title: Site-specific N-glycosylation and oligosaccharide structures of recombinant HIV-1 gp120 derived from a baculovirus expression system.
abstract: We report the complete structures of the N-linked oligosaccharides and the site-specificity of the N-glycosylation of recombinant gp120 (rgp120) of the HIV-1 BH8 isolate produce by a baculovirus expression system. Glycopeptides derived from the tryptic digests of intact rgp120 or of cyanogen bromide-generated fragments of rgp120 were isolated by their binding to concanavalin A-Sepharose and were purified by reversed-phase HPLC. The isolated glycopeptides were treated with PNGase F, releasing the carbohydrate moiety while converting Asn to Asp, and identified by amino acid analysis and/or peptide sequencing. Our results indicate that all 22 potential N-glycosylation sites in the rgp120 sequence are utilized. We did not detect N-acetylgalactosamine in rgp120, indicating that the glycoprotein lacks typical O-linked oligosaccharides. To investigate the oligosaccharide structures at the sites of glycosylation, we determined the carbohydrate composition for each site and characterized the oligosaccharides by 1H-NMR spectroscopy and by oligosaccharide mapping using high pH anion-exchange chromatography. Mannose and N-acetylglucosamine were the only sugars observed in the intact rgp120 and likewise in individual glycopeptides. All glycopeptides derived from rgp120 contained high mannose-type N-linked oligosaccharides, ranging from GlcNAc2Man5 to GlcNAc2Man9. However, different glycosylation sites showed varied degrees of processing of the high mannose-type oligosaccharides, as characterized by the ratio of GlcNAc2Man8-9 to GlcNAc2Man5-7. These results demonstrate that N-glycosylation of rgp120 in the baculovirus expression system occurs at all potential sites and is site specific in terms of oligosaccharide structures.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
authors.forename: J C
authors.initials: JC
authors.lastname: Yeh
authors.forename: J R
authors.initials: JR
authors.lastname: Seals
authors.forename: C I
authors.initials: CI
authors.lastname: Murphy
authors.forename: H
authors.initials: H
authors.lastname: van Halbeek
authors.forename: R D
authors.initials: RD
authors.lastname: Cummings
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001665:Binding Sites; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002852:Chromatography, Ion Exchange; D003208:Concanavalin A; D003488:Cyanogen Bromide; D006031:Glycosylation; D015699:HIV Envelope Protein gp120 / Q000737:chemistry* / Q000235:genetics; D015497:HIV-1; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009036:Moths; D009844:Oligosaccharides / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism
chemical_list: D015699:HIV Envelope Protein gp120; D009844:Oligosaccharides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D003208:Concanavalin A; D014357:Trypsin; D003488:Cyanogen Bromide