1 Institute of Biochemistry, University of Giessen, Friedrichstrasse 24, D-35392 Giessen, Germany
2 Institute of Medical Virology, University of Giessen, Frankfurter Strasse 107, D-35392 Giessen, Germany
Correspondence
Wolfram H. Gerlich
Wolfram.H.Gerlich{at}viro.med.uni-giessen.de
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ABSTRACT |
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Additional experiments are supplied as supplementary data, available in JGV Online.
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INTRODUCTION |
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The function of M protein is still not clear. However, M protein glycosylation at Asn-4 of pre-S2 and subsequent trimming of this N-glycan are important for the secretion of HBV (Block et al., 1994; Lu et al., 1995
; Sheu & Lo, 1994
). Proper folding and trafficking of the M glycoprotein, assisted by calnexin in a carbohydrate-dependent manner (Mehta et al., 1997
; Werr & Prange, 1998
), may support the assembly of virions. The pre-S2 N-glycan of the M protein has been considered as a promising target for antiviral therapy of hepatitis B, because viraemia could be suppressed by
-glucosidase I inhibitors in experimentally infected woodchucks (Block et al., 1998
).
Our previous studies on the pre-S2 N-glycan revealed the presence of partially sialylated diantennary complex-type oligosaccharides. Furthermore, Thr-37 of this protein is, in part, O-glycosylated by a Neu5Ac(23)Gal(
13)GalNAc
(sialyl-T-antigen), Gal(
13)GalNAc
(Thomsen-Friedenreich antigen, T-antigen) or GalNAc
residue (Tn-antigen) (Schmitt et al., 1999
). O-glycosylation is more extensive in WHV M surface protein (Tolle et al., 1998
) and in HBsAg particles secreted from non-hepatic cells (Werr & Prange, 1998
) or in particles carrying certain pre-S2 deletions (Tai et al., 2002
). The exact structures of these O-glycans are not known.
The pre-S2 domain of HBsAg displays several T- and B-cell epitopes (Milich et al., 1990; Sobotta et al., 2000
), which are capable of inducing immune protection (Emini et al., 1989
; Itoh et al., 1986
; Neurath et al., 1986a
). Hence, M protein is a vaccine against HBV, which might (i) override non-responsiveness to the standard HBV vaccine consisting only of S protein (Zuckerman et al., 1997
), (ii) allow immunotherapeutic treatment of chronic HBV infections (Michel et al., 2001
) and (iii) prevent selection of escape mutants with mutations in the S protein (Carman, 1997
). Hepatitis B vaccines containing pre-S2 have been generated in transfected yeast cells (DeWilde et al., 1991
), mouse fibroblasts (Young et al., 2001
) or Chinese hamster ovary cells (Jungers et al., 1994
; Leroux-Roels et al., 1997
; Shapira et al., 2001
; Young et al., 2001
), and studied with variable success in humans. Pre-S2-containing HBsAg from yeast cells was found to be heavily modified by O-glycans and virually ineffective as vaccine (DeWilde et al., 1991
). Studies on the O-glycosylation of pre-S2-containing vaccines expressed in rodent cell cultures have not been reported but, in view of the data observed by Werr & Prange (1998)
and Tolle et al. (1998)
, it is likely that these vaccines are also more strongly O-glycosylated than natural HBsAg.
The usefulness of the pre-S2 component in these vaccines is not yet proven, particularly because the anti-pre-S response induced by these vaccines in human recipients is rather weak and difficult to measure (W. H. Gerlich, unpublished). One reason for this observation may reside in the modifications by O-glycans. Since natural HBsAg is the target of neutralizing antibodies in vivo, knowledge on potential modification of the pre-S2 sequence in vivo would be important for the design and evaluation of future hepatitis B vaccines.
The O-glycosylation patterns of patient-derived HBsAg have only been studied for HBV genotype D, but eight genotypes (AH) of human HBV and seven or more in apes exist (Bartholomeusz & Schaefer, 2004). All contain an O-glycosylation motif around Thr-37 of pre-S2, but genotype A does not have this motif. In order to compare the in vivo pre-S2 glycosylation patterns of M protein from HBV genotypes A, C and D (Fig. 1c
), HBsAg particles were isolated from the plasma of chronically infected HBV carriers. Pre-S2 (glyco)peptides were released by trypsin digestion and subjected to carbohydrate structure analysis.
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METHODS |
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Isolation of tryptic L and M protein-derived peptides.
Digestion of purified native HBsAg particles (between 1 and 4 mg) was carried out with trypsin as described previously (Schmitt et al., 1999). In the case of genotype D, separation of tryptic peptides by reversed-phase HPLC was also carried out as published previously (Schmitt et al., 1999
), whereas peptides derived from genotypes C (2 mg) or A (1 mg) were separated employing slightly different conditions: C18-column (particle size 3 µm, pore size 9 nm, 2·1x250 mm; MZ Analysentechnik), 0·1 % (v/v) aqueous trifluoroacetic acid (TFA) with an acetonitrile gradient (060 % in 60 min) and a flow rate of 120 µl min1 at 28 °C.
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 on a Vision 2000 mass spectrometer (Finnigan MAT, Bremen). For analysis of released oligosaccharides, 50 or 15 pmol of glycans in aqueous solution was used for genotypes D and C or genotype A, respectively (Schmitt et al., 1999). For external calibration of the peptide mass spectra, human angiotensin and bovine insulin (both from Sigma) or Sequazyme (Perseptive Biosystems) peptide mass standards were used.
Liberation of N-linked oligosaccharides from glycopeptides.
rHPLC fractions containing the N-glycosylated pre-S2 tryptic peptides (20 µg, i.e. about 10 nmol for genotype D) were digested with peptide-N4-(N-acetyl--glucosaminyl)asparagine amidase F (PNGase F) from Flavobacterium meningosepticum (Roche Molecular Biochemicals) as described previously (Schmitt et al., 1999
). Glycopeptides derived from genotypes C (6·5 µg) and A (1·6 µg) were digested with recombinant PNGase F from Escherichia coli (Roche Molecular Biochemicals) as described above using 25 mU enzyme per µg substrate. Oligosaccharides were separated from peptide residues by rHPLC as described previously (Pfeiffer et al., 1993
).
High-pH anion-exchange chromatography (HPAEC).
Separation of released oligosaccharides (genotype D 2 µg, genotype C 1 µg and genotype A 0·5 µg) was carried out at room temperature on a Dionex (Sunnyvale) BioLC system using a CarboPac PA-100 column (4·6x250 mm) in series with a CarboPac PA guard column as described in detail previously (Schmitt et al., 1999). A sodium acetate (210 mM) gradient (0100 % in 48 min) in 100 mM NaOH was used at a flow rate of 1 ml min1. Oligosaccharides were monitored by pulsed amperometric detection, employing detection ranges of 300, 100 and 30 nA for genotypes D, C and A, respectively.
Peptide sequencing, carbohydrate constituent and methylation analysis, Western blotting, lectin analysis and immunostaining of HBsAg proteins, on-target sequential enzymic digestion of glycopeptides with glycosidases in combination with MALDI-TOF analysis, carboxypeptidase digestion of peptides and nano-LC-ESI-IT-MS and -MS/MS of glycopeptides are described in supplementary material in JGV Online.
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RESULTS |
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Structural analysis of pre-S2 N-glycans
Pre-S2 N-glycans were released from the amino-terminal glycopeptides 116 by treatment with PNGase 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 for genotype D, 3·3 : 3·0 : 3·4 for genotype C and 3·6 : 3·0 : 1·6 for genotype A, which is typical for complex-type N-glycans. Sialic acid residues were not registered by the methods employed.
The presence of diantennary complex-type glycans (Gal4GlcNAc
2Man
3[Gal
4GlcNAc
2Man
6]Man
4GlcNAc
4GlcNAc) was substantiated for genotype D by sequential exoglycosidase digestion in combination with MALDI-TOF-MS and by methylation analysis for all three genotypes.
In order to determine the degree of sialylation, the released oligosaccharides were separated by HPAEC (Fig. 3), which revealed differences in sialic acid substitution for the genotypes examined. N-Glycans of genotype D particles comprised disialylated (50 %), monosialylated (45 %) as well as unsialylated (5 %) oligosaccharide species, the latter of which were not found in the case of genotype C- and A-derived N-linked sugar chains. Instead, pre-S2 glycans from subviral particles of these genotypes were dominated by disialylated species, amounting to 88 (genotype C) and 93 % (genotype A) of total oligosaccharides. Hence, the N-glycans of the different genotypes examined differed in the degree of sialylation and, in part, in the linkage position of sialic acid.
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To elucidate and compare the structures of the O-glycans of genotype D and C subviral particles, glycopeptides carrying the trisaccharide unit were treated sequentially with sialidase from Arthrobacter ureafaciens and O-glycosidase from Diplococcus pneumoniae, specifically releasing Gal(13)GalNAc
chains from Ser/Thr residues, directly on the MALDI target (Fig. 4
). Resulting products were analysed by MALDI-TOF-MS. The results revealed mass shifts of about 291 and 365 Da, reflecting the release of one sialic acid and one Gal(
13)GalNAc
unit, respectively, yielding the molecular mass of the unsubstituted peptide. The glycopeptides containing only the disaccharide unit were similarly susceptible to digestion with O-glycosidase. Furthermore, the galactose residue could be removed by treatment with
-galactosidase from bovine testes, known to release preferentially (
13)-linked galactose (data not shown).
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In order to allocate the observed O-glycans to M and/or L proteins, HBsAg filaments of genotypes A, C and D were separated by SDS-PAGE and either silver stained or subjected to blotting and lectin analysis or immunostaining (Fig. 5), before and after treatment with A. ureafaciens sialidase alone or in combination with O-glycosidase, using peanut agglutinin (PNA), specifically binding the T-antigen, or a specific monoclonal anti-M protein antibody (Q19/10). Genotype A proteins did not react with PNA (Fig. 5a
, part A), confirming that this genotype is not O-glycosylated. Native genotype C and D M proteins (lanes Ø) exhibited only slight signals for lectin binding, which increased after digestion with sialidase (lanes S) and weakened strongly after additional treatment with O-glycosidase (lanes SO). The results for genotypes C and D revealed both M protein subspecies to be O-glycosylated. In contrast to our previous report (Schmitt et al., 1999
), clearly detectable binding of the lectin was also found with the L proteins from genotype D and more weakly with genotype C. This may be explained by more sensitive detection by the lectin. Monoclonal antibody Q19/10 recognition of M proteins was partly diminished after digestion with glycosidases in the case of genotypes A and C, suggesting a contribution of the sialic acid residues in the N-glycans to its epitope (Fig. 5b
). The presence of all HBsAg proteins in the preparation was verified by silver staining (Fig. 5c
). Treatment with sialidase decreased the size and heterogeneity of the M and L proteins in SDS-PAGE as expected.
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In order to identify the site of O-glycosylation chemically, glycopeptides I and II or I, II and III of the M proteins from genotypes C or D, respectively, as well as the non-glycosylated peptides IV (Figs 2a, b) were digested individually with carboxypeptidases. Resulting products were analysed by MALDI-TOF-MS. Treatment of the unglycosylated peptides 1948 generated a ladder of signals due to the removal of between 1 and 20 aa. In the case of genotype D glycopeptides carrying the disaccharide or the trisaccharide, only up to 10 aa were removed, whereas the peptide substituted solely by GalNAc allowed the release of up to 11 aa (Schmitt et al., 1999
). In the case of the respective glycopeptides from genotype C virus, exhaustive carboxypeptidase digestion stopped after the removal of 9 aa (data not shown). These results pointed to Thr-37 for genotype D and Thr-38 for genotype C being, most likely, the O-glycosylation site, since the carboxypeptidases used release only unmodified amino acids from the carboxy terminus of peptides.
To substantiate further this assignment, the glycopeptide carrying the T-antigen derived from genotype D was studied by nanospray ESI-MS/MS analysis as reported earlier (Schmitt et al., 1999), thus identifying Thr-37 as O-glycosylation site. In the case of rHPLC-purified pre-S2 glycopeptides (1948) derived from genotype C virus, analogous nano-LC-ESI-IT-MS and -MS/MS analyses were performed, yielding similar results for glycopeptides carrying a Gal-GalNAc disaccharide or a Neu5Ac-Gal-GalNAc trisaccharide unit. Data obtained for the latter peptide are presented as a Supplementary Figure. Sufficient glycosylated species were recovered to allow the exclusive assignment of Thr-37 of the pre-S2 domain as glycosylation site.
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DISCUSSION |
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Amino acid and sequence analyses as well as MALDI-TOF-MS of individual pre-S-derived (glyco)peptides revealed at least about 90 % of the M protein to be acetylated at the amino terminus. This acetylation of the amino terminus that is observed in all genotypes examined may protect against proteolytic degradation, which may be particularly relevant for the pre-S2 domain, which is known to be highly sensitive towards proteolysis (Stibbe & Gerlich, 1983). In this context, it is interesting to note that M protein of WHV is not blocked at the amino terminus (Tolle et al., 1998
).
The third known post-translational modification of the pre-S2 domain comprises O-glycosylation. Genotypes C and D carry, at least in part, a single O-linked carbohydrate substituent, which could be identified as Tn-antigen, T-antigen or sialyl-T-antigen. The degree of O-glycosylation in genotypes C and D differed in so far as that the genotype D in the M protein was glycosylated to a higher extent (63 %) than in the respective genotype C polypeptide (40 %). Genotype A HBsAg particles were not found to be O-glycosylated at all.
For exact allocation of the O-glycosylation site in natural HBsAg, two different strategies were used: (i) exhaustive digestion of the glycopeptide with carboxypeptidases in conjunction with MALDI-TOF-MS and (ii) tandem mass spectrometry. Only the latter technique unambiguously revealed a specific substitution at Thr-37. This is in agreement with data from Werr & Prange (1998) 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 (Hansen et al., 1998
) for prediction of potential O-glycosylation sites, for genotype D, Thr-37 had the highest score (Fig. 1c
), whereas, for genotype C, the highest probability was predicted at Thr-38. Thus, the assignments turned out to be correct in vivo only in the case of genotype D. Likewise, the data obtained by treatment with carboxypeptidases were misleading, since, for genotype C, results pointed to Thr-38 as the most likely glycosylation site, possibly due to steric hindrance and/or specific features of the peptide sequence. Tandem mass spectrometry with an ion-trap instrument not only allowed the assignment of the O-glycosylation site but also ensured a good coverage of the peptide sequence.
Exact quantitative estimation of the degree of M protein O-glycosylation is not possible since unglycosylated peptides 1948 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 1948, ruling out a complete O-glycosylation of this protein for genotype D (data not shown).
Since Thr-37, embedded in a sequence context favourable for O-glycosylation, is highly conserved in the pre-S2 domains of HBV genotypes BF, in all primate hepatitis B virus genomes (Bartholomeusz & Schaefer, 2004) including the virus from the New World woolly monkey (Lanford et al., 1998
) and even in the otherwise highly divergent pre-S2 sequence of WHV, respective threonine residues might be similarly glycosylated. The O-glycosylation at Thr-37 of pre-S2, however, cannot be essential for HBV genotype A, which exhibits no Thr within the range from aa 32 to 55, but contains numerous Ser residues in this region, which, as shown in this study, are not O-glycosylated.
Remarkable was the detection of O-glycan in L protein. This finding suggests that the post-translational translocation of its internal pre-S domain to the surface occurs at a site where O-glycosylation is possible, probably in the Golgi apparatus before secretion. Previously, we reported that the pre-S can be translocated at a mildly acidic pH in vitro (Bruss et al., 1994). Such conditions may possibly also occur in vivo during export of HBsAg particles. Due to the small amounts of L protein, a more accurate analysis of the O-glycan in the L proteins was not possible.
Although the number or structures of the O-glycan(s) occurring in M protein expressed in rodent cell culture (Werr & Prange, 1998) has not been investigated so far, the size increase of this protein, as evidenced by SDS-PAGE, was significantly larger than expected for the type of O-linked side chain described here. Likewise, the increase in size, due to O-glycosylation, was higher for WHV than for HBV M protein (Tolle et al., 1998
). From these data, it may be concluded that O-glycanic substituents exist in rodent cell-derived M proteins in larger numbers and/or as enlarged structures. Consequently, the carbohydrate contents of the pre-S2 domains in the two studied genotype D (Jungers et al., 1994
; Young et al., 2001
) vaccines may be higher than in natural M proteins, which may cause lower immunogenicity (DeWilde et al., 1991
). Recently, two pre-S2 deletion variants of genotype C have been identified in human hepatocellular carcinoma patients, which showed a reduced HBsAg secretion. The secreted portion of HBsAg contained a larger hypermodified M protein (Tai et al., 2002
), the nature of which is most likely extensive O-glycosylation, whereas the intracellular M protein had the expected size of the polypeptide without O-gylcosylation (P.-C. Tai, P. K. Chua, W. H. Gerlich and C. Shih, unpublished). In their studies on M protein (genotype D), Werr & Prange (1998)
also found expression in COS cells, the secreted portion of the protein to be <1?show=[to]>O-glycosylated, whereas the intracellular portion was smaller and not O-gylcosylated. Thus, O-glycosylation may support secretion of certain HBV genotypes or variants.
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ACKNOWLEDGEMENTS |
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Received 23 December 2003;
accepted 25 February 2004.
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