title: The recombinant prepro region of TvCP4 is an inhibitor of cathepsin L-like cysteine proteinases of Trichomonas vaginalis that inhibits trichomonal haemolysis.
abstract: Trichomonas vaginalis expresses multiple proteinases, mainly of the cysteine type (CPs). A cathepsin L-like 34kDa CP, designated TvCP4, is synthesized as a 305-amino-acid precursor protein. TvCP4 contains the prepro fragment and the catalytic triad that is typical of the papain-like CP family of clan CA. The aim of this work was to determine the function of the recombinant TvCP4 prepro region (ppTvCP4r) as a specific inhibitor of CPs. We cloned, expressed, and purified the recombinant TvCP4 prepro region. The conformation of the purified and refolded ppTvCP4r polypeptide was verified by circular dichroism spectroscopy and fluorescence emission spectra. The inhibitory effect of ppTvCP4r was tested on protease-resistant extracts from T. vaginalis using fluorogenic substrates for cathepsin L and legumain CPs. In 1-D zymograms, 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 inhibited by ppTvCP4r were identified. A clear reduction in the proteolytic activity of several cathepsin L-like protein 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 concentration dependent manner. These results confirm that the recombinant ppTvCP4 is a specific inhibitor of the proteolytic activity of cathepsin L-like T. vaginalis CPs that is useful for inhibiting virulence properties depending on clan CA papain-like CPs.
authors.affiliation: Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Av. IPN # 2508, Col. San Pedro Zacatenco, Delg. Gustavo A. Madero, CP 07360 México, DF, Mexico.
authors.forename: Rosa Elena
authors.initials: RE
authors.lastname: Cárdenas-Guerra
authors.affiliation: Departamento de Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Av. IPN # 2508, Col. San Pedro Zacatenco, Delg. Gustavo A. Madero, CP 07360 México, DF, Mexico. Electronic address: jortega@cinvestav.mx.
authors.forename: Jaime
authors.initials: J
authors.lastname: Ortega-López
authors.affiliation: Departamento de Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Av. IPN # 2508, Col. San Pedro Zacatenco, Delg. Gustavo A. Madero, CP 07360 México, DF, Mexico.
authors.forename: Claudia Ivonne
authors.initials: CI
authors.lastname: Flores-Pucheta
authors.affiliation: Laboratorio de Investigación Bioquímica, Programa Institucional en Biomedicina Molecular ENMyH-Instituto Politécnico Nacional, Guillermo Massieu Helguera No. 239, La Escalera Ticomán, CP 07320 México, DF, Mexico.
authors.forename: Claudia Guadalupe
authors.initials: CG
authors.lastname: Benítez-Cardoza
authors.affiliation: Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Av. IPN # 2508, Col. San Pedro Zacatenco, Delg. Gustavo A. Madero, CP 07360 México, DF, Mexico. Electronic address: rarroyo@cinvestav.mx.
authors.forename: Rossana
authors.initials: R
authors.lastname: Arroyo
mesh_terms: D000595:Amino Acid Sequence; D056668:Cathepsin L / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005260:Female; D005656:Fungal Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification; D006461:Hemolysis / Q000187:drug effects*; D006801:Humans; D008969:Molecular Sequence Data; D058849:Protein Refolding; D017433:Protein Structure, Secondary; D059748:Proteolysis / Q000187:drug effects; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D016415:Sequence Alignment; D021241:Spectrometry, Mass, Electrospray Ionization; D014246:Trichomonas vaginalis / Q000201:enzymology*
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D056668:Cathepsin L
keywords: CP inhibitor; Haemolysis; Prepro region; Trichomonas vaginalis; TvCP4