title: Trichocystatin-2 (TC-2): an endogenous inhibitor of cysteine proteinases in Trichomonas vaginalis is associated with TvCP39.
abstract: The causal agent of trichomoniasis is a parasitic protist, Trichomonas vaginalis, which is rich in proteolytic activity, primarily carried out by cysteine proteases (CPs). Some CPs are known virulence factors. T. vaginalis also possesses three genes encoding endogenous cystatin-like CP inhibitors. The aim of this study was to identify and characterize one of these CP inhibitors. Using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS), a cystatin-like peptidase inhibitor dubbed Trichocystatin-2 (TC-2) was identified in the T. vaginalis active degradome in association with TvCP39, a 39kDa CP involved in cytotoxicity. To characterize the TC-2 inhibitor, we cloned and expressed the tvicp-2 gene, purified the recombinant protein (TC-2r), and produced a specific polyclonal antibody (α-TC-2r). This antibody recognized a 10kDa protein band by western blotting. An indirect immunofluorescence assay (IFA) and cell fractionation assays using the α-TC-2r antibody showed that TC-2 was localized in the cytoplasm and lysosomes and that it colocalized with TvCP39. TC-2r showed inhibitory activity against papain, cathepsin-L, and TvCP39 in trichomonad extracts and live parasites but not legumain-like CPs. Live trichomonads treated with TC-2r showed reduced trichomonal cytotoxicity to HeLa cell monolayers in a TC-2r-concentration-dependent manner. In this study, we identified and characterized an endogenous cystatin-like inhibitor in T. vaginalis, TC-2, which is associated with TvCP39 and appears to regulate the cellular damage caused by T. vaginalis.
authors.affiliation: Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del IPN, Apdo. Postal 14-740, México, D.F. 07360, Mexico.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Puente-Rivera
authors.affiliation: Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del IPN, Apdo. Postal 14-740, México, D.F. 07360, Mexico.
authors.forename: Lucero de los Ángeles
authors.initials: Lde L
authors.lastname: Ramón-Luing
authors.affiliation: Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del IPN, Apdo. Postal 14-740, México, D.F. 07360, Mexico.
authors.forename: Elisa Elvira
authors.initials: EE
authors.lastname: Figueroa-Angulo
authors.affiliation: Departamento de Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados del IPN, Apdo. Postal 14-740, México, D.F. 07360, Mexico.
authors.forename: Jaime
authors.initials: J
authors.lastname: Ortega-López
authors.affiliation: Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del IPN, Apdo. Postal 14-740, México, D.F. 07360, Mexico. Electronic address: rarroyo@cinvestav.mx.
authors.forename: Rossana
authors.initials: R
authors.lastname: Arroyo
mesh_terms: D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D015153:Blotting, Western; D056668:Cathepsin L / Q000037:antagonists & inhibitors; D049109:Cell Proliferation; D002478:Cells, Cultured; D003001:Cloning, Molecular; D015891:Cystatins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D003546:Cysteine Endopeptidases / Q000737:chemistry; D057056:Cysteine Proteases / Q000737:chemistry* / Q000378:metabolism; D003593:Cytoplasm / Q000201:enzymology; D015180:Electrophoresis, Gel, Two-Dimensional; D006367:HeLa Cells; D006801:Humans; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D008247:Lysosomes / Q000201:enzymology; D008297:Male; D008969:Molecular Sequence Data; D010802:Phylogeny; D011480:Protease Inhibitors / Q000276:immunology / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053719:Tandem Mass Spectrometry; D014245:Trichomonas Infections / Q000188:drug therapy* / Q000378:metabolism / Q000382:microbiology; D014246:Trichomonas vaginalis / Q000201:enzymology* / Q000235:genetics; D000099240:Asparaginyl Endopeptidase
chemical_list: D056668:Cathepsin L; D015891:Cystatins; D003546:Cysteine Endopeptidases; D057056:Cysteine Proteases; D011480:Protease Inhibitors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000099240:Asparaginyl Endopeptidase
keywords: Cystatin; Cysteine proteinases (CPs); Cytotoxicity; Trichocystatin-2; Trichomonas vaginalis