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© The Rockefeller University Press, 0021-9525/1997//609 $5.00
The Journal of Cell Biology, Volume 136, Number 3, , 1997 609-620

Article

A Cytosolic Serine Endopeptidase from Trypanosoma cruzi Is Required for the Generation of Ca2+ Signaling in Mammalian Cells



Barbara A. Burleigh, Elisabet V. Caler, Paul Webster, and Norma W. Andrews

Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520-8002

An early event in the Trypanosoma cruzi cell invasion process, the recruitment of host lysosomes, led us to investigate the involvement of signal transduction. Infective trypomastigotes were found to contain a soluble Ca2+-signaling activity for mammalian cells that is sensitive to protease inhibitors. Inhibitor and substrate utilization profiles were used to purify a candidate peptidase for involvement in this process, from which we isolated a full-length cDNA clone. The sequence revealed a novel enzyme, denominated T. cruzi oligopeptidase B, which is homologous to members of the prolyl oligopeptidase family of serine hydrolases, known to participate in the maturation of biologically active peptides. The T. cruzi oligopeptidase B was expressed as a fully active product in Escherichia coli, and antibodies to the recombinant enzyme inhibited both peptidase activity and Ca2+ signaling induced in normal rat kidney cells by trypomastigote extracts. Our data suggest that the T. cruzi oligopeptidase B participates in processing events in the cytoplasm of the parasites, generating a factor with Ca2+-signaling activity for mammalian cells.

Abbreviations used in this paper: AFC, 7-amino-4-trifluoromethyl coumarin; AMC, 7-amino-4-methyl coumarin; Z-Phe-Arg-AMC, Z-phenylalanyl-arginyl-7-amino-4-methyl coumarin; [Ca2+]i, intracellular free calcium concentration; E-64, N-[N-(l-3-trans-carboxirane-2-carbonyl)-l-leucyl]- agmatine; IL, interleukin; NRK, normal rat kidney; rPEP, recombinant T. cruzi oligopeptidase B; RT, reverse transcriptase; TSE, trypomastigote soluble extract.

This work was funded by National Institutes of Health grant R01AI32056 to N.W. Andrews. B. Burleigh was supported by a postdoctoral fellowship from the Medical Research Council of Canada.

Address all correspondence to Norma W. Andrews, Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8002. Tel.: (203) 785-4314. Fax: (203) 785-7226. E-mail: norma_andrews{at}qm.yale.edu


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