PMC:7108637 / 23551-27410 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/7108637","sourcedb":"PMC","sourceid":"7108637","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7108637","text":"N-glycoproteomics\nTandem mass spectrometry has been widely used for characterization of N-glycosylation sites on viral proteins, with respect to individual site occupancy status (macroheterogeneity) and site-specific structural diversity (microheterogeneity), as these are important features that can affect protein–protein interactions and immunogenicity. Comprehensive glycoproteomic analysis has, for example, mapped N-linked glycosylation of seasonal influenza A virus H3N2 HA, identifying more than 90 % site occupancy of all putative N-linked glycan sites (An et al. 2015). Moreover, the globular head glycosites, associated with host immune receptor interaction, were strictly high-mannose type (An et al. 2015). In a separate study on the highly pathogenic H5N1 influenza A virus, it was also shown that all potential N-glycosites were consistently occupied between several different strains (Blake et al. 2009), suggesting that N-glycosylation of HA is conserved between the different isolates of the same virus subtype, with a high occupancy of potential N-glycosites. In a similar way, it has been demonstrated that all predicted N-glycan sequons were utilized in Hepatitis C virus E2 and Murray Valley encephalitis virus NS1 (Blitvich et al. 2001; Iacob et al. 2008), where the majority of HCV E2 glycosites were modified with high-mannose type glycans (Iacob et al. 2008). On the densely glycosylated HIV-1 envelope glycoprotein several strategies have been employed to characterize the nature and location of the many glycans, with up to 27 mostly highly occupied N-glycosites identified, some of which were exclusively high-mannose type (Pabst et al. 2012; Go et al. 2013; Yang W et al. 2014). Tandem mass spectrometry has also been used for addressing differences in cell-type specific glycosylation and influence of protein conformation. Out of convenience, soluble HIV-1 gp120 or gp140 preparations are often analyzed. HIV-1 gp120 produced in CHO and 293 T cells had very similar occupancy, degree of fucosylation, sialylation, and glycan maturation, with a larger share of glycosites predominantly carrying hybrid and complex-type N-glycans (Go et al. 2013). The positions of some of the exclusively high-mannose N-glycans were located within the intrinsic mannose patch of gp120 (Go et al. 2013; Behrens et al. 2016). In contrast, a much higher proportion of the N-glycosites on gp140 expressed in CHO, recombinant trimers, or those on gp120 purified from native virions were modified with high-mannose type N-glycans (Pabst et al. 2012; Behrens et al. 2016; Panico et al. 2016; Go et al. 2017). This again signifies the importance of analyzing the native protein conformation for generation of relevant glycosylation patterns. Glycosylation in different cell lines was also investigated for Hendra virus recombinant glycoprotein G, where all seven potential N-glycan sites were occupied in HeLa cells (Colgrave et al. 2012). In contrast, only four sites were N-glycosylated in HEK293, although the degree of glycan maturation was similar in both cell lines (Colgrave et al. 2012). To summarize the results obtained from various N-glycoprofiling and glycoproteomic studies, it seems that most of the putative N-glycosylation sequons are glycosylated with high occupancy on viral proteins. However, the site occupancy of viral protein N-glycosylation can vary in different producer cell lines. Moreover, high-mannose type N-glycans may constitute a substantial, if not the major, proportion of viral N-glycosites, particularly when native protein structure and oligomerization is taken into account. Thus, results obtained from analysis of recombinant monomeric proteins should be interpreted with caution also when considering the occupancy of individual N-glycosylation sites as well as complexity of glycan structures as discussed 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