Functional changes in the conformation of thrombospondin-1 during complexation with fibronectin or heparin.
Thrombospondin-1 (TSP-1) interacts specifically with heparin and fibronectin in vitro and colocalizes with fibronectin and heparan sulfate in the extracellular matrix (ECM). Its conformation is strongly dependent on Ca2+ concentration. We have previously shown that both heparin and fibronectin have two binding sites on the TSP-1 subunit which may require conformational change for their occupancy (R. Dardik and J. Lahav, 1987, Eur. J. Biochem. 168, 347; ibid 1989, 185, 581). To investigate the effect of TSP-1 binding to fibronectin and heparin on its functional conformation, TSP-1 was subjected to proteolysis in the presence and absence of ligands and of Ca2+. We found that while trypsin cleavage of free TSP-1 resulted in the inactivation of ligand binding, TSP-1 bound to either fibronectin or heparin remained stably associated with these ligands. Cleavage by thrombin or tissue plasminogen activator (tPA) showed that Ca2+-depleted TSP-1, when bound to fibronectin or to heparin, yielded proteolytic cleavage patterns typical of the Ca2+-containing form. Cleavage by chymotrypsin was not affected by binding to fibronectin or heparin; hence loss of proteolytic susceptibility was not due to steric hindrance by the ligands. Taken together, these results indicate that: (A) binding of TSP-1 to fibronectin or heparin is a two-step mechanism where binding to one site leads to conformational changes that enable binding to the second site; (B) TSP-1 in complex with fibronectin or heparin adopts the Ca2+-containing conformation in the absence of Ca2+; and (C) such complexes are highly resistant to cleavage by tPA and, if cleaved by other enzymes, the TSP-1 fragments remain bound to other ECM components. These characteristics have profound significance for platelet adhesion and cell migration into wounds where Ca2+ concentrations are reduced.
Dardik
R
R
National Center for Hemophilia, Sheba Medical Center, Tel Hashomer.
Lahav
J
J
D000818:Animals; D002118:Calcium; D002417:Cattle; D005353:Fibronectins / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D019700:Thrombospondin 1 / Q000737:chemistry* / Q000378:metabolism*
D005353:Fibronectins; D010446:Peptide Fragments; D019700:Thrombospondin 1; D006493:Heparin; D002118:Calcium