Skip to main content
SpringerLink
Log in

Calcium-dependent proteolytic activity of a cysteine protease caldonopain is detected during Leishmania infection

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

A calcium-activated protease caldonopain in the cytosolic fraction of Leishmania donovani has been found to digest different endogenous proteins when subjected to SDS-PAGE. Gelatin-embedded gel electrophoresis confirms presence of calcium-dependent protease activity. Ca2+ affects proteolytic activity after 10 h. When host–parasite interaction was conducted in vitro, caldonopain was found to be active after 10 h of incubation with calcium. A 67-kDa protein is specifically digested during this time and two new proteins of 45 and 36 kDa appeared in SDS-PAGE electrophoregram. This belated action of calcium towards protease activity may be pre-requisite to facilitate invasion of host tissues and thereby mediate protein metabolism during survival of this pathogen both independently and intracellularly. It is likely that calcium metabolism in promastigotes and amastigotes does not propagate in the same manner. Involvement of calcium to initiate caldonopain activity may be critically associated with signal transduction pathways which may be responsible for the pathobiological action of this parasite. We propose that caldonopain could be a potential target to develop new chemotherapeutic approach against leishmaniasis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  1. Nuck IH, Sharma JD: Factors in host–parasite interactions and immunological unresponsiveness in leishmaniasis. Curr Sci 61: 460–466, 1991

    Google Scholar 

  2. Gries KD, Turco SJ: Purification and characterization of an extracellular phosphoglycan from Leishmania donovani. J Biol Chem 267: 5876–5881, 1992

    Google Scholar 

  3. Williams RJ: Calcium. Meth Mol Biol 172: 21–49, 2002

    CAS  Google Scholar 

  4. Carafoli E: Calcium signalling: a tale for all seasons. Proc Natl Acad Sci USA 99: 1115–1122, 2002

    Article  PubMed  CAS  Google Scholar 

  5. Crifo C, Capuozzo E, Ovadi J, Salemo C: ATP-triggered calcium signals in human. Adv Exp Med Biol 486: 197–200, 2000

    PubMed  CAS  Google Scholar 

  6. Poenie M, Alderton J, Steinhardt R, Tsien R: Calcium rises abruptly and briefly throughout the cell at the onset of anaphase. Science 233: 886–889, 1986

    PubMed  CAS  Google Scholar 

  7. Philosolph H, Zilberstein D: Regulation of intracellular calcium in promastigotes of the human protozoan parasite Leishmania donovani. J Biol Chem 264: 10420–10424, 1989

    Google Scholar 

  8. Schettino PMS, Majumdar S, Kierszenbaum F: Regulatory effect of the level of free calcium on the host cell and on the capacity of Trypanosoma cruzi to invade and multiply intracellularly. J Parasitol 81: 597–602, 1995

    PubMed  CAS  Google Scholar 

  9. Olivier M, Baimbridge KG, Reiner NE: Stimulus–response coupling in monocytes infected with Leishmania, attenuation of calcium transients is related to defective agonist-induced accumulation of inositol phosphates. J Immunl 148: 1188–1196, 1992

    CAS  Google Scholar 

  10. Bhattacharya J, Dey R, Datta SC: Calcium dependent thiol protease caldonopain and its specific endogenous inhibitor in Leishmania donovani. Mol Cell Biochem 126: 9–16, 1993

    Article  PubMed  CAS  Google Scholar 

  11. Streit JA, Donelson JE, Agey MW, Wilson ME: Developmental changes in the expression of Leishmania chagasi gp63 and heat shock protein in a human macrophage. Infect Immunol 64: 1810–1818, 1996

    CAS  Google Scholar 

  12. Bartholomeu DC, Batista J, Vainstein MH, Lima BD, de Sa MC: Molecular cloning & characterization of a gene encoding the 29 kDa proteosome subunit from Trypanosoma cruzi. Mol Genet Genom 265: 986–992, 2001

    CAS  Google Scholar 

  13. Rosenthal PJ: Cysteine proteases of malaria parasites. Int J Parasitol 341: 489–499, 2004

    Google Scholar 

  14. Ascenzi P, Bocedi A, Gentile M, Visca P, Gradoni L: Inactivation of parasite cysteine proteinases by the NO-donor 4-(phenylsulfonyl)-3-((2-(dimethylamino)ethyl)thio)-furoxan oxalate. Biochim Biophys Acta 1703: 69–77, 2004

    PubMed  CAS  Google Scholar 

  15. Choe Y, Brinen LS, Price MS, Engel JC, Lange M, Grisostomi C, Weston SG, Pallai PV, Cheng H, Hardy LW, Hartsough DS, McMakin M, Tilton RF, Baldino CM, Craik CS: Development of alpha-keto-based inhibitors of cruzain, a cysteine protease implicated in Chagas disease. Bioorg Med Chem 13: 2141–2156, 2005

    PubMed  CAS  Google Scholar 

  16. Zeng Y, Li Q, Hanzlik RP, Aube J: Synthesis of a small library of diketopiperazines as potential inhibitors of calpain. Bioorg Med Chem Lett 15: 3034–3038, 2005

    PubMed  CAS  Google Scholar 

  17. Mottram JC, Coombs GH, Alexander J: Cysteine peptidases as virulence factors of Leishmania. Curr Opin Microbiol 7: 375–381, 2004

    Article  PubMed  CAS  Google Scholar 

  18. Besteiro S, Coombs GH, Mottram JC: A potential role for ICP, a leishmanial inhibitor of cysteine peptidases, in the interaction between host and parasite. Mol Microbiol 54: 1224–1236, 2004

    Article  PubMed  CAS  Google Scholar 

  19. de Souza Dias S, da Costa Pinheiro PH, Katz S, dos Santos MR, Barbieri CL: A recombinant cysteine proteinase from Leishmania (Leishmania) chagasi suitable for serodiagnosis of American visceral leishmaniasis. Am J Trop Med Hygiene 72: 126–132, 2005

    Google Scholar 

  20. Frame MJ, Mottram JC, Coombs GH: Analysis of the roles of cysteine proteinases of Leishmania mexicana in the host–parasite interaction. Parasitology 121: 367–377, 2000

    Article  PubMed  CAS  Google Scholar 

  21. Ueda-Nakamura T, da Conceicao Rocha Sampaio M, Cunha-e-Silva NL, Traub-Cseko YM, de Souza W: Expression and processing of megasome cysteine proteinases during Leishmania amazonensis differentiation. Parasitol Res 88: 332–337, 2002

    Article  PubMed  Google Scholar 

  22. Ray JC: Cultivation of various Leishmania parasites on solid medium. Ind J Med Res 2: 355–357, 1932

    Google Scholar 

  23. Jaffe CL, Gimaldi G, McMohan-Pratt: Genes and Antigens of Parasites, A Laboratory Manual, 2nd edn, 1984

  24. Lockwood BC, North HG, Scott KI, Bremner AF, Coombs GH: The use of a highly sensitive electrophoretic method to compare the proteinases in trichomonads. Mol Biochem Parasitol 24: 88–95, 1987

    Article  Google Scholar 

  25. Hosfield CM, Elce JS, Jia Z: Activation of calpain by Ca2+ roles of the large subunit N-terminal and domain III–IV linker peptides. J Mol Biol 343: 1049–1053, 2004

    Article  PubMed  CAS  Google Scholar 

  26. Coomb GH: Proteinases of Leishmania mexicana and other flagellar protozoa. Parasitology 84: 149–155, 1982

    Google Scholar 

  27. Bordier C: The promastigote surface protease of Leishmania. Parasitol Today 5: 151–153, 1987

    Google Scholar 

  28. Parkes C, Embhavi AA, Barett AJ: Calpain inhibition by peptide epoxides. Biochem J 230: 509–516, 1985

    PubMed  CAS  Google Scholar 

  29. Frame MJ, Mottram JC, Coombs GH: Analysis of the roles of cysteine proteinases of Leishmania mexicana in the host–parasite interaction. Parasitology 121: 367–377, 2000

    Article  PubMed  CAS  Google Scholar 

  30. Bandopadhyay K, Karmakar S, Ghosh A, Das PK: Role of 67 kDa cell surface laminin binding protein of Leishmania donovni in pathogenesis. J Biochem 130: 141–148, 2001

    Google Scholar 

  31. Carafoli E: Intracellular calcium homeostatis. Ann Rev Biochem 56: 395–433, 1987

    PubMed  CAS  Google Scholar 

  32. Philips DR, Jacabova M: Calcium dependent protease in human platelets. J Biol Chem 252: 5602–5605, 1977

    Google Scholar 

  33. Melloni E, Pentremoli S, Michetti M, Sacco O, Sparatore B, Horecker BL: The involvement of calpain in the activation of protein kinase C in neutrophils stimulated by phorbol myristic acid. J Biol Chem 261: 4101–4116, 1986

    PubMed  CAS  Google Scholar 

  34. Rubin H: Serine protease inhibitors (SERPINS): where mechanism meets medicine. Nat Med 2: 632–633, 1996

    Article  PubMed  CAS  Google Scholar 

  35. Dominquez JN, Lopez S, Charris J, Larruso L, Lobo G, Semenov A, Olison JE, Rosenthal PJ: Synthesis and antimalarial effects of phenothazine inhibitors of a Plasmodium falciparum cysteine protease. J Med Chem 40: 2726–2732, 1997

    Google Scholar 

  36. Blackman MJ: Proteases involved in erythrocytic invasion by the malaria parasite function and potential as chemotherapeutic targets. Curr Drug Targets 1: 59–83, 2000

    Article  PubMed  CAS  Google Scholar 

  37. Urbina JA: Chemotherapy of Chagas disease. Curr Pharm Design 8: 287–295, 2002

    Article  CAS  Google Scholar 

  38. Boss C, Richard-Bildstein S, Weller T, Fischli W, Meyer S, Binkert C: Inhibitors of the Plasmodium falciparum parasite aspartic protease Plasmepsin II as potential antimalarial agents. Curr Med Chem 10: 883–927, 2003

    Article  PubMed  CAS  Google Scholar 

  39. McKerrow JM: Parasite proteases. Exp Parasitol 68: 111–115, 1989

    Article  PubMed  CAS  Google Scholar 

  40. Del Nery E, Juliano M, Lima AP, Scharfstein J, Juliano L: Cysteinyl proteinases (cruzipain) from Trypanosoma cruzi. J Biol Chem 272: 2571–2578, 1997

    Article  Google Scholar 

  41. Lima AP, dos Reis FC, Serveau C, Lalmanach G, Juliano L, Menard R, Vernet T, Thomas DY, Storer AC, Scharfstein J: Cysteine protease isoforms from Trypanosoma cruzi, cruzipain 2 and cruzain, present different substrate preference and susceptibility to inhibitors. Mol Biochem Parasitol 114: 41–52, 2001

    Article  PubMed  CAS  Google Scholar 

  42. McGrath ME, Eakin AE, Engel JC, McKerrow JH, Craik CS, Fletterick J: The crystal structure of cruzain: A therapeutic target for Chagas' disease. J Mol Biol 247: 251–259, 1995

    Article  PubMed  CAS  Google Scholar 

  43. Lee BJ, Singh A, Chiang P, Kemp SJ, Goldman EA, Weinehouse MI, Vlasuk GP, Rosenthal PJ: Antimalarial activities of novel synthetic cysteine protease inhibitors. Antimicrob Agents Chemother 47: 3810–3814, 2003

    PubMed  CAS  Google Scholar 

  44. Buxbaum LU, Denise H, Coombs GH, Alexender J, Mottram JC, Scott P: Cysteine protease B of Leishmania mexicana inhibits host Th1 responses and protective immunity. J Immunol 171: 3711–3717, 2004

    Google Scholar 

  45. Na BK, Shenai BR, Sijwali PS, Choe Y, Pandey KC, Singh A, Craik CS, Rosenthal PJ: Identification and biochemical characterization of vivapains, cysteine proteases of the malaria parasite Plasmodium vivax. Biochem J 378: 529–538, 2004

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Author notes

  1. Runu Dey

    Present address: CpG, Institute of Molecular Medicine, BIPL, Salt Lake Electronics Complex, Kolkata, India

  2. Jharna Bhattacharya

    Present address: Division of Biotechnology, Indian Institute of Chemical Biology, Kolkata, India

Authors and Affiliations

  1. Department of Biological Chemistry, Infectious Diseases Group, Indian Institute of Chemical Biology, Kolkata, India

    Runu Dey, Jharna Bhattacharya & Salil C. Datta

  2. Department of Biological Chemistry, Infectious Diseases Group, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata, 700032, India

    Salil C. Datta

Authors
  1. Runu Dey
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jharna Bhattacharya
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Salil C. Datta
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Salil C. Datta.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Dey, R., Bhattacharya, J. & Datta, S.C. Calcium-dependent proteolytic activity of a cysteine protease caldonopain is detected during Leishmania infection. Mol Cell Biochem 281, 27–33 (2006). https://doi.org/10.1007/s11010-006-0171-y

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11010-006-0171-y

Keywords

Search

Navigation