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 glycosyla ted 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 th e 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 antivi ral therapy. For characterization of the pre-S2 glycosylation, pre-SE (glyc o)peptides were released from native, patient-derived hepatitis B virus sub viral particles by tryptic digestion, separated from remaining particles, p urified by reversed-phase high performance liquid chromatography, and ident ified by amino acid and N-terminal sequence analysis as well as matrix-assi sted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TO F-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 s ialylated diantennary complex-type oligosaccharides. In addition, the pre-S a domain of M protein, but not that of L protein, was found to be partially O-glycosylated by a Gal(beta 1-3)Gal-NAc alpha-, Neu5Ac(alpha 2-3)Gal(beta 1-3)GalNAc alpha-, or GalNAc alpha-residue. The respective O-glycosylation site was assigned to Thr-37 by digestion with carboxypeptidases in combina tion with MALDI-TOF-MS and by quadrupole time-of-flight electrospray mass s pectrometry, Analytical data further revealed that about 90% of M protein i s N-terminally acetylated.