IDENTIFICATION OF POSSIBLE INHIBITORY REACTIVE CENTERS IN THROMBOSPONDIN-1 THAT MAY BIND CATHEPSIN-G AND NEUTROPHIL ELASTASE

Thrombospondin 1 is a multidomain trimeric glycoprotein from platelets and a variety of normal and transformed cells of both mesenchymal and epithelial origin, which functions in cell adhesion and cell-cell int eractions. We have recently shown that human thrombospondin 1 binds an d inhibits the neutrophil enzymes, neutrophil elastase [Hogg, P. J., O wensby, D. A., Mosher, D. F., Misenheimer, T. M., & Chesterman, C. N. (1993a) J. Biol. Chem. 268, 7139-7146] and cathepsin G [Hogg, P. J., O wensby, D. A., & Chesterman, C. N. (1993b) J. Biol. Chem. 268, 21811-2 1818]. One mole of thrombospondin 1 trimer binds 3 mol of neutrophil e lastase or up to 6 mol of cathepsin G, with site-binding dissociation constants around the nanomolar range, and the enzymes have been shown to interact with thrombospondin 1 in the vicinity of the calcium-bindi ng type 3 repeats. None of the protein modules in this region; or with in the whole thrombospondin 1 molecule, have previously been implicate d in the inhibition of proteinases. We noted that there are two stretc hes of eight amino acids each in the human thrombospondin 1 type 3 rep eats, residues 735-742 and 794-801, that have striking similarity to a reactive-site consensus sequence derived from selected members of the Kazal and Streptomyces subtilisin inhibitor families. Synthetic pepti des corresponding to the putative P-5 through P-4' residues of both pr oposed reactive centers interacted efficiently with the active site of cathepsin G and were competitive inhibitors of the fibronectin-degrad ing and platelet-activating activities of this enzyme, while only the peptide corresponding to residues 793-801 efficiently interacted with the active site of neutrophil elastase and competitively inhibited its fibronectin-degrading activity. These findings are in accordance with the neutrophil enzyme:thrombospondin 1 molar stoichiometries measured kinetically. We suggest that residues 735-742 and 794-801 represent r eactive centers in thrombospondin 1 and define a novel proteinase inhi bitor.