Skip to main content Accessibility help
×
Home
Hostname: page-component-6f6fcd54b-6rwzl Total loading time: 0.272 Render date: 2021-05-11T11:55:09.906Z Has data issue: true Feature Flags: {}

Article contents

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
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
E-mail address:

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.

Type
Research Article
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.

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, 339344.CrossRefGoogle Scholar
Berti, P. J. and Storer, A. C. (1995). Alignment/phylogeny of the papain superfamily of cysteine proteases. Journal of Molecular Biology 246, 273283.CrossRefGoogle ScholarPubMed
Blackman, M. (2000). Proteases involved in erythrocyte invasion by the malaria parasite: function and potential as chemotherapeutic targets. Current Drug Targets 1, 5983.CrossRefGoogle ScholarPubMed
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, 81498157.CrossRefGoogle ScholarPubMed
Chen, X. M., Keithly, J. S., Paya, C. V. and LaRusso, N. F. (2002). Cryptosporidosis. New England Journal of Medicine 346, 17231731.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, 54925503.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, 225231.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, 10621068.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, 889892.CrossRefGoogle ScholarPubMed
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, 63116318.CrossRefGoogle Scholar
Hoff, E. F. and Carruthers, V. B. (2002). Is Toxoplasma egress the first step in invasion? Trends in Parasitology 18, 251255.CrossRefGoogle ScholarPubMed
Kim, K. (2004). Role of proteases in host cell invasion by Toxoplasma gondii and other Apicomplexa. Acta Tropica 91, 6981.CrossRefGoogle ScholarPubMed
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, 3845.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, 529538.CrossRefGoogle ScholarPubMed
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, 493502.CrossRefGoogle ScholarPubMed
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, 7788.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, 34843491.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, 381385.CrossRefGoogle ScholarPubMed
Peterson, C. (1992). Cryptosporidiosis in patients with human immunodeficiency virus. Clinical Infectious Diseases 15, 903909.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, 117126.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, 2579125797.CrossRefGoogle ScholarPubMed
Rosenthal, P. J. (2002). Hydrolysis of erythrocyte proteins by proteases of malaria parasites. Current Opinions in Hematology 9, 140145.CrossRefGoogle ScholarPubMed
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, 119132.CrossRefGoogle ScholarPubMed
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, 2900029010.CrossRefGoogle ScholarPubMed
Shinde, U. and Inouye, M. (2000). Intramolecular chaperones: polypeptide extensions that modulate protein folding. Seminars in Cell and Developmental Biology 11, 3544.CrossRefGoogle ScholarPubMed
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, 481489.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, 1491014915.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, 2048720495.Google 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, 679688.CrossRefGoogle ScholarPubMed
Tzipori, S. and Ward, H. (2002). Cryptosporidosis: biology, pathogenesis and disease. Microbes and Infection 4, 10471058.CrossRefGoogle 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, 1083810846.CrossRefGoogle ScholarPubMed
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, 167178.3.0.CO;2-Z>CrossRefGoogle ScholarPubMed

Send article to Kindle

To send this article to your Kindle, first ensure no-reply@cambridge.org is added to your Approved Personal Document E-mail List under your Personal Document Settings on the Manage Your Content and Devices page of your Amazon account. Then enter the ‘name’ part of your Kindle email address below. Find out more about sending to your Kindle. Find out more about sending to your Kindle.

Note you can select to send to either the @free.kindle.com or @kindle.com variations. ‘@free.kindle.com’ emails are free but can only be sent to your device when it is connected to wi-fi. ‘@kindle.com’ emails can be delivered even when you are not connected to wi-fi, but note that service fees apply.

Find out more about the Kindle Personal Document Service.

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

Send article to Dropbox

To send this article to your Dropbox account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Dropbox.

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

Send article to Google Drive

To send this article to your Google Drive account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Google Drive.

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

Reply to: Submit a response


Your details


Conflicting interests

Do you have any conflicting interests? *