The recombinant prepro region of TvCP4 is an inhibitor of cathepsinL-like cysteine proteinases of Trichomonas vaginalis that inhibits trichomonal haemolysis

Abstract

tTrichomonas vaginalis expresses multiple proteinases, mainly of the cysteine type (CPs). A cathepsin L-like34 kDa CP, designated TvCP4, is synthesized as a 305-amino-acid precursor protein. TvCP4 contains theprepro fragment and the catalytic triad that is typical of the papain-like CP family of clan CA. The aim ofthis work was to determine the function of the recombinant TvCP4 prepro region (ppTvCP4r) as a specificinhibitor of CPs. We cloned, expressed, and purified the recombinant TvCP4 prepro region. The conforma-tion of the purified and refolded ppTvCP4r polypeptide was verified by circular dichroism spectroscopyand fluorescence emission spectra. The inhibitory effect of ppTvCP4r was tested on protease-resistantextracts from T. vaginalis using fluorogenic substrates for cathepsin L and legumain CPs. In 1-D zymo-grams, the inhibitory effect of ppTvCP4r on trichomonad CP proteolytic activity was observed in the ∼97,65, 39, and 30 kDa regions. By using 2-D zymograms and mass spectrometry, several of the CPs inhibitedby ppTvCP4r were identified. A clear reduction in the proteolytic activity of several cathepsin L-like pro-tein spots (TvCP2, TvCP4, TvCP4-like, and TvCP39) was observed compared with the control zymogram.Moreover, pretreatment of live parasites with ppTvCP4r inhibited trichomonal haemolysis in a concen-tration dependent manner. These results confirm that the recombinant ppTvCP4 is a specific inhibitor ofthe proteolytic activity of cathepsin L-like T. vaginalis CPs that is useful for inhibiting virulence propertiesdepending on clan CA papain-like CPs.