title: Characterization of glycosylation profiles of HIV-1 transmitted/founder envelopes by mass spectrometry.
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.
authors.affiliation: Department of Chemistry, University of Kansas, Lawrence, Kansas, USA.
authors.forename: Eden P
authors.initials: EP
authors.lastname: Go
authors.forename: Geetha
authors.initials: G
authors.lastname: Hewawasam
authors.forename: Hua-Xin
authors.initials: HX
authors.lastname: Liao
authors.forename: Haiyan
authors.initials: H
authors.lastname: Chen
authors.forename: Li-Hua
authors.initials: LH
authors.lastname: Ping
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Anderson
authors.forename: David C
authors.initials: DC
authors.lastname: Hua
authors.forename: Barton F
authors.initials: BF
authors.lastname: Haynes
authors.forename: Heather
authors.initials: H
authors.lastname: Desaire
mesh_terms: D000595:Amino Acid Sequence; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D057809:HEK293 Cells; D015658:HIV Infections / Q000635:transmission / Q000821:virology; D015497:HIV-1 / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D016415:Sequence Alignment; D014357:Trypsin; D054299:env Gene Products, Human Immunodeficiency Virus / Q000737:chemistry*
chemical_list: D054299:env Gene Products, Human Immunodeficiency Virus; D006026:Glycoside Hydrolases; D014357:Trypsin