Detection of orientation-specific anti-gp120 antibodies by a new N-glycanase protection assay.
Several functions have been assigned to the extensive glycosylation of HIV-1 envelope glycoprotein gp120, especially immune escape mechanisms, but the intramolecular interactions between gp120 and its carbohydrate complement are not well understood. To analyse this phenomenon we established a new microwell deglycosylation assay for determining N-linked glycan accessibility after binding of gp120-specific agents. Orientation-specific exposition of gp120 in ELISA microplates was achieved by catching with either anti-C5 antibody D7324 or anti-V3 antibody NEA-9205. We found that soluble CD4 inhibited the deglycosylation of gp120 only when gp120 was caught by D7324 and not by NEA9205. In contrast, antibodies from HIV-infected individuals inhibited the deglycosylation best when gp120 was caught by NEA9205. These results demonstrated that both the CD4-binding site and the epitopes recognised by antibodies from HIV-infected individuals have N-glycans in the close vicinity. However, the difference in gp120 orientation indicates that antibodies in HIV-infected individuals, at least partly, bind to epitopes different from the CD4-binding site. Finally, we determined the structural class of the glycan of one V1 glycosylation site of prototype HIV-1 LAI gp120, which remained unsolved from previous studies, and found that it belonged to the complex type of glycans.
Gram
G J
GJ
Department of Clinical Virology, University of Göteborg, Sweden.
Bolmstedt
A
A
Schønning
K
K
Biller
M
M
Hansen
J E S
JE
Olofsson
S
S
D000581:Amidohydrolases; D001665:Binding Sites; D015704:CD4 Antigens / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D015483:HIV Antibodies / Q000032:analysis*; D015699:HIV Envelope Protein gp120 / Q000276:immunology*; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D009500:Neutralization Tests; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011134:Polysaccharides
D015704:CD4 Antigens; D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120; D011134:Polysaccharides; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase