Hostname: page-component-586b7cd67f-dsjbd Total loading time: 0 Render date: 2024-11-26T16:56:46.773Z Has data issue: false hasContentIssue false
  • English
  • Français

Cryptopain-1, a cysteine protease of Cryptosporidium parvum, does not require the pro-domain for folding

Published online by Cambridge University Press:  18 December 2008

B.-K. NA ,
J.-M. KANG ,
H.-I. CHEUN ,
S.-H. CHO ,
S.-U. MOON ,
T.-S. KIM  and
W.-M. SOHN
B.-K. NA*
Affiliation:
Department of Parasitology and Institute of Health Sciences, College of Medicine, Gyeongsang National University, Jinju660-751, Korea
J.-M. KANG
Affiliation:
Department of Parasitology and Institute of Health Sciences, College of Medicine, Gyeongsang National University, Jinju660-751, Korea
H.-I. CHEUN
Affiliation:
Department of Malaria and Parasitic Diseases, National Institute of Health, Centers for Disease Control and Prevention, Seoul122-701, Korea
S.-H. CHO
Affiliation:
Department of Malaria and Parasitic Diseases, National Institute of Health, Centers for Disease Control and Prevention, Seoul122-701, Korea
S.-U. MOON
Affiliation:
Department of Malaria and Parasitic Diseases, National Institute of Health, Centers for Disease Control and Prevention, Seoul122-701, Korea
T.-S. KIM
Affiliation:
Department of Parasitology and Inha Research Institute for Medical Sciences, Inha University College of Medicine, Incheon400-751, Korea
W.-M. SOHN
Affiliation:
Department of Parasitology and Institute of Health Sciences, College of Medicine, Gyeongsang National University, Jinju660-751, Korea
*
*Corresponding author: Department of Parasitology and Institute of Health Sciences,College of Medicine, Gyeongsang National University, Jinju660-751, Korea. Tel: +82 55 751 8822. Fax: +82 55 759 4022. E-mail: [email protected]
Get access Rights & Permissions [Opens in a new window]

Summary

Cryptosporidium parvum is an intracellular protozoan parasite that causes cryptosporidiosis in mammals including humans. In the current study, the gene encoding the cysteine protease of C. parvum (cryptopain-1) was identified and the biochemical properties of the recombinant enzyme were characterized. Cryptopain-1 shared common structural properties with cathepsin L-like papain family enzymes, but lacked a typical signal peptide sequence and contained a possible transmembrane domain near the amino terminus and a unique insert in the front of the mature domain. The recombinant cryptopain-1 expressed in Escherichia coli and refolded to the active form showed typical biochemical properties of cathepsin L-like enzymes. The folding determinant of cryptopain-1 was characterized through multiple constructs with or without different lengths of the pro-domain of the enzyme expressed in E. coli and assessment of their refolding abilities. All constructs, except one that did not contain the full-length mature domain, successfully refolded into the active enzymes, suggesting that cryptopain-1 did not require the pro-domain for folding. Western blot analysis showed that cryptopain-1 was expressed in the sporozoites and the enzyme preferentially degraded proteins, including collagen and fibronectin, but not globular proteins. This suggested a probable role for cryptopain-1 in host cell invasion and/or egression by the parasite.

Keywords

Cryptosporidium parvumcysteine proteasecryptopain-1pro-domainfolding
Type
Research Article
Information
Parasitology , Volume 136 , Issue 2 , February 2009 , pp. 149 - 157
Copyright
Copyright © 2008 Cambridge University Press

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Baker, D., Silen, J. L. and Agard, D. A. (1992). Protease proregion required for folding is a potent inhibitor of the mature enzyme. Proteins-Structure Function and Bioinformatics 12, 339–344.CrossRefGoogle Scholar
Berti, P. J. and Storer, A. C. (1995). Alignment/phylogeny of the papain superfamily of cysteine proteases. Journal of Molecular Biology 246, 273–283.Google Scholar
Blackman, M. (2000). Proteases involved in erythrocyte invasion by the malaria parasite: function and potential as chemotherapeutic targets. Current Drug Targets 1, 59–83.Google Scholar
Carmona, E., Dufour, E., Plouffe, C., Takebe, S., Mason, P., Mort, J. and Menard, R. (1996). Potency and selectivity of the cathepsin L propeptide as an inhibitor of cysteine proteases. Biochemistry 35, 8149–8157.CrossRefGoogle ScholarPubMed
Chen, X. M., Keithly, J. S., Paya, C. V. and LaRusso, N. F. (2002). Cryptosporidosis. New England Journal of Medicine 346, 1723–1731.CrossRefGoogle Scholar
Coulombe, R., Grochulski, P., Sivaraman, J., Menard, R., Mort, J. S. and Cygler, M. (1996). Structure of human procathepsin L reveals the molecular basis of inhibition by the pro-segment. The EMBO Journal 15, 5492–5503.CrossRefGoogle Scholar
Ctrnacta, V., Ault, J. G., Stejskal, F. and Keithly, J. S. (2006). Localization of pyruvate: NADP+ oxidoreductase in sporozoites of Cryptosporidium parvum. The Journal of Eukaryotic Microbiology 53, 225–231.CrossRefGoogle ScholarPubMed
Dasaradhi, P. V., Mohmmed, A., Kumar, A., Hossain, M. J., Bhatnagar, R. K., Chauhan, V. S. and Malhotra, P. (2005). A role of falcipain-2, principal cysteine proteases of Plasmodium falciparum in merozoite egression. Biochemical and Biophysical Research Communications 336, 1062–1068.CrossRefGoogle ScholarPubMed
Forney, J. R., Yang, S. and Healey, M. C. (1996). Protease activity associated with excystation of Cryptosporidium parvum oocysts. The Journal of Parasitology 82, 889–892.Google Scholar
Guay, J., Falgueyret, J. P., Ducret, A., Percival, M. D. and Mancini, J. A. (2000). Potency and selectivity of inhibition of cathepsin K, L and S by their respective propeptides. European Journal of Biochemistry 267, 6311–6318.CrossRefGoogle Scholar
Hoff, E. F. and Carruthers, V. B. (2002). Is Toxoplasma egress the first step in invasion? Trends in Parasitology 18, 251–255.CrossRefGoogle ScholarPubMed
Kim, K. (2004). Role of proteases in host cell invasion by Toxoplasma gondii and other Apicomplexa. Acta Tropica 91, 69–81.Google Scholar
Kumar, A., Dasaradhi, P. V., Chauhan, V. S. and Malhotra, P. (2004). Exploring the role of putative active site amino acids and pro-region motif of recombinant falcipain-2: a principal hemoglobinase of Plasmodium falciparum. Biochemical and Biophysical Research Communications 317, 38–45.CrossRefGoogle ScholarPubMed
Na, B. K., Shenai, B. R., Sijwali, P. S., Choe, Y., Pandey, K. C., Singh, A., Craik, C. S. and Rosenthal, P. J. (2004). Identification and biochemical characterization of vivapains, cysteine proteases of the malaria parasite Plasmodium vivax. The Biochemical Journal 378, 529–538.Google Scholar
Na, B. K., Kang, J. M. and Sohn, W. M. (2008). CsCF-6, a novel cathepsin F-like cysteine protease for nutrient uptake of Clonorchis sinensis. International Journal for Parasitology 38, 493–502.Google Scholar
Nesterenko, M. V., Tilley, M. and Upton, S. J. (1995). A metallo-dependent cysteine proteinase of Cryptosporidium parvum associated with the surface of sporozoites. Microbios 83, 77–88.Google ScholarPubMed
Pandey, K. C., Sijwali, P. S., Singh, A., Na, B. K. and Rosenthal, P. J. (2004). Independent intramolecular mediators of folding, activity, and inhibition for the Plasmodium falciparum cysteine protease falcipain-2. Journal of Biological Chemistry 279, 3484–3491.CrossRefGoogle ScholarPubMed
Pasquier, C., Promponas, V. J., Palaios, G. A., Hamodrakas, J. S. and Hamodrakas, S. J. (1999). A novel method for predicting transmembrane segments in proteins based on a statistical analysis of the SwissProt database: the PRED-TMR algorithm. Protein Engineering 12, 381–385.Google Scholar
Peterson, C. (1992). Cryptosporidiosis in patients with human immunodeficiency virus. Clinical Infectious Diseases 15, 903–909.CrossRefGoogle Scholar
Priest, J. W., Xie, L., Arrowood, M. J. and Lammie, P. J. (2001). The immunodominant 17 kDa antigen from Cryptosporidium parvum is glycosylphosphatidylinositol-anchored. Molecular and Biochemical Parasitology 13, 117–126.CrossRefGoogle Scholar
Que, X., Ngo, H., Lawton, J., Gray, M., Liu, Q., Engel, J., Brinen, L., Ghosh, P., Joiner, K. A. and Reed, S. L. (2002). The cathepsin B of Toxoplasma gondii, toxopain-1, is critical for parasite invasion and rhoptry protein processing. Journal of Biological Chemistry 277, 25791–25797.CrossRefGoogle ScholarPubMed
Rosenthal, P. J. (2002). Hydrolysis of erythrocyte proteins by proteases of malaria parasites. Current Opinions in Hematology 9, 140–145.Google Scholar
Shaw, M. K., Roos, D. S. and Tilney, L. G. (2002). Cysteine and serine protease inhibitors block intracellular development and disrupt the secretory pathway of Toxoplasma gondii. Microbes and Infection 4, 119–132.Google Scholar
Shenai, B. R., Sijwali, P. S., Singh, A. and Rosenthal, P. J. (2000). Characterization of native and recombinant falcipain-2, a principal trophozoite cysteine protease and essential hemoglobinase of Plasmodium falciparum. Journal of Biological Chemistry 275, 29000–29010.CrossRefGoogle ScholarPubMed
Shinde, U. and Inouye, M. (2000). Intramolecular chaperones: polypeptide extensions that modulate protein folding. Seminars in Cell and Developmental Biology 11, 35–44.Google Scholar
Sijwali, P. S., Shenai, B. R., Gut, J., Singh, A. and Rosenthal, P. J. (2001). Expression and characterization of the Plasmodium falciparum haemoglobinase falcipain-3. The Biochemical Journal 360, 481–489.CrossRefGoogle ScholarPubMed
Sijwali, P. S., Shenai, B. R. and Rosenthal, P. J. (2002). Folding of the Plasmodium falciparum cysteine protease falcipain-2 is mediated by a chaperone-like peptide and not the prodomain. Journal of Biological Chemistry 277, 14910–14915.CrossRefGoogle Scholar
Smith, S. M. and Gottesman, M. M. (1989). Activity and deletion analysis of recombinant human cathepsin L expressed in Escherichia coli. Journal of Biological Chemistry 264, 20487–20495.CrossRefGoogle ScholarPubMed
Teo, C. F., Zhou, X. W., Bogyo, M. and Carruthers, V. B. (2007). Cysteine protease inhibitors block Toxoplasma gondii microneme secretion and cell invasion. Antimicrobial Agents and Chemotherapy 51, 679–688.CrossRefGoogle ScholarPubMed
Tzipori, S. and Ward, H. (2002). Cryptosporidosis: biology, pathogenesis and disease. Microbes and Infection 4, 1047–1058.Google Scholar
Vernet, T., Berti, P. J., de Montigny, C., Musil, R., Tessier, D. C., Menard, R., Magny, M. C., Storer, A. C. and Thomas, D. Y. (1995). Processing of the papain precursor. Journal of Biological Chemistry 270, 10838–10846.Google Scholar
Yamamoto, Y., Watabe, S., Kageyama, T. and Takahashi, S. Y. (1999). Proregion of Bombyx mori cysteine proteinase functions as an intramolecular chaperone to promote proper folding of the mature enzyme. Archives of Insect Biochemistry and Physiology 42, 167–178.3.0.CO;2-Z>CrossRefGoogle ScholarPubMed