of the glycans contained two sialic acids, 45% contained one, and
about 5% contained no sialic acid substituent. The detailed structure
of the carbohydrate chain is shown in Fig. 4B.
In order to compare pre-S2 N-glycans with the known
structure of the N-glycan present in the S domain (49, 50),
trypsin-treated subviral particles, obtained from sera of two different
patients, from which the pre-S2 segments were completely removed (as
confirmed by SDS-PAGE and Edman sequencing; see Fig. 1C, and
see above), were subjected to carbohydrate constituent and methylation
analysis. In both cases, the results obtained were compatible with the
published diantennary complex-type structure carrying two, one, or zero terminal sialic acids (see Fig. 4B). In detail, Man, Gal,
and GlcNAc were identified as neutral oligosaccharide components by carbohydrate constituent analysis in a molar ratio of 3.0:2.2:3.9. Furthermore, methylation analysis revealed the presence of
3,6-disubstituted Man, 2-substituted Man, 4-substituted GlcNAc,
terminal Gal, and 6-substituted Gal in a molar ratio of about
1.3:2.0:1.5:0.3:1.6, respectively. The published low degree in
fucosylation of about 5%, however, could not be confirmed by the
methods used. Furthermore, we could exclude O-glycosylation
of the S domain due to the absence of GalNAc.
Identification and Structural Analysis of Pre-S2
O-Glycans--
The pre-S2 tryptic peptides (19-48),
identified by amino acid analysis and Edman sequencing, were found to
elute in four different peaks from the rHPLC column (Fig. 2,
brackets IIa and IIb), each one of
which displayed a different molecular mass in MALDI-TOF-MS (see Fig.
5 and Table I). The mass differences were
consistent with the mass increments of monosaccharide components such
as HexNAc, Hex, and NeuAc (cf. Table I). Carbohydrate
constituent analysis of the glycopeptides (fraction IIa peptides in
Fig. 2) confirmed the peptides to be modified by an
O-glycosidically linked glycan, comprising GalNAc and Gal as
neutral monosaccharide constituents. Sialic acid was not detectable by
the procedure used. The extent of glycosylation was calculated from the
peak areas in the rHPLC chromatogram (Fig. 2), resulting in about 40%
to be not O-glycosylated, 5% to contain only one
GalNAc-residue (Tn-antigen), 30% to contain the
disaccharide Gal-GalNAc (Thomsen-Friedenreich antigen, TF-antigen, T-antigen), and about 25% containing the trisaccharide
Neu5Ac-Gal-GalNAc (sialosyl T-antigen) (see Fig. 9).
To elucidate the structure of the O-glycan, glycopeptides
carrying the trisaccharide unit were treated sequentially with
sialidase from A. ureafaciens and O-glycosidase
from D. pneumoniae, specifically releasing
Gal(
In order to verify these conclusions, the O-glycopeptides
were subjected to methylation analysis. The results supported the assignment, that the structure of the sialylated glycan is
Neu5Ac(
In order to allocate the observed O-glycosylation to M
and/or L proteins, HBsAg filaments (cf. Fig. 1B,
lane 4) were separated by SDS-PAGE and subjected
to blotting and lectin analysis or immunostaining, before and after
treatment with A. ureafaciens sialidase, using peanut
agglutinin, specifically binding Gal( Localization of the Pre-S2 O-Glycan--
The sequence of the
pre-S2 peptide (19-48) contains seven serine and three
threonine residues. Using this peptide for prediction of the
O-glycosylation site by the NetOGlyc 2.0 Prediction Server,
which produces neural network predictions of mucin type GalNAc
O-glycosylation sites in mammalian proteins (51), three threonine (Thr-31, -37, and
-38) and two serine residues (Ser-43 and
-47) were predicted as potential O-glycosylation
sites (see Fig. 1C), with Thr-37 having
the highest potential of all. Similar results appeared by using the
complete M or L protein sequences.
In order to chemically identify the site of O-glycosylation,
glycopeptides 15, 16, and 17 as well as the nonglycosylated peptide 19 (see Fig. 2 and Table I) were digested individually with
carboxypeptidases in combination with MALDI-TOF-MS analysis. Treatment
of the unglycosylated peptide 19-48 (fraction 19;
cf. Fig. 2 and Table I) led to a ladder of signals due to
the removal of from one up to 18-20 amino acids.
In the case of the glycopeptides carrying the disaccharide or the
trisaccharide, only up to 10 amino acids were removed, whereas the
peptide modified with GalNAc only allowed the release of up to 11 amino
acids (Fig. 7). These results pointed to
Thr-37 being most likely the O-glycosylation
site, since the carboxypeptidases used cleave only unmodified amino
acids from the C terminus of peptides.
To further substantiate this assignment, the glycopeptide carrying the
Gal-GalNAc disaccharide unit was studied by QTOF analysis (Fig.
8). In the nanospray ESI-QTOF MS/MS
experiment, both carbohydrate and peptide/glycopeptide sequence data
were obtained in a single experiment. The triply charged molecular ion
[M + 3H]3+ at m/z = 1129.7 was used as a
precursor ion. It depicted the general composition of the glycopeptide,
the peptide chain (19-48) with the attached
disaccharide Hex-HexNAc. The predominant fragment ion formation by
the cleavage of the peptide chain was observed to be of the b and y
type, which was essential for the full assignment of the
O-glycosylation site. The N- and the C-terminal sequences were documented by the singly charged b3-b7 and y2-y8 arrays of the peptide fragment ions. The carbohydrate portion was characterized by the disaccharide Hex-HexNAc B-ion at m/z = 366, beside the diagnostic HexNAc glycan ion at m/z = 204. Glycopeptide ions were detected as fully and partially glycosylated
singly, doubly, and triply charged fragment ions. The partial
deglycosylation in the gas phase occurs due to the relative lability of
the glycosidic bonds in comparison with the peptide bonds. However,
enough glycosylated species were detected in order to assign the
Thr-19 (i.e. Thr-37 of the pre-S2
domain) as the sole glycosylation site, according to the sequence
overlapping ions b19 + HexNAc2+ at m/z = 996.9, b19 + HexNAc+ at m/z = 1992.7, y13 + HexNAc2+ at m/z = 817.8, and y13 + HexNAc+ at m/z 1634.6 (see appropriate
enlargements in Fig. 8B). In general, excellent
coverage of the sequence ions was obtained for this rather large
peptide stretch, probably due to the high dynamic range of the QTOF
instrument.
As outlined in the Introduction, pre-S2 N-glycosylation
of HBV M protein as well as proper trimming of the respective
carbohydrate chain appear to play a crucial role in the secretion of
virions. Since the N-glycan of HBV M protein is considered
to be a potential target for antiviral therapy, elucidation of its
exact structure is of high interest. Analyses of the
N-glycans attached to the pre-S2 peptide region, however,
are complicated by the fact that HBsAg particles, usually used as
starting material, contain the S protein in vast excess, which is
itself N-glycosylated, in part, at Asn-146. Hence, it is
difficult to exclude cross-contamination of the pre-S2 glycan fraction
by S protein-derived oligosaccharides. In order to overcome this
problem, pre-S2-derived (glyco)peptides were selectively released from
intact HBsAg particles by trypsin treatment, leaving the S protein
intact. Furthermore, resulting (glyco)peptides were separated by rHPLC
and identified by amino acid and sequence analysis as well as
MALDI-TOF-MS prior to carbohydrate analysis, thus providing for the
first time unambiguous information on the pre-S2
N-glycosylation of M protein obtained from patient-derived HBsAg particles. Carbohydrate structure analyses revealed that pre-S2
N-glycans represented exclusively partially sialylated, diantennary complex-type oligosaccharides. Since the HBsAg particles used have been exposed to serum conditions for varying periods of time,
the possibility cannot be excluded that monosialylated and unsialylated
glycan species may, at least in part, be due to naturally occurring
desialylation. In contrast to data reported for N-glycans of
(a) HBsAg particles obtained from HBV DNA-transfected HepG2.2.15 cells (16), (b) patient-derived HBsAg subtype adw particles (50), or (c) recombinant hepatitis B pre-S2
surface antigen (20), our analyses detected no fucosylated
oligosaccharide species. It is noteworthy that our analytical results
also provided no evidence for the presence of a high mannose- or
hybrid-type glycan, as suggested primarily on the basis of lectin
binding studies (2, 21). The question as to whether the differences in
fucosylation reflect cell type- or donor-specific variations in
glycosylation remains to be investigated.
Amino acid and sequence analyses, as well as MALDI-TOF-MS, of
individual pre-S-derived tryptic and chymotryptic (glyco)peptides revealed that about 90% of the M protein is N-terminally acetylated. Possibly, acetylation of the amino-terminal end protects against proteolytic degradation. This may be particularly relevant for the
pre-S2 domain, which is known to be highly sensitive toward proteolysis
(46). This finding is remarkable in so far that it could be
demonstrated that the recently identified M protein of WHV is not
N-terminally blocked (9).
The third post-translational modification of the pre-S2 domain
comprises O-glycosylation, which has similarly been found to be restricted to the M protein. Although there is evidence that HBV M
protein expressed in mammalian cell culture (8) or WHV M protein (9)
may be O-glycosylated, information on the number of
carbohydrate substituents, their structures, and their linkage position(s) is still lacking. Hence, our results represent the first
example for a detailed structural analysis of HBV M protein O-glycosylation. It is demonstrated that the M proteins
under study carried, at least in part, a single O-linked
carbohydrate substituent, which could be identified as GalNAc Since Thr-37, embedded in a sequence context
favorable for O-glycosylation, is highly conserved in the
pre-S2 domains of HBV genotypes B-F, in all primate HBV
genomes4 including the
recently described virus from the new world woolly monkey (52), and
even in the otherwise highly divergent pre-S2 sequence of WHV,
respective threonine residues might be similarly glycosylated, thus
pointing to an as yet unknown function of potential evolutionary
advantage. The two isolates of HBsAg analyzed in this study are typical
but not totally identical representatives of HBV genotype D2 (53),
which is prevalent in Europe and the Middle East. The complete identity
of the results, even in the proportions of the various glycan
structures, suggests that the detected modifications of pre-S2 may be
present in many, possibly most, HBV isolates. The
O-glycosylation at Thr-37 of pre-S2, however, cannot be essential because genotype A of HBV has no Thr from position
32 to 55 of pre-S2 but contains numerous Ser
residues in this region.
Glycoproteins with O-linked glycans have been found in a
number of enveloped viruses including, for example, the M (E1) protein of murine corona virus, carrying exclusively O-glycans (54), and herpes simplex virus glycoproteins (55), Friend murine leukemia virus glycoprotein 71 (56), respiratory syncytial virus G protein (57),
and Marburg virus glycoprotein (58), all of which contain both
O- and N-glycans. Distinct functions of the
O-linked glycans of viral glycoproteins are still obscure.
It is speculated, however, that they may influence the antigenicity of
the virion surface structure and protect the polypeptide against degradation.
One putative function of the pre-S2 O-glycan may reside in
the partial masking of the respective peptide sequence. This assumption is corroborated by the finding that anti-pre-S2 antibodies are not
readily detectable in recipients of pre-S2-containing vaccines and can
be only transiently registered in patients with acute hepatitis
B.5 In particular, mouse
monoclonal antibodies directed against an epitope involving
Thr-37 or neighboring amino acids have never been
identified so far, whereas mouse antibodies recognizing an epitope
encompassing the N-glycan linked to Asn-4 are
readily available.6 In
addition to modulation of pre-S2 immunogenicity, O-glycans of M protein might also prevent interaction of hepadnaviral surface proteins with cells of the innate immune system or endothelial cells.
In agreement with earlier qualitative data on
N-glycosylation (4), this study definitely proves that HBV L
protein is not N-glycosylated in its pre-S region, thus
supporting the assumption that the pre-S(1 + 2) domain of L protein is
not translocated into the lumen of the endoplasmic reticulum during
biosynthesis (13). The absence of O-glycans in L protein
might suggest that the translocation of the pre-S(1 + 2) domain occurs
in the medial to trans-Golgi or later. Alternatively, the
Thr-37 of pre-S2 may be masked by pre-S1 sequences.
The cytosolic orientation of the pre-S domains of the L protein allows
alternative post-translational modifications such as myristoylation
(59) or phosphorylation as has been reported for the duck hepatitis B
virus (60). The latter modification has also been observed within the
HBV pre-S2 domain, if it is exposed into the cytosol (34). In its
phosphorylated state, L protein of avian hepadnaviruses acts as a
transcriptional activator and mediates intracellular signaling (61).
Structural analyses, performed in this study, provided no evidence for
the presence of phosphorylated pre-S2 (glyco)peptides. It is,
therefore, concluded that either pre-S2 phosphorylation may not occur
in HBV-infected liver cells or phosphorylated L protein may not be
incorporated into secreted HBsAg particles.
1-3)GalNAc
-,
Neu5Ac(
2-3)Gal(
1-3)GalNAc
-, or GalNAc
-residue. The
respective O-glycosylation site was assigned to Thr-37 by
digestion with carboxypeptidases in combination with MALDI-TOF-MS and
by quadrupole time-of-flight electrospray mass spectrometry. Analytical
data further revealed that about 90% of M protein is N-terminally acetylated.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glucosidase I and II
similarly impaired virion secretion (17, 18). The pre-S2
N-glycan mediates an association of the M protein with the
chaperone calnexin, whereas the N-glycan linked to Asn-146
of the S domain is not involved in calnexin binding (8, 16). It is
concluded that proper folding and trafficking of the M glycoprotein,
assisted by calnexin in a carbohydrate-dependent manner,
may play a crucial role in the assembly of virions, whereas secretion
of subviral particles is not prevented by glycosylation- and
trimming-inhibitors (15-19). The pre-S2 N-glycan of the M
protein may be considered as a promising target for antiviral therapy of hepatitis B, because viremia can be suppressed by
-glucosidase I
inhibitors in experimentally infected woodchucks (18). Previous studies
on the structure(s) of the N-glycan at Asn-4 of
HBV M protein provided contradictory data proposing either partially fucosylated, complex-type (16, 20) or high mannose- and hybrid-type species (21). The presence of terminal mannose would have been biologically interesting, since a genetic linkage between polymorphism of the mannose-binding protein and the persistence of HBV infection was
observed (22).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C.
20 °C.
4GlcNAc
2Man
3(Gal
4GlcNAc
2Man
6)Man
4GlcNAc
4GlcNAc; Oxford GlycoSciences, Abingdon Oxfordshire,
United Kingdom) and bovine insulin B (Sigma) were used. The accuracy of
mass determination was about 0.03% for free oligosaccharides and (glyco)peptides.
-glucosaminyl)asparagine
amidase F from Flavobacterium meningosepticum (Roche
Molecular Biochemicals) for 24 h at 37 °C. The digestion was
repeated with 50 milliunits of enzyme. Oligosaccharides were separated
from peptide residues by rHPLC as described previously (37).
-Galactosidase from bovine testes or Diplococcus
pneumoniae,
-N-acetylglucosaminidase from D. pneumoniae, and
-mannosidase from jack beans (all from Roche Molecular Biochemicals) and O-glycosidase from D. pneumoniae (Calbiochem, Bad Soden, Germany) were dialyzed against
20 mM ammonium acetate buffer adjusted to the suggested pH
for each enzyme (i.e. pH 6 for
-galactosidase from
D. pneumoniae and O-glycosidase; pH 5 for
-N-acetylglucosaminidase and
-mannosidase; pH 4 for
-galactosidase from bovine testes) on a floating membrane (Millipore
VS) with a pore size of 0.025 µm. Sialidase from Arthrobacter
ureafaciens (Calbiochem) was redissolved in water and not dialyzed
before usage. 50 pmol (1 µl) of released N-glycans or 2 pmol (0.4 µl) of rHPLC-purified O-glycosylated peptide
(19-48) were mixed with an equal volume of
6-aza-2-thiothymine matrix (Sigma; 5 mg/ml in water) directly on the
MALDI target, air-dried, and analyzed by MALDI-TOF-MS as described
above. Spectra were recorded both in reflectron and linear modes. For
glycosidase digestion, the analyte spot was resuspended in 1 µl of 20 mM ammonium acetate buffer (pH 6.0), and 0.4 milliunits of
sialidase (0.4 µl) was added. The target was incubated overnight in a
moist chamber at 37 °C. Spots were air-dried and directly analyzed
by MALDI-TOF-MS without adding new matrix. Thereafter, the next enzyme
was added, and the reaction was similarly allowed to proceed. The mass
profile was determined after each step of digestion. Thus, in the case of the released N-glycans, four cycles were performed with
the same analyte spot using 0.4 milliunits of sialidase and 0.5 milliunits of
-galactosidase,
-N-acetylglucosaminidase, and
-mannosidase each, and
two cycles were performed in the case of the O-glycosylated peptide using 0.4 milliunits of sialidase and 0.5 milliunits of O-glycosidase.
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Organization of the surface proteins and the
pre-S2 domain from HBV. A, schematic organization of
the domain structure of HBV surface proteins. N- and
O-glycosylation sites are shown; parenthesis
indicate partial glycosylation. B, protein pattern of
purified HBsAg observed in SDS-PAGE after silver staining. S, M, and L
proteins show the indicated apparent molecular masses in kDa.
St, molecular weight protein marker; lane
1, 1.8 µg of HBsAg (mainly spherical particles), purified
by sucrose density gradient centrifugation and KBr flotation;
lane 2, of the sample volume as in
lane 1 after 10-fold concentration and desalting
by ultrafiltration; lane 3, 3.2 µg of HBsAg
after digestion with trypsin and removal of pre-S peptides by
ultrafiltration; lane 4, protein pattern of
purified HBsAg filamentous particles containing higher M and L protein
quantities than spheres. C, amino acid sequences of the
pre-S2 domains of the L and M proteins deduced from sequencing of the
HBV-DNA isolated from the two chronically infected patients. The
arrows indicate trypsin cleavage sites; glycosylated amino
acids as shown in this study are marked by boldface
letters; asterisks qualify predicted
O-glycosylation sites.
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Fig. 2.
Separation of tryptic peptides derived from
native HBsAg particles. Peptides were fractionated by rHPLC on a
C18-column (5 µm; 4.6 × 250 mm), using a gradient
of acetonitrile in 0.1% aqueous trifluoroacetic acid as indicated. The
N-terminal pre-S2 glycopeptide from the M protein (pre-S2 positions
1-16) eluted within a time interval of about 5 min, marked
by bracket Ia. The corresponding peptide of the L
protein ( 6 to 16; see Fig. 1C) was
not glycosylated and eluted mainly as peak 11 (bracket
Ib). The unglycosylated C-terminal tryptic peptides of the
pre-S2 domains (positions 19-48) from M and L proteins
eluted as peak 19 (bracket IIb), whereas
bracket IIa indicates the elution positions of
O-glycosylated peptides 19-48. For further
explanations, see also Table I.
Characterization and assignment of tryptic peptides from HBsAg after
separation by rHPLC
6 to 16)
was also partially oxidized but not glycosylated at Asn-4.
The tryptic peptide 19-48 of the pre-S2 domain was found
to be partially O-glycosylated. Peaks 12-14 were not
assigned.
6 to 16;
cf. Fig. 1, peptides 8 and 11 in Fig. 2, and Table I). By a
similar line of evidence, the pre-S2 tryptic peptide
(19-48) was found to be partially modified by
O-glycosylation (see below).
-glucosaminyl)asparagine
amidase F and isolated by rHPLC. Neutral carbohydrate constituent
analysis revealed a molar ratio of GlcNAc, Man, and Gal of 3.4:3.0:2.2,
which is typical for diantennary complex-type N-glycans.
Sialic acid residues were not registered by the method employed.
-galactosidase from D. pneumoniae, cleaving only Gal(
1-4) linkages, caused a shift in the molecular mass approaching 324 Da, demonstrating the release of two hexose residues (Fig. 3C). Further digestion with
-N-acetylglucosaminidase from D. pneumoniae,
known to split exclusively GlcNAc(
1-2) bonds, resulted in the loss
of two GlcNAc residues and corresponding mass shifts of roughly 406 Da
(Fig. 3D), yielding the molecular mass of the common
pentasaccharide core of N-linked oligosaccharides. It is
noteworthy that these experiments were performed with only 50 pmol of
released N-glycans in a single spot directly on the MALDI
target without intermediate purification or desalting procedures. The
results were confirmed by methylation analysis of the total fraction of
released N-glycans, which demonstrated the presence of
3,6-disubstituted Man, 2-substituted Man, 4-substituted GlcNAc, terminal Gal, and 6-substituted Gal in a molar ratio of about 0.8:2.0:1.3:0.3:1.3, respectively.
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Fig. 3.
MALDI-TOF mass spectra of the products of
sequential exoglycosidase digestion. A, native
oligosaccharide fraction (about 50 pmol); B-D, products
obtained after digestion with 0.4 milliunits of sialidase from A. ureafaciens (B), 0.5 milliunits of -galactosidase
from D. pneumoniae (C), and 0.5 milliunits of
-N-acetylglucosaminidase from D. pneumoniae
(D). All digestions were carried out directly on the MALDI
target in a moist chamber at 37 °C overnight. Neutral
oligosaccharides are recorded as [M + Na]+ and [M + K]+ pseudomolecular ions (A-D); acidic
oligosaccharides are registered as [M
H + 2Na]+
and [M
2H + 3Na]+ in the case of glycans with one
and two sialic acids (A). Mass spectra were recorded in the
positive ion reflectron mode using 6-aza-2-thiothymine as matrix.
,
GlcNAc,
, Man;
, Gal;
, Neu5Ac.
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Fig. 4.
Separation of oligosaccharides released from
Asn-4 of the M protein by HPAEC and structures of the HBV
N-glycans. A, released oligosaccharides, obtained after
treatment of isolated glycopeptides with
peptide-N4-(N-acetyl- -glucosaminyl)asparagine
amidase F, were fractionated by HPAEC using a CarboPac PA-100 column
(4.6 × 250 mm) and a gradient of sodium acetate in 0.1 M NaOH as indicated. Oligosaccharides were monitored by
pulsed amperometric detection. B, structures proposed for
the N-linked carbohydrate chains of the pre-S2 and S domains
of HBsAg.
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Fig. 5.
MALDI-TOF mass spectra of the rHPLC fractions
containing the tryptic peptide 19-48 of the pre-S2
domain. Aliquots of rHPLC-purified peptide fractions
(i.e. peak 19 (A), peak 17 (B), peak
16 (C), and peak 15 (D) (cf. Fig. 2
and Table I)) were analyzed by MALDI-TOF-MS. Mass spectra were recorded
in the positive ion reflectron mode using 2,5-dihydroxybenzoic acid as
matrix. (Glyco)peptides are recorded mainly as [M + H]+
pseudomolecular ions. Symbols are as described in the legend
to Fig. 3. , GalNAc. For comparison with molecular masses see Fig.
9A.
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Fig. 9.
O-Glycosylation of M proteins in
the pre-S2 domain. A, extent of
O-glycosylation of the peptide 19-48 and
calculated average masses of the different glycopeptides. B,
summarized structure of the O-glycans linked to the peptide
19-48 of M proteins, proposed on the basis of data
obtained by exo- and endoglycosidase digestion, methylation analysis,
rHPLC, and MALDI-TOF as well as QTOF mass spectrometry.
1-3)GalNAc
-chains from Ser/Thr residues, directly on the
MALDI target. Resulting products were analyzed by MALDI-TOF-MS. The
results revealed mass shifts of about 293 and 365 Da, reflecting the
release of one sialic acid and one Gal(
1-3)GalNAc
unit, respectively, yielding the molecular mass of the unsubstituted peptide
(data not shown; see also Ref. 43). The glycopeptide containing only
the disaccharide unit was similarly susceptible to digestion with
O-glycosidase.
2-3)Gal(
1-3)GalNAc
-Ser/Thr, i.e. the so
called sialosyl-T-antigen (see Fig. 9B). The assumption that
the reducing GalNAc-residue is
-glycosidically linked is based on
the reported substrate specificity of the enzyme
O-glycosidase, whereas the
-anomeric configuration of
galactosyl residues was confirmed by on target digestion with
-galactosidase from bovine testes in combination with MALDI-TOF-MS
(data not shown). Notably, sialic acid was found to be linked to C-3 of
the subterminal Gal, although A. ureafaciens sialidase is
known to release preferentially (
2-6)-linked sialic acid residues.
Possibly, the kinetic properties of this enzyme are influenced by the
presence of matrix during on target digestion of the glycopeptide.
1-3)GalNAc-units or specific
monoclonal anti-HBsAg antibodies directed against M or L proteins. The
results revealed both M protein subspecies to be
O-glycosylated, whereas L proteins did not react with the lectin, demonstrating that this type of O-glycosylation is
obviously a specific feature of the HBV M protein (Fig.
6).
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Fig. 6.
Localization of O-glycans to
M proteins. The assignment was performed by lectin analysis of
HBsAg filaments with the lectin peanut agglutinin (PNA).
Purified antigens were either left untreated or digested with sialidase
before subjection to 12% SDS-PAGE and Western blotting. A,
20 µg of blotted proteins of filaments were incubated with the lectin
peanut agglutinin; B, immunostaining of 0.5 µg of blotted
HBsAg proteins with the monoclonal pre-S1-specific antibody MA18/7;
C, immunostaining with an M protein-specific antibody
Q19/10. D, protein pattern observed by silver staining after
Western blotting of 20 µg HBsAg filaments.
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Fig. 7.
MALDI-TOF mass spectra of
O-glycosylated peptide 19-48 before and
after digestion with carboxypeptidases. A, 15 pmol of
native glycopeptide carrying one GalNAc residue (peak
17 in Fig. 2 and Table I); B, product obtained
after extensive digestion (2 × 24 h) with carboxypeptidases
P and Y from P. janthinellum and yeast. 11 amino acids are
removed from the C terminus of the peptide, suggesting
Thr-37 to be the linkage position of the glycan chain
(cf. Fig. 1C). Conditions for MALDI-TOF-MS were
as in Fig. 5.
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Fig. 8.
ESI-QTOF analysis of the pre-S2 peptide
19-48 carrying a Gal-GalNAc disaccharide.
A, spectrum obtained by (+) nano-ESI-MS/MS of the
Gal-GalNAc-substituted glycopeptide 19-48 in the QTOF
mode using the triply charged molecular ion at m/z = 1129.7 as a precursor. The fragment ions from the C-terminal
yn and N-terminal bm ions are in accordance
with the peptide sequence and assigned regarding their status of
glycosylation. The linkage position of the O-glycan is
deduced from the fragment ions at the two cleavage sites, b19 + HexNAc
(found as the singly and as the doubly charged ion) as well as y13 + HexNAc (singly and doubly charged) and y13 + HexNAc + Hex (only singly
charged) ions. These two glycopeptide fragment ions overlap at exactly
one potential glycosylation site, i.e. at the
Thr-37, providing direct evidence for glycan
attachment. The Hex-HexNAc sugar chain structure is documented by the
ion at m/z = 366.11 Da, besides the HexNAc diagnostic
ion at m/z = 204.08. B, enlarged mass range
details from the spectrum depicted in A: m/z = 810-1000 (top), m/z = 1550-1660
(middle), and m/z = 1820-2010
(bottom).
DISCUSSION
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-
(Tn-antigen), Gal(
1-3)GalNAc
- (T-antigen), or
Neu5Ac(
2-3)Gal(
1-3)GalNAc
-unit (sialosyl T-antigen).
Although neither the number nor the structures of the
O-glycan(s) in M protein expressed in rodent cell culture (8) were investigated, the size increase of this protein, as evidenced
by SDS-PAGE, was significantly larger than expected for the
O-linked side chain described here. Likewise, the increase in size, due to O-glycosylation, was higher for WHV than for
HBV M protein (9). Possibly, O-glycan substituents exist in
rodent cell-derived M proteins in higher numbers and/or enlarged
structures. Thus, the carbohydrate moieties, antigenicity and
immunogenicity of the pre-S2 domains in certain candidate vaccines may
differ from that of natural M proteins. For exact allocation of the
O-glycosylation site, two different strategies were adopted:
(a) exhaustive digestion of the glycopeptide with
carboxypeptidases in conjunction with MALDI-TOF-MS and (b)
ESI-QTOF-MS/MS analysis. Although the C-terminal half of the pre-S2
region is rich in Ser and Thr, both techniques revealed a specific
substitution of the protein at Thr-37. This is consistent
with recent findings (8) that M protein expressed in COS-7 cells is
O-glycosylated between positions 27 and
47 of its amino acid sequence. Using the NetOGlyc 2.0 Prediction Server (51) for prediction of potential
O-glycosylation sites, five Ser/Thr residues of the
C-terminal pre-S2 peptide sequence are highlighted, with
Thr-37 having the highest potential. It is interesting to
note that in vivo only this amino acid is, in fact,
glycosylated. Exact quantitative estimation of the degree of M protein
O-glycosylation is not possible, since unglycosylated
peptides 19-48 may be derived from both M and L proteins.
Preliminary analyses of pre-S2-derived (glyco)peptides from M protein,
isolated by preparative SDS-PAGE and in situ trypsin
digestion, similarly revealed the presence of unglycosylated peptides
19-48, ruling out a complete O-glycosylation of
this protein (data not shown).
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ACKNOWLEDGEMENTS |
---|
We thank G. Caspari and R. Repp for providing HBV plasma, A. Berting for HBV DNA sequencing; S. Broehl and P. Dotzauer for technical assistance; H.-G. Welker and M. Dreisbach for Edman sequencing and amino acid analysis; and W. Mink, P. Kaese, and S. Kühnhardt for carbohydrate constituent and methylation analysis.
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FOOTNOTES |
---|
* This work was supported by Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 535, Projects Z1 (to R. G. and D. L.) and A2 (to W. H. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This paper is in partial fulfillment of the requirements for the
degree of Dr. Rer. Physiol. at Marburg University.
To whom correspondence should be addressed: Institute of
Biochemistry, University of Giessen, Friedrichstrasse 24, D-35392 Giessen, Germany. Tel.: 49-641-99-47400; Fax: 49-641-99-47409; E-mail:
Rudolf.Geyer{at}biochemie.med.uni-giessen.de.
2 Amino acid residues derived from the pre-S2 region are underlined throughout.
3 W. H. Gerlich, unpublished observations.
4 S. Schaefer and W. H. Gerlich, unpublished observations.
5 W. H. Gerlich, unpublished results.
6 D. Sobotta and W. H. Gerlich, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
HBV, human hepatitis
B virus;
BSA, bovine serum albumin;
ESI, electrospray ionization;
HBsAg, hepatitis B virus surface antigen;
Hex, hexose;
HexNAc, N-acetylhexosamine;
HPAEC, high pH anion exchange
chromatography;
MALDI, matrix-assisted laser desorption/ionization;
TOF, time-of-flight;
MS, mass spectrometry;
QTOF, quadrupole
time-of-flight;
HPLC, high-performance liquid chromatography;
rHPLC, reversed-phase HPLC;
S, M, and L proteins, small, middle and large
hepatitis B surface proteins, respectively;
sialosyl T-antigen, Neu5Ac(2-3)Gal(
1-3)GalNAc
-Ser/Thr;
T-antigen, Thomsen-Friedenreich antigen Gal(
1-3)GalNAc
-Ser/Thr;
Tn-antigen, GalNAc
-Ser/Thr;
WHV, woodchuck hepatitis B
virus;
PAGE, polyacrylamide gel electrophoresis.
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