title: Leishmanolysin (gp63 metallopeptidase)-like activity extracellularly released by Herpetomonas samuelpessoai.
abstract: In previous studies, we showed that Herpetomonas samuelpessoai produced a large amount of a surface-located metallopeptidase that presented similar biochemical properties to that of gp63 from Leishmania spp., which is a well-known virulence factor expressed by these digenetic parasites. The present study aims to identify the proteolytic activity released by living H. samuelpessoai cells. In this context, the parasites were incubated in phosphate buffer up to 4 h, and the supernatants were obtained by centrifugation and filtration steps and were then applied on SDS-PAGE to determine the secretory protein profile and on gelatin-SDS-PAGE to identify the proteolytic activity. The results demonstrated that H. samuelpessoai secreted at least 12 polypeptides and an extracellular peptidase of 66 kDa. This enzyme had its activity diminished by 1,10-phenanthroline, EDTA and EGTA. This metallopeptidase was active in a broad spectrum of pH, showing maximum activity at pH 6.0 at 37 degrees C. Casein was also cleaved by this secretory proteolytic enzyme, while bovine serum albumin and haemoglobin were not degraded under these conditions. Fluorescence microscopy and flow cytometry using anti-gp63 antibody against leishmanolysin of L. amazonensis demonstrated the presence of similar molecules on the cell-surface of H. samuelpessoai. Moreover, immunoblot analysis showed the presence of a reactive polypeptide in the cellular extract and in the supernatant fluid of H. samuelpessoai, which suggests immunological similarities between these two distinct trypanosomatids. The zinc-metallopeptidase inhibitor 1,10-phenanthroline was able to inhibit the secretion of the 66 kDa metallopeptidase in a dose-dependent manner, while the phospholipase C inhibitor (p-CMPS) did not alter the secretion pattern. Additionally, anti-cross-reacting determinant (CRD) antibody failed to recognize any secreted polypeptide from H. samuelpessoai. Collectively, these results suggest that the gp63-like molecule was released from the H. samuelpessoai surface by proteolysis instead of phospholipolysis, in a similar mechanism to that observed in Leishmania.
authors.affiliation: Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Góes, IMPPG, Centro de Ciências da Saúde, CCS, Universidade Federal do Rio de Janeiro, UFRJ, Ilha do Fundão, Rio de Janeiro, RJ 21941-590, Brazil.
authors.forename: C G R
authors.initials: CG
authors.lastname: Elias
authors.forename: F M
authors.initials: FM
authors.lastname: Pereira
authors.forename: B A
authors.initials: BA
authors.lastname: Silva
authors.forename: C S
authors.initials: CS
authors.lastname: Alviano
authors.forename: R M A
authors.initials: RM
authors.lastname: Soares
authors.forename: A L S
authors.initials: AL
authors.lastname: Santos
mesh_terms: D000818:Animals; D000913:Antibodies, Protozoan / Q000032:analysis*; D002364:Caseins / Q000378:metabolism; D003429:Cross Reactions; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006454:Hemoglobins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010447:Peptide Hydrolases; D010618:Phenanthrolines / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology*; D012709:Serum Albumin / Q000378:metabolism; D013696:Temperature; D014351:Trypanosomatina / Q000201:enzymology* / Q000472:pathogenicity*; D014774:Virulence
chemical_list: D000913:Antibodies, Protozoan; D002364:Caseins; D006454:Hemoglobins; D010618:Phenanthrolines; D011480:Protease Inhibitors; D012709:Serum Albumin; D010447:Peptide Hydrolases; D008666:Metalloendopeptidases; C055146:glycoprotein gp63, Leishmania; C025205:1,10-phenanthroline