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Detection of orientation‐specific anti‐gp120 antibodies by a new N‐glycanase protection assay
Author(s) -
Gram G. J.,
Bolmstedt A.,
Schønning K.,
Biller M.,
Hansen J.E. S.,
Olofsson S.
Publication year - 2002
Publication title -
apmis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.909
H-Index - 88
eISSN - 1600-0463
pISSN - 0903-4641
DOI - 10.1034/j.1600-0463.2002.100202.x
Subject(s) - epitope , glycan , antibody , glycoprotein , glycosylation , v3 loop , chemistry , human immunodeficiency virus (hiv) , biology , biochemistry , virology , immunology
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.