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

Abstract

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(a2-3)Gal(b1-3)GalNAca-or Gal(b1-3)GalNAca-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.

N-Linked Glycosylation at an Appropriate Position in the Pre-S2 Domain Is Critical for Cellular and Humoral Immunity against Middle HBV Surface Antigen

“…Therefore, MHBs, which also carries the pre-S2 sequences, has been considered to offer an improvement over the HBV vaccine currently in use. The N-linked glycosylation site (Asn4) within the pre-S2 domain is conserved among all serotypes of HBV, which reflects an important role for glycosylation at this position (Schmitt et al 2004). In the present study, we analyzed the properties of modified MHBs with respect to assembly, secretion, antigenicity, and immunogenicity, using transient expression in 293T cells and immunization of mice.…”

“…The O-linked glycosylation motif is not conserved across all HBV genotypes of HBV. However, the N-linked glycosylation site is highly conserved across all HBV serotypes, which might reflect its importance (Schmitt et al 2004). MHBs possesses two N-linked glycosylation sites: Asn4 in the pre-S2 domain and Asn146 in the S domain.…”

Host factors involved in hepatitis B virus maturation, assembly, and egress

“…In addition to N-glycans, the preS2 domain of many HBV genotypes contains O-glycans at Thr-37 ( Fig. 1b) [10,19]. Interestingly, the secretion of subviral and viral particles depends on the preS2-linked N-glycans that mediate a productive interaction with calnexin, a key component of the ER quality control machinery [10,20,21].…”

Hepatitis B virus morphogenesis

“…The M protein is N-glycosylated at asn 4 [55] . In addition, the preS2 domain is O-glycosylated in some but not all HBV genotypes [56] . Glycine residue 2 of the L protein is myristylated [57] .…”