title: Cysteine peptidases in the tomato trypanosomatid Phytomonas serpens: influence of growth conditions, similarities with cruzipain and secretion to the extracellular environment.
abstract: We have characterized the cysteine peptidase production by Phytomonas serpens, a tomato trypanosomatid. The parasites were cultivated in four distinct media, since growth conditions could modulate the synthesis of bioactive molecules. The proteolytic profile has not changed qualitatively regardless the media, showing two peptidases of 38 and 40kDa; however, few quantitative changes were observed including a drastic reduction (around 70%) on the 40 and 38kDa peptidase activities when parasites were grown in yeast extract and liver infusion trypticase medium, respectively, in comparison with parasites cultured in Warren medium. The time-span of growth did not significantly alter the protein and peptidase expression. The proteolytic activities were blocked by classical cysteine peptidase inhibitors (E-64, leupeptin, and cystatin), being more active at pH 5.0 and showing complete dependence to reducing agents (dithiothreitol and l-cysteine) for full activity. The cysteine peptidases were able to hydrolyze several proteinaceous substrates, including salivary gland proteins from Oncopeltus fasciatus, suggesting broad substrate utilization. By means of agglutination, fluorescence microscopy, flow cytometry and Western blotting analyses we showed that both cysteine peptidases produced by P. serpens share common epitopes with cruzipain, the major cysteine peptidase of Trypanosoma cruzi. Moreover, our data suggest that the 40kDa cysteine peptidase was located at the P. serpens cell surface, attached to membrane domains via a glycosylphosphatidylinositol anchor. The 40kDa peptidase was also detected in the cell-free culture supernatant, in an active form, which suggests secretion of this peptidase to the extracellular environment.
authors.affiliation: Laboratório de Estudos Integrados em Bioquímica Microbiana, Departamento de Microbiologia Geral, IMPPG, CCS, Bloco I-subsolo, Universidade Federal do Rio de Janeiro (UFRJ), Cidade Universitária, Rio de Janeiro, RJ, Brazil.
authors.forename: Camila G R
authors.initials: CG
authors.lastname: Elias
authors.forename: Fernanda M
authors.initials: FM
authors.lastname: Pereira
authors.forename: Felipe A
authors.initials: FA
authors.lastname: Dias
authors.forename: Thiago L A
authors.initials: TL
authors.lastname: Silva
authors.forename: Angela H C S
authors.initials: AH
authors.lastname: Lopes
authors.forename: Claudia M
authors.initials: CM
authors.lastname: d'Avila-Levy
authors.forename: Marta H
authors.initials: MH
authors.lastname: Branquinha
authors.forename: André L S
authors.initials: AL
authors.lastname: Santos
mesh_terms: D000818:Animals; D015153:Blotting, Western; D003470:Culture Media; D015891:Cystatins / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D006430:Hemiptera / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D018551:Solanum lycopersicum / Q000469:parasitology; D008856:Microscopy, Fluorescence; D015800:Protozoan Proteins; D019163:Reducing Agents / Q000494:pharmacology; D012471:Salivary Proteins and Peptides / Q000378:metabolism; D014351:Trypanosomatina / Q000201:enzymology* / Q000254:growth & development
chemical_list: D003470:Culture Media; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D015800:Protozoan Proteins; D019163:Reducing Agents; D012471:Salivary Proteins and Peptides; D003546:Cysteine Endopeptidases; C063214:cruzipain; D007930:Leucine; C032854:leupeptin; C024974:E 64