Simplified procedure for fractionation and structural characterisation of complex mixtures of N-linked glycans, released from HIV-1 gp120 and other highly glycosylated viral proteins.

Abstract

HIV-1 gp120 is heavily glycosylated containing 24 N-glycosylation sites, and this makes elucidation of the significance of glycans at individual glycosylation sites a difficult task. A procedure is described where a complex mixture of biologically radiolabelled glycans of gp120, derived from a relatively small number of virus-infected cells may be characterized by a combination of N-glycanase release, single lectin separation, and normal phase HPLC (NP-HPLC). The method was applied in analysis of three N-linked glycosylation sites essential for the in vivo priming of T-cells, specific for an epitope in their vicinity (Sjölander, S., Bolmstedt, A., Åkerblom, 1996. Virology 215, 124–133.). The carbohydrate compositions of wild type gp120 and of mutant variants gp120 lacking one, two, or all of these three active N-linked glycans were analysed. Cells were infected with r-vaccinia virus expressing wild-type gp120 or mutated gp120, or were infected with HIV-1 BRU (wild type) or mutant virus variants. HIV-1 glycoproteins were purified by immunosorbent affinity chromatography and released glycans were separated on lectins, then analysed with NP-HPLC. Our data showed that the structural composition of glycans occupying two of the three glycosylation sites was heterogeneous but the site located adjacent to the T-cell epitope was equipped with one large, high mannose-type structure (>11 units) with the capacity to cover a substantial part of the gp120 surface.