Abstract
Trypanosoma brucei contains a soluble serine oligopeptidase (OP-Tb) that is released into the host bloodstream during infection, where it has been postulated to participate in the pathogenesis of African trypanosomiasis. Here, we report the identification of a single copy gene encoding the T. brucei oligopeptidase and a homologue from the related trypanosomatid pathogen Leishmania major. The enzymes encoded by these genes belong to an emerging subgroup of the prolyl oligopeptidase family of serine hydrolases, referred to as oligopeptidase B. The trypanosomatid oligopeptidases share 70% amino acid sequence identity with oligopeptidase B from the intracellular pathogen Trypanosoma cruzi, which has a demonstrated role in mammalian host cell signaling and invasion. OP-Tb exhibited no activity toward the prolyl oligopeptidase substrate H-Gly-Pro-7-amido-4-methylcoumarin. Instead, it had activity toward substrates of trypsin-like enzymes, particularly those that have basic amino acids in both P(1) and P(2) (e.g. benzyloxycarbonyl-Arg-Arg-7-amido-4-methylcoumarin k(cat)/K(m) = 529 s(-1) microM(-1)). The activity of OP-Tb was enhanced by reducing agents and by polyamines, suggesting that these agents may act as in vivo regulators of OP-Tb activity. This study provides the basis of the characterization of a novel subgroup of serine oligopeptidases from kinetoplastid protozoa with potential roles in pathogenesis.
Highlights
Trypanosoma brucei contains a soluble serine oligopeptidase (OP-Tb) that is released into the host bloodstream during infection, where it has been postulated to participate in the pathogenesis of African trypanosomiasis
Four peptides generated by digestion of OP-Tb with endoproteinase Lys-C (Fig. 1) were sequenced and revealed 54% similarity to the deduced amino acid sequences of corresponding peptides in T. cruzi oligopeptidase B and 30% similarity to corresponding peptides in the E. coli and Moraxella lacunata enzymes
The amino acid sequence of the OP-Tb-derived peptides precisely matched peptide sequences found in the deduced amino acid sequence of the T. brucei oligopeptidase B gene (Fig. 1), and we conclude that the T. brucei enzyme (OP-Tb) is encoded by the gene for oligopeptidase B
Summary
Trypanosoma congolense [27,28,29]. If these enzymes are homologues of the T. cruzi oligopeptidase B, this raises intriguing questions regarding the roles of these enzymes in parasites with such widely different lifestyles. One goal of this study was to confirm or refute the contention that the trypsinlike enzymes found in African trypanosomes belong to the oligopeptidase B subfamily of prolyl oligopeptidases. Our studies have indicated that the T. brucei oligopeptidase, called OP-Tb, may play an important direct role in the pathogenesis of African trypanosomiasis. The disturbed hormonal pulsatility and endocrine rhythms [31], the unusual cleavage of peptide hormones in the blood of T. brucei-infected rats [32], the diminished levels of regulatory peptides such as atrial natriuretic factor (which is a substrate for OP-Tb [28]) [33], and many of the generalized symptoms of trypanosomiasis [34] all point to the possible role of oligopeptidase B in the disruption of host hormone metabolism during trypanosome infection. The oligopeptidase B enzymes define a new subgroup of the prolyl oligopeptidase family
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