Analysis of the pre-S2 N- and O-linked glycans of the M surface protein from human hepatitis B virus.
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(beta1-3)GalNAcalpha-, Neu5Ac(alpha2-3)Gal(beta1-3)GalNAcalpha-, or GalNAcalpha-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.
Schmitt
S
S
Institute of Biochemistry, University of Giessen, Friedrichstrasse 24, D-35392 Giessen, Germany.
Glebe
D
D
Alving
K
K
Tolle
T K
TK
Linder
M
M
Geyer
H
H
Linder
D
D
Peter-Katalinic
J
J
Gerlich
W H
WH
Geyer
R
R
D000595:Amino Acid Sequence; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006020:Glycopeptides / Q000737:chemistry; D006514:Hepatitis B Surface Antigens / Q000737:chemistry*; D006515:Hepatitis B virus / Q000737:chemistry* / Q000276:immunology; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011134:Polysaccharides / Q000032:analysis*; D011498:Protein Precursors / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D006020:Glycopeptides; D006514:Hepatitis B Surface Antigens; D011134:Polysaccharides; D011498:Protein Precursors; C050807:presurface protein 2, hepatitis B surface antigen