Leishmanolysin (gp63 metallopeptidase)-like activity extracellularly released byHerpetomonas samuelpessoai

In previous studies, we showed thatHerpetomonas samuelpessoaiproduced a large amount of a surface-located metallopeptidase that presented similar biochemical properties to that of gp63 fromLeishmaniaspp., which is a well-known virulence factor expressed by these digenetic parasites. The present study aims to identify the proteolytic activity released by livingH. samuelpessoaicells. 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 thatH. samuelpessoaisecreted 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 °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 ofL. amazonensisdemonstrated the presence of similar molecules on the cell-surface ofH. samuelpessoai. Moreover, immunoblot analysis showed the presence of a reactive polypeptide in the cellular extract and in the supernatant fluid ofH. 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 fromH. samuelpessoai. Collectively, these results suggest that the gp63-like molecule was released from theH. samuelpessoaisurface by proteolysis instead of phospholipolysis, in a similar mechanism to that observed inLeishmania.