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Characterization of Glycosylation Profiles of HIV-1 Transmitted/Founder Envelopes by Mass Spectrometry

2011; American Society for Microbiology; Volume: 85; Issue: 16 Linguagem: Inglês

10.1128/jvi.05053-11

1098-5514

Eden P. Go, Geetha S. Hewawasam, Hua‐Xin Liao, Haiyan Chen, Li‐Hua Ping, Jeffrey A. Anderson, David Hua, Barton F. Haynes, Heather Desaire,

Monoclonal and Polyclonal Antibodies Research

ABSTRACT The analysis of HIV-1 envelope carbohydrates is critical to understanding their roles in HIV-1 transmission as well as in binding of envelope to HIV-1 antibodies. However, direct analysis of protein glycosylation by glycopeptide-based mass mapping approaches involves structural simplification of proteins with the use of a protease followed by an isolation and/or enrichment step before mass analysis. The successful completion of glycosylation analysis is still a major analytical challenge due to the complexity of samples, wide dynamic range of glycopeptide concentrations, and glycosylation heterogeneity. Here, we use a novel experimental workflow that includes an up-front complete or partial enzymatic deglycosylation step before trypsin digestion to characterize the glycosylation patterns and maximize the glycosylation coverage of two recombinant HIV-1 transmitted/founder envelope oligomers derived from clade B and C viruses isolated from acute infection and expressed in 293T cells. Our results show that both transmitted/founder Envs had similar degrees of glycosylation site occupancy as well as similar glycan profiles. Compared to 293T-derived recombinant Envs from viruses isolated from chronic HIV-1, transmitted/founder Envs displayed marked differences in their glycosylation site occupancies and in their amounts of complex glycans. Our analysis reveals that the glycosylation patterns of transmitted/founder Envs from two different clades (B and C) are more similar to each other than they are to the glycosylation patterns of chronic HIV-1 Envs derived from their own clades.