Hostname: page-component-cd9895bd7-p9bg8 Total loading time: 0 Render date: 2024-12-28T10:27:36.765Z Has data issue: false hasContentIssue false
  • English
  • Français

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

Published online by Cambridge University Press:  18 March 2011

J.-M. KANG ,
H.-L. JU ,
W.-M. SOHN  and
B.-K. NA
J.-M. KANG
Affiliation:
Department of Parasitology and Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju 660-751, Korea
H.-L. JU
Affiliation:
Department of Parasitology and Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju 660-751, Korea
W.-M. SOHN
Affiliation:
Department of Parasitology and Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju 660-751, Korea
B.-K. NA*
Affiliation:
Department of Parasitology and Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju 660-751, Korea
*
*Corresponding author: Tel: +82 55 751 8822. Fax: +82 55 759 4022. E-mail: [email protected]
Get access Rights & Permissions [Opens in a new window]

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.

Keywords

Cryptosporidium parvumM17 leucine aminopeptidaserecombinant proteindrug target
Type
Research Article
Information
Parasitology , Volume 138 , Issue 6 , May 2011 , pp. 682 - 690
Copyright
Copyright © Cambridge University Press 2011

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

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, 29–33.CrossRefGoogle ScholarPubMed
Blackman, M. J. (2008). Malarial proteases and host cell egress: an ‘emerging’ cascade. Cellular Microbiology 10, 1925–1934.CrossRefGoogle ScholarPubMed
Dalal, S. and Klemba, M. (2007). Roles for two aminopeptidases in vacuolar hemoglobin catabolism in Plasmodium falciparum. The Journal of Biological Chemistry 282, 35978–35987.CrossRefGoogle ScholarPubMed
Fayer, R., Morgan, U. and Upton, S. J. (2000). Epidemiology of Cryptosporidium transmission, detection and identification. International Journal for Parasitology 30, 1305–1322.CrossRefGoogle ScholarPubMed
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, 619–626.CrossRefGoogle ScholarPubMed
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, 638–640.CrossRefGoogle Scholar
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, 1741–1745.CrossRefGoogle ScholarPubMed
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, 37–48.CrossRefGoogle ScholarPubMed
Goldberg, D. E. (2005). Hemoglobin degradation. Current Topics of Microbiology and Immunology 295, 275–291.Google ScholarPubMed
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), D419–D422.CrossRefGoogle ScholarPubMed
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, 1317–1326.CrossRefGoogle ScholarPubMed
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, 1–6.CrossRefGoogle ScholarPubMed
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, 945–952.CrossRefGoogle ScholarPubMed
Kim, K. (2004). Role of proteases in host cell invasion by Toxoplasma gondii and other Apicomplexa. Acta Tropica 91, 69–81.CrossRefGoogle ScholarPubMed
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, 5006–5010.CrossRefGoogle ScholarPubMed
Kosek, M., Alcantara, C., Lima, A. A. and Guerrant, R. L. (2001). Cryptosporidiosis: an update. Lancet Infectious Diseases 1, 262–269.CrossRefGoogle ScholarPubMed
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, 45–48.CrossRefGoogle ScholarPubMed
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, 5435–5439.CrossRefGoogle ScholarPubMed
Matsui, M., Fowler, J. H. and Walling, L. L. (2006). Leucine aminopeptidases: diversity in structure and function. Biological Chemistry 387, 1535–1544.CrossRefGoogle ScholarPubMed
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, 2449–2454.CrossRefGoogle ScholarPubMed
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, 149–157.CrossRefGoogle Scholar
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, 552–558.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, 77–88.Google ScholarPubMed
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
Rawlings, N. D., Morton, F. R. and Barrett, A. J. (2006). MEROPS: the peptidase database. Nucleic Acids Research 34(Database issue), D270–D272.CrossRefGoogle ScholarPubMed
Roiko, M. S. and Carruthers, V. B. (2009). New roles for perforins and proteases in apicomplexan egress. Cellular Microbiology 11, 1444–1452.CrossRefGoogle ScholarPubMed
Rosenthal, P. J. (2002). Hydrolysis of erythrocyte proteins by proteases of malaria parasites. Current Opinions in Hematology 9, 140–145.CrossRefGoogle ScholarPubMed
Schorlemmer, H. U., Bosslet, K. and Sedlacek, H. H. (1983). Ability of the immunomodulating dipeptide bestatin to activate cytotoxic mononuclear phagocytes. Cancer Research 49, 4148–4153.Google Scholar
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, 798–804.Google Scholar
Scornik, O. A. and Botbol, V. (2001). Bestatin as an experimental tool in mammals. Current Drug Metabolism 2, 67–85.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, 119–132.CrossRefGoogle ScholarPubMed
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, 53–61.CrossRefGoogle ScholarPubMed
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, 2069–2080.CrossRefGoogle ScholarPubMed
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, 14792–14800.CrossRefGoogle ScholarPubMed
Taylor, A. (1993). Aminopeptidases: structure and function. FASEB Journal 7, 290–298.CrossRefGoogle ScholarPubMed
Tzipori, S. and Ward, H. (2002). Cryptosporidosis: biology, pathogenesis and disease. Microbes and Infection 4, 1047–1058.CrossRefGoogle Scholar
Umezawa, H. (1980). Low-molecular weight immunomodulators produced by microorganisms. Biotechnology and Bioengineering 22, 99–110.Google ScholarPubMed
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, 470–477.CrossRefGoogle ScholarPubMed
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, 346–367.CrossRefGoogle ScholarPubMed
Wilson, R. J., Williamson, D. H. and Preiser, P. (1994). Malaria and other Apicomplexans: the “plant” connection. Infectious Agents and Disease 3, 29–37.Google ScholarPubMed