PMC:7108637 / 27430-31260 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7108637","sourcedb":"PMC","sourceid":"7108637","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7108637","text":"For decades very little information has been available regarding site-specific O-glycosylation of viral proteins. Some of the first described virus-derived site-specific O-glycans were on isoform M of HBV surface antigen purified from patient-derived viral particles (Schmitt et al. 1999). The O-glycosylation site was identified by combining MALDI analysis of exoglycosidase-treated glycopeptide and Edman sequencing of the underlying peptide. The position of the glycan attachment site was deduced by carboxypeptidase digestion and confirmed by collision-induced dissociation tandem mass spectrometry, representing some of the early glycoproteomic experiments of viral proteins (Schmitt et al. 1999). The recent advances in mass spectrometry-based proteomics have resulted in numerous studies addressing O-glycosylation of individual viral glycoproteins. O-glycoproteomic analyses have been performed for several recombinant or isolated viral glycoproteins, including HIV-1 gp120, influenza A virus HA1, HCV E2, HSV-1 gC and Hendra virus glycoprotein G (Colgrave et al. 2012; Brautigam et al. 2013; Go et al. 2013; Yang W et al. 2014; Norden et al. 2015; Stansell et al. 2015). Recombinantly expressed gp120 and HA1 were glycosylated at a single position each, which in both cases was modified with core 1 or core 2 elongated O-glycans (Stansell et al. 2015). Interestingly, site occupancy was much lower in recombinant gp140 undergoing proteolytic cleavage to gp120, assuming equivalent ionization efficiencies of nonmodified and glycosylated peptides. Recombinant gp140 possessed shorter less sialylated, predominantly core 1 O-glycans, again underlining the importance of native protein conformation for analogous studies (Stansell et al. 2015). In contrast, gp120 purified from T-cell derived virions was devoid of the single site found glycosylated in recombinantly expressed gp120 (Stansell et al. 2015). This, however, might be related to the viral strain used, as the single site was reported to be glycosylated in a separate study using virions from a different HIV-1 strain (Yang W et al. 2014). In addition, treatment of passaged or plasma-derived HIV-1 virions with antibodies against O-linked carbohydrate structures resulted in inhibition of cell entry, and virus neutralization (Hansen et al. 1990, 1991), suggesting that HIV-1 gp120 can indeed be O-glycosylated in vivo. Comparison of recombinant gp120 O-glycosylation in two different cell lines revealed predominant core 1 O-glycosylation in CHO cells, compared to core 1, core 2 and core 4 in 293 T cells (Go et al. 2013). In a similar manner, Hendra virus glycoprotein G expressed in HeLa and HEK293 cells, differed considerably with different numbers of O-glycosites identified and carrying different core structures (Colgrave et al. 2012). Recombinant HCV E2 was O-glycosylated at six positions, with predominantly core 1 and core 2 O-glycan structures (Brautigam et al. 2013). More than 80 % occupancy was estimated for five of the six sites, whereas one site had very low occupancy. Moreover, a high level of structural heterogeneity was observed for the O-glycans localized at the individual sites, with up to 14 different structures identified (Brautigam et al. 2013). A recent study on O-linked glycosylation of HSV-1 mucin-like protein gC provided some insight into O-glycan synthesis, suggesting that the eleven O-glycosites were added in an orderly fashion, before elongation took place (Norden et al. 2015). These studies underscore the high heterogeneity of O-glycan structures, which are both cell type and protein specific, and the need for careful selection of candidates and expression cell lines for clinical applications. Moreover, comprehensive analysis of immune responses mounted by these different structures would be highly 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