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Characterization of an extracellular serine protease of Leishmania (Leishmania) amazonensis

Published online by Cambridge University Press:  28 February 2005

R. E. da SILVA-LOPEZ ,
M. G. PINTO COELHO  and
S. G. De SIMONE
R. E. da SILVA-LOPEZ
Affiliation:
Laboratório de Bioquímica de Proteínas e Peptídeos, Departamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz, Rio de Janeiro, RJ, Brasil
M. G. PINTO COELHO
Affiliation:
Departamento de Bioquímica, Instituto de Biologia, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ, Brasil
S. G. De SIMONE
Affiliation:
Laboratório de Bioquímica de Proteínas e Peptídeos, Departamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz, Rio de Janeiro, RJ, Brasil Departamento de Bioquímica e Biologia Molecular, Instituto de Biologia, Universidade Federal Fluminense, Rio de Janeiro, Niterói, Brasil
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Abstract

A serine protease was purified 942-fold from culture supernatant of L. amazonensis promastigotes using (NH4)2SO4 precipitation followed by affinity chromatography on aprotinin-agarose and continuous elution electrophoresis by Prep Cell, yielding a total recovery of 61%. The molecular mass of the active enzyme estimated by SDS-PAGE under conditions of reduction was 56 kDa and 115 kDa under conditions of non-reduction, suggesting that the protease is a dimeric protein. Additionally, it was found to be a non-glycosylated enzyme, with a pI of 5·0. The optimal pH and temperature of the enzyme were 7·5 and 28 °C respectively, using α-N-ρ-tosyl-L-arginine-methyl ester (L-TAME) as substrate. Assays of thermal stability indicated that 61% of the enzyme activity was preserved after 1 h of pre-treatment at 42 °C. Haemoglobin, bovine serum albumin (BSA), ovalbumin, fibrinogen, collagen, gelatin and peptide substrates containing arginine in an ester bond and amide substrates containing hydrophobic residues at the P1 site were hydrolysed by this extracellular protease. The insulin β-chain was also hydrolysed by the enzyme and many peptidic bonds were susceptible to the protease action, and 4 of them (L11-V12, E13-A14, L15-Y16 and Y16-L17) were identified. Inhibition studies suggested that the enzyme belongs to the serine protease class inhibited by calcium and manganese and activated by zinc. These findings show that this enzyme of L. amazonensis is a novel serine protease, which differs from all known flagellate proteases characterized.

Keywords

Leishmania amazonensisextracellular serine proteasepurificationcharacterization
Type
Research Article
Information
Parasitology , Volume 131 , Issue 1 , July 2005 , pp. 85 - 96
Copyright
© 2005 Cambridge University Press

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References

REFERENCES

ABRAHAM, L. D., CHOW, D. T. & BREUIL, C. ( 1995). Characterization of the cleavage specificity of a subtilisin-like serine proteases from Ophistoma piceae by liquid chromatography/mass spectrometry and tandem MS. FEBS Letters 374, 208–210.CrossRefGoogle Scholar
ALEXANDER, J. & RUSSEL, D. G. ( 1992). The interaction of Leishmania species and macrophages. Advances in Parasitology 31, 175–254.CrossRefGoogle Scholar
ALVES, C. R., MARZOCHI, M. C. A. & De SIMONE, S. G. ( 1993). Heterogeneity of cysteine protease in Leishmania braziliensis and Leishmania major. Brazilian Journal of Medical Biology Research 26, 167–171.Google Scholar
BARANKIEWICZ, J., DOSH, H. M. & COHEN, A. ( 1988). Extracellular nucleotide catabolism in human B and T lymphocytes. Journal of Biological Chemistry 263, 7094–7098.Google Scholar
BEREZEIN, I. & MARTINEK, K. ( 1970). Specificity of [alfa]-chymotrypsin. FEBS Letters 8, 261–267.CrossRefGoogle Scholar
BURLEIGH, B. A. & ANDREWS, N. A. ( 1995). 120-kDa alkaline peptidase from Trypanosoma cruzi is involved in the generation of a novel calcium-signaling factor for mammalian cells. Journal of Biological Chemistry 270, 5172–5180.CrossRefGoogle Scholar
BURLEIGH, B. A. & WOOLSEY, A. M. ( 2002). Cell signaling and Trypanosoma cruzi invasion. Cellular Microbiology 4, 701–711.CrossRefGoogle Scholar
CHANG, K. P., REED, S. G., McGWIRE, B. S. & SOONG, L. ( 2003). Leishmania model for microbial virulence: the relevance of parasite multiplication and pathoantigenicity. Acta Tropica 85, 375–390.CrossRefGoogle Scholar
CHUNG, L., DINAKARPANDIAN, D., YOSHIDA, N., LAUER-FIELDS, J. L., FIELDS, G. B., VISSE, R. & NAGASE, H. ( 2004). Collagenase unwinds triple-helical collagen prior to peptide bond hydrolysis. EMBO Journal 23, 3020–3030.CrossRefGoogle Scholar
CIBRELEUS, P., PRECIGOUT, E., SERENO, D., CARCY, B., LEMERSE, J. L. & GORENFLOT, A. ( 1999). Secreted antigens of the amastigote and promastigote forms of Leishmania infantum inducing a humoral response in human and dogs. Parasite 6, 121–129.CrossRefGoogle Scholar
CLARK, D. J., HAWRYLIK, S. J., KAVANAGH, E. & OPHEIN, D. J. ( 2000). Purification and characterization of a unique alkaline elastase from Micrococcus luteus. Protein Experimental Purification 18, 46–55.CrossRefGoogle Scholar
CONSEIL, V., SOÊTE, M. & DUBREMETZ, J. F. ( 1999). Serine protease inhibitors block invasion of host cells by Toxoplasma gondii. Antimicrobial agents and Chemotherapy 46, 1358–1361.Google Scholar
COOMBS, G. H. & MOTTRAM, J. C. ( 1997). Proteases in trypanosomatids. In Trypanosomiasis and Leishmaniasis (ed. Hide, G., Mottram, J. C., Coombs, G. H. & Holmes, P. H.), pp. 176–197. CAB International, London.
ERLANGER, B. F., KOKOWSKY, N. & COHEN, W. ( 1961). The preparation and properties of two new chromogenic substrates of trypsin. Archives of Biochemistry and Biophysics 95, 271–278.CrossRefGoogle Scholar
De SIMONE, S. G., AGUIAR, A. S., GIMENEZ, A. R., NOVELINO, K. A. & MOURA, R. S. ( 1997). Purification, properties and N-terminal amino acid sequence of a kalikrein-like enzyme from the venom of Lachesis Muta rhombeata (Bushmaster). Journal of Protein Chemistry 16, 809–818.CrossRefGoogle Scholar
HERWALDT, B. L. ( 1999). Leishmaniasis. Lancet 354, 1191–1199.CrossRefGoogle Scholar
HEU, M. S., KIM, H. R. & PYEUN, J. H. ( 1995). Comparison of trypsin and chymotrypsin from the viscera of anchovy, Engraulis japonic. Comparative Biochemical Physiology B 112, 557–567.CrossRefGoogle Scholar
JAFFE, C. L. & DWYER, D. M. ( 2003). Extracellular releases of the metalloproteases, gp63, from Leishmania and insect trypanosomatids. Parasitology Research 91, 229–237.CrossRefGoogle Scholar
JANG, H. J., KIM, B. C., PYU, Y. R. & KIM, Y. S. ( 2002). A novel subtilisin-like serine protease from Thermoanaerobacter yonseiensis KB-1: its cloning, expression, and biochemical properties. Extremophiles 6, 233–243.CrossRefGoogle Scholar
JOSHI, M. B., MALLINSON, D. J. & DWYER, D. M. ( 2004). The human pathogen Leishmania donovani secretes a histidine acid phosphatase activity that is resistant to proteolytic degradation. Journal Eukaryotic Microbiology 51, 108–112.CrossRefGoogle Scholar
KONG, H. H., KIM, T. H. & CHUNG, D-II. ( 2000). Purification and characterization of a secretory serine protease of Acanthamoeba healyi isolated from GAE. Journal of Parasitology 86, 12–17.CrossRefGoogle Scholar
KUSSMAN, M., NORDHOFF, E., RAHBEK-NIELSEN, H., HAEBEL, S., LARSEN, M. R., JAKBOSEN, L., GOBOM, J., MIRGORODSKAYA, E., KROLL-KRISTENSEN, A., PALM, L. & ROEPSTORFF, P. ( 1997). Matrix-associated laser desorption/ionization mass spectrometry sample preparation techniques designed for various peptide and protein analytes. Journal of Mass Spectrometry 32, 593–601.3.0.CO;2-D>CrossRefGoogle Scholar
LAEMMLI, U. K. ( 1970). Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature, London 227, 680–685.CrossRefGoogle Scholar
LIM, K. C., BAHGAT, M., BUCKS, D., GUY, R., HINZ, R. S., CULLANDER, C. & Mc KERROW, J. H. ( 1999). Blockage of skin invasion by schistosome cercariae by serine protease inhibitors. American Journal of Tropical Medicine and Hygiene 60, 487–492.CrossRefGoogle Scholar
LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. ( 1951). Protein measurement with the Folin reagent. Journal of Biological Chemistry 193, 264–275.Google Scholar
McKERROW, J. H., SUN, E., ROSENTHAL, P. J. & BOUVIER, J. ( 1993). The proteases and the pathogenicity of parasitic protozoa. Annual Review Microbiology 47, 821–853.CrossRefGoogle Scholar
MISRA, S., NASKAR, K., SARKAR, D. & GHOSH, D. K. ( 1991). Role of Ca2+ ion on Leishmania-macrophage attachment. Molecular Cellular Biochemistry 27, 13–18.CrossRefGoogle Scholar
MOCZON, T. ( 1996). A serine protease in the penetration glands of the cercariae of Plagiorchis elegans (Trematoda, Plagioochiidae). Parasitology Research 82, 72–76.CrossRefGoogle Scholar
OVERATH, P., STIERHOF, Y. D. & WIESE, M. ( 1997). Endocytosis and secretion in trypanosomatid parasites-tumultuous traffic in a pocket. Trends in Cell Biology 7, 27–33.CrossRefGoogle Scholar
PIEPER, J. S, VAN DER KRAAN, P. M., HAFMANS, T., KAMP, J., BUMA, P., VAN SUSANTE, J. L., VAN DER BERG, W. B, VEERKAMP, J. H. & VAN KUPPEVELT, T. H. ( 2002). Crosslinked type II collagen matrices: preparation, characterization, and potential for cartilage engineering. Biomaterials 23, 3183–3192.CrossRefGoogle Scholar
PRASAD, A., KAUR, S., MALLA, N., GANGULY, N. K. & MAHAJAN, R. C. ( 2001). Ca++ signaling in the transformation of promastigotes to axenic amastigotes of Leishmania donovani. Molecular Cellular Biochemistry 224, 39–44.CrossRefGoogle Scholar
PUPKINS, M. E., TETLEY, L. & COOMBS, G. H. ( 1986). Leishmania mexicana: Amastigote hydrolases in unusual lysosomes. Experimental Parasitology 62, 29–39.CrossRefGoogle Scholar
RHOADS, M. L. & FETTERER, R. H. ( 1997). Extracellular matrix: a tool for defining the extracorporeal function of parasite proteases. Parasitology Today 13, 119–122.CrossRefGoogle Scholar
RIBEIRO de ANDRADE, A., SANTORO, M. M., NORMA de MELO, M. & MARES-GUIA, M. ( 1998). Leishmania (Leishmania) amazonensis: purification and enzyme characterization of a soluble serine oligopeptidase from promastigotes. Experimental Parasitology 89, 153–160.CrossRefGoogle Scholar
RODRIGUES MACEDO, M. L., MACHADO FREIRE, M. G., CABRINI, E. C., TOYAMA, M. H., NOVELLO, J. C. & MARANGONI, S. ( 2003). A trypsin inhibitor from Peltophorum dubium seeds active against pest proteases and its effect on the survival of Anagasta kuehniella (Lepidoptera: Pyralidae). Biochimica et Biophysica Acta 2, 170–182.CrossRefGoogle Scholar
ROSENTHAL, P. J. ( 1999). Proteases of protozoan parasites. Advances in Parasitology 43, 105–159.CrossRefGoogle Scholar
ROUGGWILLE, E., BÉTOULLE, M. E. M, BLISNICK, T. & BRAUN-BRETON, C. ( 1996). A role for erythrocyte band 3 by the parasite gp76 serine protease in the formation of the parasitophorous vacuole during the invasion of erythrocytes by Plasmodium falciparum. Molecular Biochemical Parasitology 82, 13–24.CrossRefGoogle Scholar
SALTER, J. P., KEE-CHONG, L., HANSELL, E., HSIEH, I. & McKERROW, J. H. ( 2000). Schistosoma invasion of human skin and degradation of dermal elastin are mediated by a single serine protease. Journal of Cellular Biology 275, 38667–38673.Google Scholar
SANTANA, J. M., GRELLIER, P., SCHERÉVEL, J. & TEIXEIRA, A. R. ( 1997). A Trypanosoma cruzi-secreted 80 kDa protease with specificity for human collagen types I and IV. Biochemical Journal 324, 129–137.CrossRefGoogle Scholar
SEGLEN, P. O. & BOHLEY, P. ( 1992). Autophagy and other vacuolar protein degradation mechanisms. Experientia 48, 158–172.CrossRefGoogle Scholar
SENGPUTA, S., TRIPATHI, T., TANDON, R., RAJE, M., ROY, R. P., BASU, S. K. & MUKHOPADHYAY, A. ( 1999). Hemoglobin endocytosis in Leishmania is mediated through a 46-kDa protein located in the flagellar pocket. Journal of Biological Chemistry 274, 2758–2765.CrossRefGoogle Scholar
SILVA-LOPEZ, R. E., MORGADO-DÍAZ, J. A., ALVES, C. R., CÔRTE-REAL, S. & GIOVANNI De SIMONE, S. ( 2004). Subcellular localization of an extracellular serine protease in Leishmania (Leishmania) amazonensis. Parasitology Research 93, 328–331.CrossRefGoogle Scholar
SOUSA, M. O., MIRANDA, T. S. L., MAIA, C. N., BITTAR, E. R., SANTORO, M. M. & FIGUEIREDO, A. F. S. ( 2002). Kinetic peculiarities of human tissue kallikrein: 1-Substrate activation in the catalyzed hydrolysis of H-D-valyl-leucyl-L-arginine 4-nitroanilide and H-D-valyl-leucyl-L-lysine 4-nitroanilide; 2-Substrate inhibition in the catalysed hydrolysis of N-ρ-tosyl-L-arginine methyl ester. Archives of Biochemistry and Biophysics 400, 7–14.CrossRefGoogle Scholar
TODORA, V. K. & STOYANOV, D. I. ( 2000). Partial characterization of a serine protease secreted by adult Trichinella spiralis. Parasitology Research 86, 684–687.CrossRefGoogle Scholar
TROEBERG, L., PIKE, R. N., MORTY, R. E., BERRY, R. K., COETHER, T. H. T. & LONSDALE-ECCLES, J. D. ( 1996). Proteases from Trypanosoma brucei brucei. European Journal of Biochemistry 238, 728–736.CrossRefGoogle Scholar
UBEIDAT, M. & RUTHERFORD, C. L. ( 2002). Expression and one-step purification of a developmentally regulated protein from Dictyostelium discoideum. Protein Expression and Purification 25, 472–480.CrossRefGoogle Scholar
WEBB, J. R., CAMPOS-NETO, A., OVENDALE, P. J., MARTIN, T. I., STROMBERG, E. J., BADARÓ, R. & REED, S. G. ( 1998). Human and murine immune responses to a novel Leishmania major recombinant protein encoded by members of a multicopy gene family. Infection and Immunity 66, 3279–3289.Google Scholar
ZHAO, J., LI, L., WU, C. & HE, R. Q. ( 2003). Hydrolysis of fibrinogen and plasminogen by immobilized earthworm fibrinolytic enzyme II from Eisenia fetida. International Journal of Biological Macromolecules 32, 165–171.CrossRefGoogle Scholar