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Serine protease activities in Leishmania (Leishmania) chagasi promastigotes

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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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Abbreviations

BSA:

bovine serum albumin

EDTA:

ethylenediaminetetraacetic acid

E-64:

l-trans-epoxysuccinyleucylamido-(4-guanidino) butane

SDS-PAGE:

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

L-TAME:

N-ρ-tosyl-l-arginine methyl ester

TLCK:

N-tosyl-lysine chloromethyl ketone

TPCK:

N-tosyl-l-phenylalanine chloromethyl ketone

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Acknowledgments

This work was supported with grants from Fundação Oswaldo Cruz (FIOCRUZ) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). We are grateful to Mr. G. A. de Souza for his technical assistance on Leishmania cultures, Mr. A. L. Antelo for his computer assistance, and Dr. M. R. Lishon for the English review and revision.

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Authors and Affiliations

  1. Laboratório de Bioquímica de Proteínas e Peptídeos, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Rio de Janeiro, Brazil

    Raquel Elisa da Silva-López, Tatiana Resende dos Santos & Salvatore Giovanni de Simone

  2. Departamento de Produtos Naturais, Farmanguinhos, FIOCRUZ, Avenida Brasil 4365, 21045-900, Rio de Janeiro, Rio de Janeiro, Brazil

    Raquel Elisa da Silva-López

  3. Grupo de Biologia Estrutural, Centro de Pesquisas, Instituto Nacional do Câncer, INCa, Rio de Janeiro, Rio de Janeiro, Brazil

    José Andrés Morgado-Díaz & Marcelo Neves Tanaka

  4. Departamento de Bioquímica e Biologia Molecular, Instituto de Biologia, UFF, Niterói, Rio de Janeiro, Brazil

    Salvatore Giovanni de Simone

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  1. Raquel Elisa da Silva-López
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  2. Tatiana Resende dos Santos
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Correspondence to Raquel Elisa da Silva-López.

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da Silva-López, R.E., dos Santos, T.R., Morgado-Díaz, J.A. et al. Serine protease activities in Leishmania (Leishmania) chagasi promastigotes. Parasitol Res 107, 1151–1162 (2010). https://doi.org/10.1007/s00436-010-1983-y

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