Evidence for a specific interaction of vitronectin with arginine: effects of reducing agents on the expression of functional domains and immunoepitopes.
Vitronectin (Vn) circulates in plasma primarily in the native, monomeric form, whereas platelet-associated Vn in conformationally altered and multimeric. Here, we report that denatured Vn specifically binds to L-Arg, whereas the L-Arg binding site is cryptic in the native form of Vn. In addition, combined treatment of disulfide-linked Vn multimers with L-Arg, urea, and reducing agent results in the formation of disperse oligomers with reduced expression of denaturation-sensitive epitopes. These results suggest that L-Arg modulates the partitioning between monomeric and multimeric Vn species and that L-Arg affinity chromatography can be employed to test for exposure of conformationally sensitive binding sites in Vn. The effects of denaturation on the exposure of conformationally sensitive epitopes in the N-terminus of Vn is controversial. Treatment of Vn with reducing agents abolished type 1 plasminogen activator inhibitor and antibody binding to the highly disulfide-linked N-terminal somatomedin B domain (amino acids 1 to 51), whereas epitopes located in the connecting region/first hemopexin-like repeat (amino acids 52 to 239) and the glycosaminoglycan binding domain (amino acids 343-379) were not affected. These observations indicate that appropriate disulfide-linkage of the N-terminal somatomedin B domain is required for ligand binding and that published differences on the effects of denaturation on the expression of binding sites are probably due to the use of reducing agents in the denaturation process.
Seiffert
D
D
Department of Vascular Biology, Scripps Research Institute, La Jolla, California, USA.
Wagner
N V
NV
D000911:Antibodies, Monoclonal / Q000378:metabolism; D001120:Arginine / Q000378:metabolism*; D001666:Binding Sites, Antibody; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D019163:Reducing Agents / Q000494:pharmacology*; D013002:Somatomedins / Q000378:metabolism; D014508:Urea / Q000494:pharmacology; D019096:Vitronectin / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D017395:Plasminogen Activator Inhibitor 1; D019163:Reducing Agents; D013002:Somatomedins; D019096:Vitronectin; C016756:somatomedin B; D014508:Urea; D001120:Arginine; D004229:Dithiothreitol