Glycosylation site-specific analysis of HIV envelope proteins (JR-FL and CON-S) reveals major differences in glycosylation site occupancy, glycoform profiles, and antigenic epitopes' accessibility.
The HIV-1 envelope (Env) is a key determinant in mediating viral entry and fusion to host cells and is a major target for HIV vaccine development. While Env is typically about 50% glycan by mass, glycosylation sites are known to evolve, with some glycosylation profiles presumably being more effective at facilitating neutralization escape than others. Thus, characterizing glycosylation patterns of Env and native virions and correlating glycosylation profiles with infectivity and Env immunogenicity are necessary first steps in designing effective immunogens. Herein, we describe a mass spectrometry-based strategy to determine HIV-1 Env glycosylation patterns and have compared two mammalian cell expressed recombinant Env immunogens, one a limited immunogen and one that induces cross-clade neutralizing antibodies. We have used a glycopeptide-based mass mapping approach to identify and characterize Env's glycosylation patterns by elucidating which sites are utilized and what type of glycan motif is present at each glycosylation site. Our results show that the immunogens displayed different degrees of glycosylation as well as a different characteristic set of glycan motifs. Thus, these techniques can be used to (1) define glycosylation profiles of recombinant Env proteins and Env on mature virions, (2) define specific carbohydrate moieties at each glycosylation site, and (3) determine the role of certain carbohydrates in HIV-1 infectivity and in modulation of Env immunogenicity.
Go
Eden P
EP
Department of Chemistry, University of Kansas, Lawrence, Kansas 66045, USA.
Irungu
Janet
J
Zhang
Ying
Y
Dalpathado
Dilusha S
DS
Liao
Hua-Xin
HX
Sutherland
Laura L
LL
Alam
S Munir
SM
Haynes
Barton F
BF
Desaire
Heather
H
D016915:AIDS Vaccines / Q000276:immunology; D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D000939:Epitopes / Q000032:analysis* / Q000276:immunology; D006020:Glycopeptides / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D019302:HIV Envelope Protein gp160 / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D015394:Molecular Structure; D025942:Oligosaccharides, Branched-Chain / Q000737:chemistry; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism
D016915:AIDS Vaccines; D000939:Epitopes; D006020:Glycopeptides; D019302:HIV Envelope Protein gp160; D025942:Oligosaccharides, Branched-Chain; D011994:Recombinant Proteins; D014357:Trypsin