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.

[1]  M. Boyd,et al.  Cyanovirin-N defines a new class of antiviral agent targeting N-linked, high-mannose glycans in an oligosaccharide-specific manner. , 2001, Molecular pharmacology.

[2]  M. Boyd,et al.  Analysis of the interaction between the HIV-inactivating protein cyanovirin-N and soluble forms of the envelope glycoproteins gp120 and gp41. , 2000, Molecular pharmacology.

[3]  M. Boyd,et al.  Multiple Antiviral Activities of Cyanovirin-N: Blocking of Human Immunodeficiency Virus Type 1 gp120 Interaction with CD4 and Coreceptor and Inhibition of Diverse Enveloped Viruses , 2000, Journal of Virology.

[4]  E. Toone,et al.  On the Meaning of Affinity:  Cluster Glycoside Effects and Concanavalin A , 1999 .

[5]  J. Sodroski,et al.  Determinants of Neutralization Resistance in the Envelope Glycoproteins of a Simian-Human Immunodeficiency Virus Passaged In Vivo , 1999, Journal of Virology.

[6]  A. Gronenborn,et al.  Crystal structure of cyanovirin-N, a potent HIV-inactivating protein, shows unexpected domain swapping. , 1999, Journal of molecular biology.

[7]  Q. Sattentau,et al.  Cyanovirin-N Binds to gp120 To Interfere with CD4-Dependent Human Immunodeficiency Virus Type 1 Virion Binding, Fusion, and Infectivity but Does Not Affect the CD4 Binding Site on gp120 or Soluble CD4-Induced Conformational Changes in gp120 , 1999, Journal of Virology.

[8]  T. Hayakawa,et al.  Analysis of carbohydrate heterogeneity in a glycoprotein using liquid chromatography/mass spectrometry and liquid chromatography with tandem mass spectrometry. , 1999, Analytical biochemistry.

[9]  S. Olofsson,et al.  Simplified procedure for fractionation and structural characterisation of complex mixtures of N-linked glycans, released from HIV-1 gp120 and other highly glycosylated viral proteins. , 1998, Journal of virological methods.

[10]  A. Gronenborn,et al.  Solution structure of cyanovirin-N, a potent HIV-inactivating protein , 1998, Nature Structural Biology.

[11]  J. Sodroski,et al.  The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens. , 1998, Science.

[12]  J. Sodroski,et al.  Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody , 1998, Nature.

[13]  L K Pannell,et al.  Discovery of cyanovirin-N, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: potential applications to microbicide development , 1997, Antimicrobial agents and chemotherapy.

[14]  K. Guthrie,et al.  HIV-1 membrane fusion mechanism: structural studies of the interactions between biologically-active peptides from gp41. , 1996, Biochemistry.

[15]  I D Campbell,et al.  The effects of variable glycosylation on the functional activities of ribonuclease, plasminogen and tissue plasminogen activator. , 1995, Biochimica et biophysica acta.

[16]  R. Dwek,et al.  Protein surface oligosaccharides and protein function , 1994, Nature Structural Biology.

[17]  D. Mandal,et al.  Thermodynamics of lectin-carbohydrate interactions. Titration microcalorimetry measurements of the binding of N-linked carbohydrates and ovalbumin to concanavalin A. , 1994, Biochemistry.

[18]  R. Cummings,et al.  Site-specific N-glycosylation and oligosaccharide structures of recombinant HIV-1 gp120 derived from a baculovirus expression system. , 1993, Biochemistry.

[19]  G. Böhm,et al.  Quantitative analysis of protein far UV circular dichroism spectra by neural networks. , 1992, Protein engineering.

[20]  J. Schneider,et al.  Carbohydrates of human immunodeficiency virus. Structures of oligosaccharides linked to the envelope glycoprotein 120. , 1988, The Journal of biological chemistry.

[21]  U. Arnold,et al.  Modification of the unfolding region in bovine pancreatic ribonuclease and its influence on the thermal stability and proteolytic fragmentation. , 1999, European journal of biochemistry.

[22]  K. Rice,et al.  Oligosaccharide valency and conformation in determining binding to the asialoglycoprotein receptor of rat hepatocytes. , 1993, Advances in enzymology and related areas of molecular biology.

[23]  M. Rossmann.  The canyon hypothesis. , 1989, Viral immunology.