Tropolysin, a new oligopeptidase from African trypanosomes

Oligopeptidases are emerging as important pathogenic factors and therapeutic targets in trypanosome infections. We describe here the purification, cloning, and biochemical analysis of a new oligopeptidase from two pathogenic African trypanosomes. This oligopeptidase, which we have called tropolysin (encoded by the trn gene), represents an evolutionarily distant member of the M3A subfamily of metallopeptidases, ancestral to thimet oligopeptidase, neurolysin, and saccharolysin. the trn gene was present as a single copy per haploid genome, was expressed in both the mammalian and insect stages of the parasite life cycle, and encoded an 84 kDa protein. Both purified and hyperexpressed tropolysin hydrolyzed bradykinin-derived fluorogenic peptide substrates at restricted sites, with an alkaline pH optimum, and were activated by dithiothreitol and reduced glutathione and by divalent metal cations, in the order Zn2+ > CO2+ > Mn2+. Under oxidizing conditions, tropolysin reversibly formed inactive multimers. Tropolysin exhibited a preference for acidic amino acid side chains in P-4, hydrophobic side chains in P-3, and hydrophobic or large uncharged side chains in P-1, P-1', and P-3('), while the S-2(') site was unselective. Highly charged residues were not tolerated in P-1('). Tropolysin was responsible for the bulk of the kinin-degrading activity in trypanosome lysates, potently (kcat approximate to 119 s(-1)) inactivated the vasoactive kinins bradykinin and kallidin, and generated angiotensin(1-7) from angiotensin I. This hydrolysis both abolished the capacity of bradykinin to stimulate the bradykinin B-2 receptor and abrogated bradykinin prohypotensive properties in vivo, raising the possibility that tropolysin may play a role in the dysregulated kinin metabolism observed in the plasma of trypanosome-infected hosts.Univ Hosp Giessen, Dept Internal Med, D-35392 Giessen, GermanyCEA Saclay, Direct Sci Vivant, F-91191 Gif Sur Yvette, FranceUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04044020 São Paulo, BrazilWeb of Scienc