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Purification and partial characterization of an extracellular serine oligopeptidase from Leishmania braziliensis
Herbert Leonel de Matos Guedes1,2; João de Mello Rezende Neto1,2; Monique Pacheco1; Cristiane Cardoso Martins de Salles1; Bartira Rossi Bergmann2 and Salvatore Giovanni de Simone1,3.
1Laboratório de Bioquímica e Biologia Molecular, DBBM, IOC, FIOCRUZ.
2Laboratório de Imunofarmacologia, IBCCF, UFRJ
3Departamento de Biologia Celular e Molecular, IB, UFF
Leishmania (V.) brasiliensis is one of the most important ethiologic agent of American tegumentary leishmaniasis. Protozoan proteases play crucial roles in the host-parasite interaction and serine oligopeptidases of tryponassomatids are important virulence factors. The enzyme was purified from cell-free culture supernatant form L. braziliensis using a combination of affinity chromatography and gel filtration chromatography. By gel filtration chromatography and in non reducing conditions the apparent molecular mass of enzyme presented as 120 kDa. The enzyme appeared as a single band by SDS-PAGE and with an apparent molecular weight of 80 kDa under nonreducing conditions and 92kDa under reducing conditions. Considering peptide substrates, L-BAPNA and L-BAEE, containing arginyl residue in P1 (in amide bound for L-BAPNA and in ester bound for L-BAEE), this enzyme preferentially cleaved ester bound. Using L-BAEE as substrate, the proteinase showed an optimum pH of 8.0 and KM= 35 µM. The enzyme did not hydrolyzed casein, albumin, hemoglobin and gelatin but was active against small peptide substrates. This fact may characterize the enzyme as an oligopeptidase. Using a panel of inhibitors against the various proteases classes, it was showed that it was inhibited by benzamidine, PMSF and aprotinin (inhibitors of serine proteases indicating that this enzyme is a serine oligopeptidase.
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