A TRYPANOSOMA CRUZI-SECRETED 80 KDA PROTEINASE WITH SPECIFICITY FOR HUMAN COLLAGEN TYPE-I AND TYPE-IV

Specific interactions between parasites and extracellular matrix compo nents are an important mechanism in the dissemination of Chagas' disea se. Binding of the extracellular matrix proteins to Trypanosoma cruzi receptors has been described as a significant step in this phenomenon. In this study, a specific proteinase activity was identified in cell- free extracts of amastigote, trypomastigote and epimastigote forms of T. cruzi using the collagenase fluorogenic substrate -Suc-Gly-Pro-Leu- Gly-Pro-7-amido-4-methylcoumarin. Isolation of this activity was achie ved by a four-step FPLC procedure. Optimal enzyme activity was found t o occur at pH 8.0 and was associated with a single T. cruzi 80 kDa pro tein (Tc 80 proteinase) on SDS/PAGE under reducing conditions. An inte rnal peptide sequence of Tc 80 proteinase was obtained (AGDNYTPPE), an d no similarity was found to previously described proteinases of T. cr uzi. This enzyme activity is strongly inhibited by HgCl2, tosyl-lysylc hloromethane ('TLCK') p-chloromercuribenzoate and benzyloxycarbonyl-Ph e-Ala-diazomethane. The purified enzyme was able to hydrolyse purified human [C-14]collagen types I and IV at neutral pH, but not C-14-label led BSA, rat laminin, rabbit IgG or small proteins such as insulin or cytochrome c. In addition, Tc 80 proteinase activity was found to be s ecreted by T. cruzi forms infective to mammalian cells. Furthermore we demonstrated that purified Tc 80 proteinase mediates native collagen type I hydrolysis in rat mesentery. This feature is compared with that of Clostridium histolyticum collagenase. These findings suggest that Tc 80 proteinase may facilitate T. cruzi host-cell infection by degrad ing the collagens of the extracellular matrix and could represent a go od target for Chagas' disease chemotherapy.