Pj. Hogg et al., IDENTIFICATION OF POSSIBLE INHIBITORY REACTIVE CENTERS IN THROMBOSPONDIN-1 THAT MAY BIND CATHEPSIN-G AND NEUTROPHIL ELASTASE, Biochemistry, 33(21), 1994, pp. 6531-6537
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.