title: Structural analysis of a highly glycosylated and unliganded gp120-based antigen using mass spectrometry.
abstract: Structural characterization of the HIV-1 envelope protein gp120 is very important for providing an understanding of the protein's immunogenicity and its binding to cell receptors. So far, the crystallographic structure of gp120 with an intact V3 loop (in the absence of a CD4 coreceptor or antibody) has not been determined. The third variable region (V3) of the gp120 is immunodominant and contains glycosylation signatures that are essential for coreceptor binding and entry of the virus into T-cells. In this study, we characterized the structure of the outer domain of gp120 with an intact V3 loop (gp120-OD8) purified from Drosophila S2 cells utilizing mass spectrometry-based approaches. We mapped the glycosylation sites and calculated the glycosylation occupancy of gp120-OD8; 11 sites from 15 glycosylation motifs were determined as having high-mannose or hybrid glycosylation structures. The specific glycan moieties of nine glycosylation sites from eight unique glycopeptides were determined by a combination of ECD and CID MS approaches. Hydroxyl radical-mediated protein footprinting coupled with mass spectrometry analysis was employed to provide detailed information about protein structure of gp120-OD8 by directly identifying accessible and hydroxyl radical-reactive side chain residues. Comparison of gp120-OD8 experimental footprinting data with a homology model derived from the ligated CD4-gp120-OD8 crystal structure revealed a flexible V3 loop structure in which the V3 tip may provide contacts with the rest of the protein while residues in the V3 base remain solvent accessible. In addition, the data illustrate interactions between specific sugar moieties and amino acid side chains potentially important to the gp120-OD8 structure.
authors.affiliation: Center for Proteomics and Bioinformatics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA.
authors.forename: Liwen
authors.initials: L
authors.lastname: Wang
authors.forename: Yali
authors.initials: Y
authors.lastname: Qin
authors.forename: Serguei
authors.initials: S
authors.lastname: Ilchenko
authors.forename: Jen
authors.initials: J
authors.lastname: Bohon
authors.forename: Wuxian
authors.initials: W
authors.lastname: Shi
authors.forename: Michael W
authors.initials: MW
authors.lastname: Cho
authors.forename: Keiji
authors.initials: K
authors.lastname: Takamoto
authors.forename: Mark R
authors.initials: MR
authors.lastname: Chance
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D004330:Drosophila; D006031:Glycosylation; D015483:HIV Antibodies / Q000378:metabolism; D015699:HIV Envelope Protein gp120 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D066298:In Vitro Techniques; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance; D053719:Tandem Mass Spectrometry
chemical_list: D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120; C059762:HIV envelope protein gp120 (305-321); D007074:Immunoglobulin G; D008024:Ligands; D010446:Peptide Fragments; D011994:Recombinant Proteins; C514793:gp120 protein, Human immunodeficiency virus 1
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