Demonstration of peripheral fucose units in N-linked glycans of human immunodeficiency virus type 1 gp 120: effects on glycoprotein conformation

Summary Fucosylated N-linked glycans are important constituents of membrane glycoproteins, owing to their significance as biologically active ligands for several selectins and their role in modulating protein conformation of viral glycoproteins. The human immunodeficiency virus type 1 (HIV-1) glycoprotein contains more than 30 different glycan structures but so far fucose was found associated solely with the innermost GlcNAc of N-linked glycans. In the present report we determined whether fucose units also were linked to the distal GlcNAc via α(1–3) or α(1–4) linkages in N-linked glycans of gp 120. [ 3 H]-fucose labelled gp 120 was subjected to endoglycosidase F digestion, releasing diantennary complex type N-linked glycans, but leaving the inner polypeptide-bound carbohydrates, GlcNAc and possibly associated fucose units, intact. Gel filtration of the digested material revealed that [ 3 H]-fucose label was released from gp 120 by this treatment, indicating presence of peripheral fucose units. Furthermore, [ 3 H]-focuse label was also released by treatment of the labelled gp 120 with an α-L-fucosidase specifically removing fucose in α(1–3) and α(1–4) linkages. Altogether the results indicated presence of fucose units linked to peripheral GlcNAc of gp 120 N-linked glycans. We have earlier shown that other peripheral carbohydrate determinants, i.e. β(1–4)-galactose on N-linked glycans, maintain a correct antigenic conformation of gp 120. Using a coupled ELISA system, where changes in antigenic behaviour of a viral glycoprotein were correlated to stepwise elimination of peripheral monosaccharides from N-linked glycans, we found that treatment of gp 120 with a pan-specific α-fucosidase as well as an enzyme specific for α(1–3)- or α(1–4)-linked fucose disclosed a hidden linear epitope situated in the gp 120 C2 region. The effects of the general fucosidase on epitope exposure was more prominent than those obtained with the enzyme with narrow specificity, suggesting that peripheral and inner fucose units co-operate in the maintenance of gp 120 conformation.