Selective Interactions of the Human Immunodeficiency Virus-Inactivating Protein Cyanovirin-N with High-Mannose Oligosaccharides on gp 120 and Other Glycoproteins

摘要

The virucidal protein cyanovirin-N (CV-N) mediates its highly potent anti-human immunodeficiency virus activity, at least in part, through interactions with the viral envelope glycop- rotein gp120. Here we dissect in further detail the mecha- nism of CV-N's glycosylation-dependent binding to gp120. Isothermal titration calorimetry (ITC) binding studies of CV-N with endoglycosidase H-treated gp120 showed that binding was completely abrogated by removal of high-mannose oli- gosaccharides from the glycoprotein. Additional ITC and circular dichroism spectral studies with CV-N and other gly- coproteins as well showed that CV-N discriminately bound only glycoproteins that contain high-mannose oligosaccha- rides. Binding experiments with RNase B indicated that the single high-mannose oligosaccharide on that enzyme medi- ated all of its binding with CV-N (Kd = 0.602 Jl,M). A finer level of oligosaccharide selectivity of CV-N was revealed in affinity chromatography-liquid chromatography-mass spectrometry experiments, which showed that CV-N preferentially bound only oligomannose-8 (Man-8) and oligomannose-9 isoforms of RNase B. Finally, we biophysically characterized the in- teraction of CV-N with a purified, single oligosaccharide, Man-8. The binding affinity of Man-8 for CV-N is unusually strong (Kd = 0.488 Jl,M), several hundredfold greater than observed for oligosaccharides and their protein lectins (Kd = 1 Jl,M-1 mM), further establishing a critical role of high- mannose oligosaccharides in CV-N binding to glycoproteins.