Serine protease activities in Leishmania (Leishmania) chagasi promastigotes |
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Authors: | Raquel Elisa da Silva-López Tatiana Resende dos Santos José Andrés Morgado-Díaz Marcelo Neves Tanaka Salvatore Giovanni de Simone |
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Institution: | 1. Laboratório de Bioquímica de Proteínas e Peptídeos, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Rio de Janeiro, Brazil 2. Departamento de Produtos Naturais, Farmanguinhos, FIOCRUZ, Avenida Brasil 4365, 21045-900, Rio de Janeiro, Rio de Janeiro, Brazil 3. Grupo de Biologia Estrutural, Centro de Pesquisas, Instituto Nacional do Cancer, INCa, Rio de Janeiro, Rio de Janeiro, Brazil 4. Departamento de Bioquímica e Biologia Molecular, Instituto de Biologia, UFF, Niterói, Rio de Janeiro, Brazil
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Abstract: | The present work reports the isolation, biochemical characterization, and subcellular location of serine proteases from aqueous,
detergent soluble, and culture supernatant of Leishmania chagasi promastigote extracts, respectively, LCSII, LCSI, and LCSIII. The active enzyme molecular masses of LCSII were about 105,
66, and 60 kDa; of LCSI, 60 and 58 kDa; and of LCSIII, approximately 76 and 68 kDa. Optimal pH for the enzymes was 7.0 for
LCSI and LCSIII and 8.5 for LCSII, and the optimal temperature for all enzymes was 37°C, using α-N-ρ-tosyl-l-arginine methyl ester as substrate. Assay of thermal stability indicated that LCSIII is the more stable enzyme. Hemoglobin,
bovine serum albumin, and ovalbumin were hydrolyzed by LCSII and LCSI but not by LCSIII. Inhibition studies suggested that
enzymes belong to the serine protease class modulated by divalent cations. Rabbit antiserum against 56-kDa serine protease
of Leishmania amazonensis identified proteins in all extracts of L. chagasi. Furthermore, immunocytochemistry demonstrated that serine proteases are located in flagellar pocket region and cytoplasmic
vesicles of L. chagasi promastigotes. These findings indicate that L. chagasi serine proteases differ from L. amazonensis proteases and all known flagellate proteases, but display some similarities with serine proteases from other Leishmania species, suggesting a conservation of this enzymatic activity in the genus. |
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