New insights into heparin binding to vitronectin: studies with monoclonal antibodies.
Vitronectin is a plasma glycoprotein that binds to a variety of ligands. There is considerable debate regarding the dependency of these binding interactions upon the conformational status of vitronectin, the role of multimerization and how the binding of different ligands can change vitronectin's conformational state. We have developed a method of capturing vitronectin directly from fresh plasma using solid-phase monoclonal antibodies. Various biotin-labelled secondary monoclonal antibodies were used to quantify the bound vitronectin and to measure its degree of denaturation. Using these tools we demonstrated that one monoclonal antibody partially denatured vitronectin without direct multimerization. Treatment of vitronectin in plasma with soluble heparin produced a similar degree of denaturation. These results led to a proposed adaptation of the unfolding/refolding pathways for chemically denatured vitronectin originally presented by Zhuang and co-workers in 1996 [Zhuang, Blackburn and Peterson (1996) J. Biol. Chem. 271, 14323-14332 and Zhuang, Li, Williams, Wagner, Seiffert and Peterson (1996) J. Biol. Chem. 271, 14333-14343]. The adapted version allows for the production of a more stable partially unfolded intermediate, resulting from the binding of particular ligands. We also demonstrated that the avidity of heparin binding to vitronectin is governed by both the conformational state of the monomer and multimerization of the molecule.
Underwood
P Anne
PA
CSIRO Molecular Science, P.O. Box 184, N Ryde, NSW 1670, Australia. anne.underwood@molsci.csro.au
Kirkpatrick
Alan
A
Mitchell
Sue M
SM
D000818:Animals; D000911:Antibodies, Monoclonal; D018604:Epitope Mapping; D006493:Heparin / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding; D011489:Protein Denaturation / Q000187:drug effects; D019096:Vitronectin / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D008024:Ligands; D019096:Vitronectin; D006493:Heparin