Selective interactions of the human immunodeficiency virus-inactivating protein cyanovirin-N with high-mannose oligosaccharides on gp120 and other glycoproteins

The virucidal protein cyanovirin-N (CV-N) mediates its highly potent anti-h uman immunodeficiency virus activity, at least in part, through interaction s with the viral envelope glycoprotein gp120. Here we dissect in further de tail the mechanism of CV-N's glycosylation-dependent binding to gp120. Isot hermal titration calorimetry (ITC) binding studies of CV-N with endoglycosi dase H-treated gp120 showed that binding was completely abrogated by remova l of high-mannose oligosaccharides from the glycoprotein. Additional ITC an d circular dichroism spectral studies with CV-N and other glycoproteins as well showed that CV-N discriminately bound only glycoproteins that contain high-mannose oligosaccharides. Binding experiments with RNase B indicated t hat the single high-mannose oligosaccharide on that enzyme mediated all of its binding with CV-N (K-d = 0.602 muM). A finer level of oligosaccharide s electivity of CV-N was revealed in affinity chromatography-liquid chromatog raphy-mass spectrometry experiments, which showed that CV-N preferentially bound only oligomannose-8 (Man-8) and oligomannose-9 isoforms of RNase B. F inally, we biophysically characterized the interaction of CV-N with a purif ied, single oligosaccharide, Man-8. The binding affinity of Man-8 for CV-N is unusually strong (K-d = 0.488 muM), several hundredfold greater than obs erved for oligosaccharides and their protein lectins (K-d = 1 muM-1 mM), fu rther establishing a critical role of high-mannose oligosaccharides in CV-N binding to glycoproteins.