The cluster of basic amino acids in vitronectin contributes to its binding of plasminogen activator inhibitor-1: evidence from thrombin-, elastase- and plasmin-cleaved vitronectins and anti-peptide antibodies.
Derivatives of vitronectin obtained by specific cleavage at its cluster of basic amino acids with thrombin, elastase and plasmin are shown to have a decreased ability to bind plasminogen activator inhibitor-1 (PAI-1). The identification and localization of the segment involved in the binding of PAI-1 (Lys348-Arg379) were carried out by purification of these cleaved vitronectins and their subsequent structural characterization (sequence analysis, phosphorylation of Ser378 with cAMP-dependent protein kinase and immunostaining with peptide-specific antibodies), then measurement of the vitronectin-PAI-1 interaction by (a) a two-phase system (ELISA); (b) co-precipitation of the vitronectin-PAI-1 complex out of solution, and (c) analysis of the stereospecific interaction between the active conformation of PAI-1 and a peptide derived from the above-mentioned cluster; this interaction occurs when the peptide is composed of all-l-amino acids but not when it is composed of all-d-amino acids. Our results explain why workers who have used immobilized vitronectin to study this interaction could not have observed the involvement of the cluster of basic amino acids in PAI-1 binding, since the immobilization of vitronectin is shown to render this cluster inaccessible for interaction. We propose that vitronectin binds active PAI-1 by interaction via amino acid residues that originate from distal locations in the N- and C-termini of vitronectin.
Gechtman
Z
Z
Department of Biological Regulation, The Weizmann Institute of Science, IL-76100 Rehovot, Israel.
Belleli
A
A
Lechpammer
S
S
Shaltiel
S
S
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005341:Fibrinolysin / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D010766:Phosphorylation; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism*; D011485:Protein Binding; D017421:Sequence Analysis; D013917:Thrombin / Q000378:metabolism; D019096:Vitronectin / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D010446:Peptide Fragments; D017395:Plasminogen Activator Inhibitor 1; D019096:Vitronectin; D006493:Heparin; D017868:Cyclic AMP-Dependent Protein Kinases; D010196:Pancreatic Elastase; D013917:Thrombin; D005341:Fibrinolysin