Analysis of the Pre-S2 N- and O-Linked Glycans of the M Surface Protein from Human Hepatitis B Virus

Abstract

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(␤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. 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 10 10 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, an...

Structure of pre-S2 N- and O-linked glycans in surface proteins from different genotypes of hepatitis B virus

“…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.…”

“…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: C 18 -column (particle size 3 mm, pore size 9 nm, 2?16250 mm; MZ Analysentechnik), 0?1 % (v/v) aqueous trifluoroacetic acid (TFA) with an acetonitrile gradient (0-60 % in 60 min) and a flow rate of 120 ml min 21 at 28 uC.…”

“…Subviral spherical and filamentous particles were purified from the sera of three chronically HBV-infected donors and quantified as described previously [for genotype D see Schmitt et al, (1999); for genotypes C and A see ]. Serum no.…”

“…Furthermore, Thr-37 of this protein is, in part, O-glycosylated by a Neu5Ac(a2-3)Gal(b1-3)GalNAca (sialyl-T-antigen), Gal(b1-3)GalNAca (Thomsen-Friedenreich antigen, T-antigen) or GalNAca residue (T n -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 second potential N-glycosylation site at Asn-4 of the pre-S2 domain is used only in the M but not in the L protein (Heermann et al, 1984). In addition to N-glycosylation, pre-S2 domains of M proteins from HBV (Schmitt et al, 1999) as well as the related woodchuck hepatitis virus (WHV) (Tolle et al, 1998) are O-glycosylated.…”