Skip to main content Accessibility help
×
Home

Molecular cloning and characterization of a M17 leucine aminopeptidase of Cryptosporidium parvum

  • J.-M. KANG (a1), H.-L. JU (a1), W.-M. SOHN (a1) and B.-K. NA (a1)

Summary

Leucine aminopeptidases (LAPs) are a group of metalloexopeptidases that catalyse the sequential removal of amino acids from the N-termini of polypeptides or proteins. They play an important role in regulating the balance between catabolism and anabolism in living cells. LAPs of apicomplexa parasitic protozoa have been intensively investigated due to their crucial roles in parasite biology as well as their potentials as drug targets. In this study, we identified an M17 leucine aminopeptidase of Cryptosporidium parvum (CpLAP) and characterized the biochemical properties of the recombinant protein. Multiple sequence alignment of the deduced amino acid sequence of CpLAP with those of other organisms revealed that typical amino acid residues essential for metal binding and active-site formation in M17 LAPs were well conserved in CpLAP. Recombinant CpLAP shared similar biochemical properties such as optimal pH, stability at neutral pHs, and metal-binding characteristics with other characterized LAPs. The enzyme showed a marked preference for Leu and its activity was effectively inhibited by bestatin. These results collectively suggest that CpLAP is a typical member of the M17 LAP family and may play an important role in free amino acid regulation in the parasite.

Copyright

Corresponding author

*Corresponding author: Tel: +82 55 751 8822. Fax: +82 55 759 4022. E-mail: bkna@gnu.ac.kr

References

Hide All
Abe, F., Alvord, G., Koyama, M., Matsuda, A. and Talmadge, J. E. (1989). Pharmacokinetics of bestatin and oral activity for treatment of experimental metastases. Cancer Immunology and Immunotherapy 28, 2933.
Blackman, M. J. (2008). Malarial proteases and host cell egress: an ‘emerging’ cascade. Cellular Microbiology 10, 19251934.
Dalal, S. and Klemba, M. (2007). Roles for two aminopeptidases in vacuolar hemoglobin catabolism in Plasmodium falciparum. The Journal of Biological Chemistry 282, 3597835987.
Fayer, R., Morgan, U. and Upton, S. J. (2000). Epidemiology of Cryptosporidium transmission, detection and identification. International Journal for Parasitology 30, 13051322.
Feng, X., Akiyoshi, D. E., Widmer, G. and Tzipori, S. (2007). Characterization of subtilase protease in Cryptosporidium parvum and C. hominis. Journal of Parasitology 93, 619626.
Forney, J. R., Yang, S., Du, C. and Healey, M. C. (1996). Efficacy of serine protease inhibitors against Cryptosporidium parvum infection in a bovine fallopian tube epithelial cell culture system. Journal of Parasitology 82, 638640.
Gardiner, D. L., Trenholme, K. R., Skinner-Adams, T. S., Stack, C. M. and Dalton, J. P. (2006). Overexpression of leucyl aminopeptidase in Plasmodium falciparum parasites. Target for the antimalarial activity of bestatin. The Journal of Biological Chemistry 281, 17411745.
Gavigan, C. S., Dalton, J. P. and Bell, A. (2001). The role of aminopeptidases in haemoglobin degradation in Plasmodium falciparum-infected erythrocytes. Molecular and Biochemical Parasitology 117, 3748.
Goldberg, D. E. (2005). Hemoglobin degradation. Current Topics of Microbiology and Immunology 295, 275291.
Heiges, M., Wang, H., Robinson, E., Aurrecoechea, C., Gao, X., Kaluskar, N., Rhodes, P., Wang, S., He, C. Z., Su, Y., Miller, J., Kraemer, E. and Kissinger, J. C. (2006). CryptoDB: a Cryptosporidium bioinformatics resource update. Nucleic Acids Research 34(Database issue), D419D422.
Iwaki, S., Nakamura, T. and Koyama, J. (1986). Inhibitory effects of various synthetic substrates for aminopeptidases on phagocytosis of immune complexes by macrophages. Journal of Biochemistry (Tokyo) 99, 13171326.
Jia, H., Nishikawa, Y., Luo, Y., Yamagishi, J., Sugimoto, C. and Xuan, X. (2010). Characterization of a leucine aminopeptidase from Toxoplasma gondii. Molecular and Biochemical Parasitology 170, 16.
Jia, H., Terkawi, M. A., Aboge, G. O., Goo, Y. K., Luo, Y., Li, Y., Yamagishi, J., Nishikawa, Y., Igarashi, I., Sugimoto, C., Fujisaki, K. and Xuan, X. (2009). Characterization of a leucine aminopeptidase of Babesia gibsoni. Parasitology 136, 945952.
Kim, K. (2004). Role of proteases in host cell invasion by Toxoplasma gondii and other Apicomplexa. Acta Tropica 91, 6981.
Kim, H. and Lipscomb, W. N. (1993). Differentiation and identification of the two catalytic metal binding sites in bovine lens leucine aminopeptidase by x-ray crystallography. Proceedings of the National Academy of Sciences, USA 90, 50065010.
Kosek, M., Alcantara, C., Lima, A. A. and Guerrant, R. L. (2001). Cryptosporidiosis: an update. Lancet Infectious Diseases 1, 262269.
Lee, J. Y., Song, S. M., Seok, J. W., Jha, B. K., Han, E. T., Song, H. O., Yu, H. S., Hong, Y., Kong, H. H. and Chung, D. I. (2010). M17 leucine aminopeptidase of the human malaria parasite Plasmodium vivax. Molecular and Biochemical Parasitology 170, 4548.
Maric, S., Donnelly, S. M., Robinson, M. W., Skinner-Adams, T., Trenholme, K. R., Gardiner, D. L., Dalton, J. P., Stack, C. M. and Lowther, J. (2009). The M17 leucine aminopeptidase of the malaria parasite Plasmodium falciparum: importance of active site metal ions in the binding of substrates and inhibitors. Biochemistry 48, 54355439.
Matsui, M., Fowler, J. H. and Walling, L. L. (2006). Leucine aminopeptidases: diversity in structure and function. Biological Chemistry 387, 15351544.
McGowan, S., Oellig, C. A., Birru, W. A., Caradoc-Davies, T. T., Stack, C. M., Lowther, J., Skinner-Adams, T., Mucha, A., Kafarski, P., Grembecka, J., Trenholme, K. R., Buckle, A. M., Gardiner, D. L., Dalton, J. P. and Whisstock, J. C. (2010). Structure of the Plasmodium falciparum M17 aminopeptidase and significance for the design of drugs targeting the neutral exopeptidases. Proceedings of the National Academy of Sciences, USA 107, 24492454.
Na, B. K., Kang, J. M., Cheun, H. I., Cho, S. H., Moon, S. U., Kim, T. S. and Sohn, W. M. (2009). Cryptopain-1, a cysteine protease of Cryptosporidium parvum, does not require the pro-domain for folding. Parasitology 136, 149157.
Nankya-Kitaka, M. F., Curley, G. P., Gavial, C. S., Bell, A. and Dalton, J. P. (1998). Plasmodium chabaudi chabaudi and P. falciparum: inhibition of aminopeptidase and parasite growth by bestatin and nitrobestatin. Parasitology Research 84, 552558.
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.
Peterson, C. (1992). Cryptosporidiosis in patients with human immunodeficiency virus. Clinical Infectious Diseases 15, 903909.
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.
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.
Rawlings, N. D., Morton, F. R. and Barrett, A. J. (2006). MEROPS: the peptidase database. Nucleic Acids Research 34(Database issue), D270D272.
Roiko, M. S. and Carruthers, V. B. (2009). New roles for perforins and proteases in apicomplexan egress. Cellular Microbiology 11, 14441452.
Rosenthal, P. J. (2002). Hydrolysis of erythrocyte proteins by proteases of malaria parasites. Current Opinions in Hematology 9, 140145.
Schorlemmer, H. U., Bosslet, K. and Sedlacek, H. H. (1983). Ability of the immunomodulating dipeptide bestatin to activate cytotoxic mononuclear phagocytes. Cancer Research 49, 41484153.
Scornik, O. A. and Botbol, V. (1997). Cellular uptake of 3H-bestatin in tissues of mice after its intravenous injection. Drug Metabolism and Disposition 27, 798804.
Scornik, O. A. and Botbol, V. (2001). Bestatin as an experimental tool in mammals. Current Drug Metabolism 2, 6785.
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.
Skinner-Adams, T. S., Stack, C. M., Trenholme, K. R., Brown, C. L., Grembecka, J., Lowther, J., Mucha, A., Drag, M., Kafarski, P., McGowan, S., Whisstock, J. C., Gardiner, D. L. and Dalton, J. P. (2009). Plasmodium falciparum neutral aminopeptidases: new targets for anti-malarials. Trends in Biochemical Sciences 35, 5361.
Stack, C. M., Lowther, J., Cunningham, E., Donnelly, S., Gardiner, D. L., Trenholme, K. R., Skinner-Adams, T. S., Teuscher, F., Grembecka, J., Mucha, A., Kafarski, P., Lua, L., Bell, A. and Dalton, J. P. (2007). Characterization of the Plasmodium falciparum M17 leucyl aminopeptidase. A protease involved in amino acid regulation with potential for antimalarial drug development. The Journal of Biological Chemistry 282, 20692080.
Sträter, N. and Lipscomb, W. N. (1995). Two metal ion mechanism of bovine lens leucine aminopeptidase: active site, solvent structure and binding mode of i-leucinal, a gem-biolato transition state analogue by X-ray crystallography. Biochemistry 34, 1479214800.
Taylor, A. (1993). Aminopeptidases: structure and function. FASEB Journal 7, 290298.
Tzipori, S. and Ward, H. (2002). Cryptosporidosis: biology, pathogenesis and disease. Microbes and Infection 4, 10471058.
Umezawa, H. (1980). Low-molecular weight immunomodulators produced by microorganisms. Biotechnology and Bioengineering 22, 99110.
Wanyiri, J. W., Techasintana, P., O'Connor, R. M., Blackman, M. J., Kim, K. and Ward, H. D. (2009). Role of CpSUB1, a subtilisin-like protease, in Cryptosporidium parvum infection in vitro. Eukaryotic Cell 8, 470477.
Wegscheid-Gerlach, C., Gerber, H. D. and Diederich, W. E. (2010). Proteases of Plasmodium falciparum as potential drug targets and inhibitors thereof. Current Topics in Medicinal Chemistry 10, 346367.
Wilson, R. J., Williamson, D. H. and Preiser, P. (1994). Malaria and other Apicomplexans: the “plant” connection. Infectious Agents and Disease 3, 2937.

Keywords

Molecular cloning and characterization of a M17 leucine aminopeptidase of Cryptosporidium parvum

  • J.-M. KANG (a1), H.-L. JU (a1), W.-M. SOHN (a1) and B.-K. NA (a1)

Metrics

Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed