Structure of pre-S2 N- and O-linked glycans in surface proteins from different genotypes of hepatitis B virus.
The middle-sized (M) surface proteins of hepatitis B virus (HBV) and other orthohepadnaviruses contain a conserved N-glycan in their pre-S2 domain, which is essential for the secretion of viral particles. Recently, we also found O-glycans in the pre-S2 domain of M protein from woodchuck hepatitis virus (WHV) and HBV genotype D. Since the O-glycosylation motif is not conserved in all genotypes of HBV, the glycosylation patterns of HBV genotypes A and C were analysed. Pre-S2 (glyco)peptides were released from HBV-carrier-derived HBV subviral particles by tryptic digestion, purified by reversed-phase HPLC and identified by amino acid and amino-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 all genotypes examined. Pre-S2 O-glycans were characterized by on-target sequential exoglycosidase digestions in combination with MALDI-TOF-MS. The pre-S2 domain of M protein and, to a minor extent, of L (large) protein from HBV genotype C and D was partially O-glycosylated by Neu5Ac(alpha2-3)Gal(beta1-3)GalNAcalpha- or Gal(beta1-3)GalNAcalpha-units at Thr-37 within a conserved sequence context. Genotype A, containing no Thr at position 37 or 38, was not O-glycosylated. Analytical data further revealed that M protein is mostly amino-terminally acetylated in all examined genotypes and that the terminal methionine is partially oxidized. The findings may be relevant for the secretion and the immunogenicity of HBV.
Schmitt
Sigrid
S
Institute of Biochemistry, University of Giessen, Friedrichstrasse 24, D-35392 Giessen, Germany.
Glebe
Dieter
D
Institute of Medical Virology, University of Giessen, Frankfurter Strasse 107, D-35392 Giessen, Germany.
Tolle
Tanja K
TK
Institute of Medical Virology, University of Giessen, Frankfurter Strasse 107, D-35392 Giessen, Germany.
Lochnit
Günter
G
Institute of Biochemistry, University of Giessen, Friedrichstrasse 24, D-35392 Giessen, Germany.
Linder
Dietmar
D
Institute of Biochemistry, University of Giessen, Friedrichstrasse 24, D-35392 Giessen, Germany.
Geyer
Rudolf
R
Institute of Biochemistry, University of Giessen, Friedrichstrasse 24, D-35392 Giessen, Germany.
Gerlich
Wolfram H
WH
Institute of Medical Virology, University of Giessen, Frankfurter Strasse 107, D-35392 Giessen, Germany.
D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D005838:Genotype; D006020:Glycopeptides / Q000737:chemistry; D006514:Hepatitis B Surface Antigens / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D006515:Hepatitis B virus / Q000235:genetics*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011134:Polysaccharides / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics
D006020:Glycopeptides; D006514:Hepatitis B Surface Antigens; D009844:Oligosaccharides; D010446:Peptide Fragments; D011134:Polysaccharides; D014759:Viral Envelope Proteins; D014357:Trypsin