Selective Interactions of the Human Immunodeficiency Virus-Inactivating Protein Cyanovirin-N with High-Mannose Oligosaccharides on gp120 and Other Glycoproteins
- 1Laboratory of Drug Discovery Research and Development, NCI Center for Cancer Research, National Cancer Institute, Frederick, Maryland (S.R.S., B.R.O., M.R.B.); 2Department of Clinical Virology, University of Göteborg, Göteborg, Sweden (A.J.B.); and 3SAIC-Frederick, Frederick, Maryland (L.K.C.)
Abstract
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 μM). 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 μM), several hundredfold greater than observed for oligosaccharides and their protein lectins (K d = 1 μM–1 mM), further establishing a critical role of high-mannose oligosaccharides in CV-N binding to glycoproteins.
Footnotes
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Send reprint requests to: Dr. Michael R. Boyd, Laboratory of Drug Discovery Research & Development, NCI Center for Cancer Research, NCI-Frederick, Bldg. 1052, Room 121, Frederick, MD 21702-1201. E-mail: boyd{at}dtpax2.ncifcrf.gov
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This work was in part supported by grants from the Swedish Medical Research Council (Grants 9083 and 10437), the National Swedish Board for Technical Development (Project 87-0256P), and the Medical Faculty, University of Göteborg. This article constitutes part 74 in the series “HIV-inhibitory Natural Products”; for part 73 seeMeragelman et al. (2001).
- Abbreviations:
- CV-N
- cyanovirin-N
- HIV
- human immunodeficiency virus
- gp
- glycoprotein
- sgp
- soluble glycoprotein
- ITC
- isothermal titration calorimetry
- Man
- oligomannose
- Endo-H
- endoglycosidase H
- CD
- circular dichroism
- LC-MS
- liquid chromatography-mass spectrometry
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- Received December 11, 2000.
- Accepted February 6, 2001.
- U.S. Government
