Role of N-linked glycans in the interaction between the envelope glycoprotein of human immunodeficiency virus and its CD4 cellular receptor. Structural enzymatic analysis.

Abstract

gp120 and CD4 are two glycoproteins that are considered to interact together to allow the binding of HIV to CD4+ cells. We have utilized enzymatic digestion by endoglycosidases in order to analyze N-linked carbohydrate chains of these proteins and their possible role in the interaction of gp120 or gp160 with CD4. SDS denaturation was not necessary to obtain optimal deglycosylation of either molecule, but deglycosylation of CD4, nonetheless, depended on the presence of 1% Triton X-100. Endo H and Endo F that cleave high mannose type and biantennary glycans diminish the molecular mass of the glycoproteins from 120 or 160 Kd to 90 or 130 Kd, respectively; but these enzymes had no action on CD4 glycans. Endo F N-glycanase mixture, which acts on all glycan species, including triantennary chains, led to complete deglycosylation of gp120/160 and of CD4. Therefore, probably half of the glycan moieties of gp120/160 are composed of high mannose and biantennary chains, the other half being triantennary species. The carbohydrate structures of CD4 seems to be triantennary chains. To analyze the binding of gp120/160 to CD4, we used a molecular assay in which an mAb (110-4) coupled to Sepharose CL4B allowed the attachment of soluble gp120/160 to the beads; 125I-sCD4 was then added to measure the binding of CD4 to different amounts of gp120/160. Binding to gp160 was not modified when using completely deglycosylated 125I-sCD4, while deglycosylation of gp120 or of gp160 resulted in the decrease of the binding to native CD4 by two- and fivefold, respectively. Native and deglycosylated gp120/160 bound to CD4+ cells with comparable affinities. In addition, deglycosylated gp120 displaced 125I-gp160 binding to CD4+ cells and inhibited fusion of fresh Molt-T4 cells with CEM HIV1- or HIV2-infected cells to the same extent. Taken together, these results indicate that carbohydrates of CD4 and of gp120/160 do not play a significant role in the in vitro interaction between these two molecules.

Partial Enzymatic Deglycosylation Preserves the Structure of Cleaved Recombinant HIV-1 Envelope Glycoprotein Trimers

“…In previous studies using monomeric gp120 or virions, many different glycosidases were tested under a range of experimental conditions, some of which are harsh enough to probably be incompatible with Env trimer stability (8). To avoid such problems, we adopted a two-stage strategy in which we first made trimers bearing a more homogeneous set of glycans that could then be digested by enzymes under mild conditions of temperature, salt, and pH (67).…”

“…Thus, when N-glycosylation is blocked by enzyme inhibitors or by eliminating the canonical sites, gp120 does not fold properly (8,(11)(12)(13). The modification or removal of carbohydrates from virions can affect Env function and virus infectivity (14 -17).…”

A Human Milk Factor Inhibits Binding of Human Immunodeficiency Virus to the CD4 Receptor

“…Because the viral envelope is heavily glycosylated, it is likely that glycosyl groups on the envelope are important determinants of viral-cellular interactions, and that exogenous glycoconjugates can modulate HIV interaction with its receptor (35)(36)(37)(38)(39)(40). The CD4 receptor molecule has been extensively characterized by means of its reactions with HIV envelope glycoprotein (gp120) and with MAb that bind at specific CD4 epitopes (3 1, 4 1, 42), including the gp 120 binding site.…”

HIV Infection «HIV Tat Protein, a Key Factor in Pathogenesis and Immune System Dysregulation: Implication of IL-10»

“…The external envelope gp120 is a highly glycosylated protein. Carbohydrates represent about 50% of its molecular weight (Fenouillet, Clerget-Raslain et al 1989). The majority of these carbohydrates are of N-type glycosylation.…”