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J. Biol. Chem., Vol. 282, Issue 3, 2069-2080, January 19, 2007

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Characterization of the Plasmodium falciparum M17 Leucyl Aminopeptidase

A PROTEASE INVOLVED IN AMINO ACID REGULATION WITH POTENTIAL FOR ANTIMALARIAL DRUG DEVELOPMENT^*

Colin M. Stack¹, Jonathan Lowther, Eithne Cunningham², Sheila Donnelly, Donald L. Gardiner^¶2, Katharine R. Trenholme^¶2, Tina S. Skinner-Adams^¶||3, Franka Teuscher^¶, Jolanta Grembecka^**, Artur Mucha, Pawel Kafarski, Linda Lua, Angus Bell¹, and John P. Dalton¹⁴

From the Institute for the Biotechnology of Infectious Diseases, University of Technology Sydney, Level 6, Building 4, Corner of Thomas and Harris Street, Ultimo, Sydney, New South Wales 2007, Australia, the Department of Microbiology, Moyne Institute of Preventive Medicine, University of Dublin, Trinity College, Dublin 2, Republic of Ireland, ^¶Malaria Biology Laboratory, The Australian Centre for International and Tropical Health and Nutrition, The Queensland Institute of Medical Research, 300 Herston Rd, Herston, Brisbane, Queensland 4006, Australia, ^||Central Medical Division, University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia, the ^**Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908, Institute of Organic Chemistry, Biochemistry, and Biotechnology, Wroclaw University of Technology, Wybrzeze, Wyspianskiego 27, 50-370 Wroclaw, Poland, and SRC Protein Expression Facility, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia

Amino acids generated from the catabolism of hemoglobin by intra-erythrocytic malaria parasites are not only essential for protein synthesis but also function in maintaining an osmotically stable environment, and creating a gradient by which amino acids that are rare or not present in hemoglobin are drawn into the parasite from host serum. We have proposed that a Plasmodium falciparum M17 leucyl aminopeptidase (PfLAP) generates and regulates the internal pool of free amino acids and therefore represents a target for novel antimalarial drugs. This enzyme has been expressed in insect cells as a functional 320-kDa homo-hexamer that is optimally active at neutral or alkaline pH, is dependent on metal ions for activity, and exhibits a substrate preference for N-terminally exposed hydrophobic amino acids, particularly leucine. PfLAP is produced by all stages in the intra-erythrocytic developmental cycle of malaria but was most highly expressed by trophozoites, a stage at which hemoglobin degradation and parasite protein synthesis are elevated. The enzyme was located by immunohistochemical methods and by transfecting malaria cells with a PfLAP-green fluorescent protein construct, to the cytosolic compartment of the cell at all developmental stages, including segregated merozoites. Amino acid dipeptide analogs, such as bestatin and its derivatives, are potent inhibitors of the protease and also block the growth of P. falciparum malaria parasites in culture. This study provides a biochemical basis for the antimalarial activity of aminopeptidase inhibitors. Availability of functionally active recombinant PfLAP, coupled with a simple enzymatic readout, will aid medicinal chemistry and/or high throughput approaches for the future design/discovery of new antimalarial drugs.

Received for publication, September 29, 2006 , and in revised form, November 8, 2006.

^* This work was supported in part by an Australian Research Council Discovery Project Grant DP0666128 and by an Australian Government Endeavour Scholarship (to E. C. and J. P. D.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

¹ Supported by Basic Research Grant SC/2003/111 from Enterprise Ireland.

² 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.

³ Recipient of a University of Queensland postdoctoral fellowship and a Ramaciotti development grant.

⁴ Recipient of New South Wales Government BioFirst Award in Biotechnology (2004). To whom correspondence should be addressed. Tel.: 61-2-95144142; Fax: 61-2-95144201; E-mail: john.dalton{at}uts.edu.au.

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