Functional changes in the conformation of thrombospondin-1 during complexation with fibronectin or heparin

Rima Dardik, Judith Lahav

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)407-414
Number of pages8
JournalExperimental Cell Research
Volume248
Issue number2
DOIs
StatePublished - 1 May 1999

Keywords

  • Conformation
  • Extracellular matrix
  • Fibronectin
  • Heparin
  • Thrombospondin

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