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-human immunodeficiency virus activity, at least in part, through interactions with the viral envelope glycoprotein gp120. Here we dissect in further detail the mechanism 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 oligosaccharides from the glycoprotein. Additional ITC and 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 that the single high-mannose oligosaccharide on that enzyme mediated all of its binding with CV-N (K(d) = 0.602 microM). 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 interaction of CV-N with a purified, single oligosaccharide, Man-8. The binding affinity of Man-8 for CV-N is unusually strong (K(d) = 0.488 microM), several hundredfold greater than observed for oligosaccharides and their protein lectins (K(d) = 1 microM--1 mM), further establishing a critical role of high-mannose oligosaccharides in CV-N binding to glycoproteins.
Shenoy
S R
SR
Laboratory of Drug Discovery Research and Development, NCI Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, USA.
O'Keefe
B R
BR
Bolmstedt
A J
AJ
Cartner
L K
LK
Boyd
M R
MR
D019380:Anti-HIV Agents / Q000494:pharmacology*; D001426:Bacterial Proteins; D002151:Calorimetry; D002352:Carrier Proteins / Q000494:pharmacology*; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D006023:Glycoproteins / Q000187:drug effects*; D015699:HIV Envelope Protein gp120 / Q000187:drug effects*; D006801:Humans; D008358:Mannose / Q000494:pharmacology; D008968:Molecular Conformation; D009844:Oligosaccharides / Q000494:pharmacology*; D012260:Ribonucleases / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013816:Thermodynamics
D019380:Anti-HIV Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D006023:Glycoproteins; D015699:HIV Envelope Protein gp120; D009844:Oligosaccharides; C107733:cyanovirin N; D012260:Ribonucleases; C029600:ribonuclease B; D008358:Mannose