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J. Biol. Chem., Vol. 281, Issue 3, 1741-1745, January 20, 2006

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Overexpression of Leucyl Aminopeptidase in Plasmodium falciparum Parasites

TARGET FOR THE ANTIMALARIAL ACTIVITY OF BESTATIN^*

Donald L. Gardiner1, Katharine R. Trenholme1, Tina S. Skinner-Adams2, Colin M. Stack3, and John P. Dalton, Recipient of a New South Wales BioFirst Award4

From the Malaria Biology Laboratory, The Australian Centre for International and Tropical Health and Nutrition, Queensland Institute of Medical Research, 300 Herston Road, Herston, Brisbane QLD 4029, Australia and Institute for the Biotechnology of Infectious Diseases, University of Technology Sydney, Westbourne Street, Gore Hill, Sydney, New South Wales 2065, Australia

Malaria aminopeptidases are important in the generation and regulation of free amino acids that are used in protein anabolism and for maintaining osmotic stability within the infected erythrocyte. The intraerythrocytic development of malaria parasites is blocked when the activity of aminopeptidases is specifically inhibited by reagents such as bestatin. One of the major aminopeptidases of malaria parasites is a leucyl aminopeptidase of the M17 family. We reasoned that, when this enzyme was the target of bestatin inhibition, its overexpression in malaria cells would lead to a reduced sensitivity to the inhibitor. To address this supposition, transgenic Plasmodium falciparum parasites overexpressing the leucyl aminopeptidase were generated by transfection with a plasmid that housed the full-length gene. Transgenic parasites expressed a 65-kDa protein close to the predicted molecule size of 67.831 kDa for the introduced leucyl aminopeptidase, and immunofluorescence studies localized the protein to the cytosol, the location of the native enzyme. The product of the transgene was shown to be functionally active with cytosolic extracts of transgenic parasites exhibiting twice the leucyl aminopeptidase activity compared with wild-type parasites. In vitro inhibitor sensitivity assays demonstrated that the transgenic parasites were more resistant to bestatin (EC₅₀ 64 µM) compared with the parent parasites (EC₅₀ 25 µM). Overexpression of genes in malaria parasites would have general application in the identification and validation of targets for antimalarial drugs.

Received for publication, August 15, 2005 , and in revised form, October 26, 2005.

^* This work was partially supported by the Australian Research Council grant. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by National Health and Medical Research Council Program Grant 290208 and by a generous donation from Mark Nicholson, Alice Hill, and the Tudor Foundation.

² Recipient of a University of Queensland postdoctoral fellowship.

³ Supported by a basic research grant obtained from Enterprise Ireland.

⁴ To whom correspondence should be addressed: Institute for the Biotechnology of Infectious Diseases, University of Technology, Sydney, Westbourne St., Gore Hill, Sydney NSW 2065, Australia. Tel.: 61-2-95144142; Fax: 61-2-95144201; E-mail: john.dalton{at}uts.edu.au.

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