title: Assignment of intrachain disulfide bonds and characterization of potential glycosylation sites of the type 1 recombinant human immunodeficiency virus envelope glycoprotein (gp120) expressed in Chinese hamster ovary cells.
abstract: This report describes the structural characterization of the recombinant envelope glycoprotein (rgp120) of human immunodeficiency virus type 1 produced by expression in Chinese hamster ovary cells. Enzymatic cleavage of rgp120 and reversed-phase high performance liquid chromatography were used to confirm the primary structure of the protein, to assign intrachain disulfide bonds, and to characterize potential sites for N-glycosylation. All of the tryptic peptides identified were consistent with the primary structure predicted from the cDNA sequence. Tryptic mapping studies combined with treatment of isolated peptides with Staphylococcus aureus V8 protease or with peptide:N-glycosidase F followed by endoproteinase Asp-N permitted the assignment of all nine intrachain disulfide bonds of rgp120. The 24 potential sites for N-glycosylation were characterized by determining the susceptibilities of the attached carbohydrate structures to peptide:N-glycosidase F and to endo-beta-N-acetylglucosaminidase H. Tryptic mapping of enzymatically deglycosylated rgp120 was used in conjunction with Edman degradation and fast atom bombardment-mass spectrometry of individually treated peptides to determine which of these sites are glycosylated and what types of structures are present. The results indicate that all 24 sites of gp120 are utilized, including 13 that contain complex-type oligosaccharides as the predominant structures, and 11 that contain primarily high mannose-type and/or hybrid-type oligosaccharide structures.
authors.affiliation: Department of Medicinal Chemistry, Genentech, Inc., South San Francisco, California 94080.
authors.forename: C K
authors.initials: CK
authors.lastname: Leonard
authors.forename: M W
authors.initials: MW
authors.lastname: Spellman
authors.forename: L
authors.initials: L
authors.lastname: Riddle
authors.forename: R J
authors.initials: RJ
authors.lastname: Harris
authors.forename: J N
authors.initials: JN
authors.lastname: Thomas
authors.forename: T J
authors.initials: TJ
authors.lastname: Gregory
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004220:Disulfides / Q000032:analysis; D005260:Female; D006031:Glycosylation; D015699:HIV Envelope Protein gp120 / Q000235:genetics* / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances; D008961:Models, Structural; D008969:Molecular Sequence Data; D010053:Ovary; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D014357:Trypsin
chemical_list: D004220:Disulfides; D015699:HIV Envelope Protein gp120; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014357:Trypsin