SITE-SPECIFIC N-GLYCOSYLATION AND OLIGOSACCHARIDE STRUCTURES OF RECOMBINANT HIV-1 GP120 DERIVED FROM A BACULOVIRUS EXPRESSION SYSTEM

We report the complete structures of the N-linked oligosaccharides and the site-specificity of the N-glycosylation of recombinant gp120 (rgp 120) of the HIV-1 BH8 isolate produced by a baculovirus expression sys tem. Glycopeptides derived from the tryptic digests of intact rgp120 o r of cyanogen bromide-generated fragments of rgp120 were isolated by t heir binding to concanavalin A-Sepharose and were purified by reversed -phase HPLC. The isolated glycopeptides were treated with PNGase F, re leasing the carbohydrate moiety while converting Asn to Asp, and ident ified by amino acid analysis and/or peptide sequencing. Our results in dicate that all 22 potential N-glycosylation sites in the rgp120 seque nce are utilized. We did not detect N-acetylgalactosamine in rgp120, i ndicating that the glycoprotein lacks typical O-linked oligosaccharide s. To investigate the oligosaccharide structures at the sites of glyco sylation, we determined the carbohydrate composition for each site and characterized the oligosaccharides by H-1-NMR spectroscopy and by oli gosaccharide mapping using high pH anion-exchange chromatography. Mann ose and N-acetylglucosamine were the only sugars observed in the intac t rgp120 and likewise in individual glycopeptides. All glycopeptides d erived from rgp120 contained high mannose-type N-linked oligosaccharid es, ranging from GlcNAc2Man5 to GlcNAc2Man9. However, different glycos ylation 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 rg p120 in the baculovirus expression system occurs at all potential site s and is site specific in terms of oligosaccharide structures.