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J. Biol. Chem., Vol. 282, Issue 42, 30817-30826, October 19, 2007

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The M18 Aspartyl Aminopeptidase of the Human Malaria Parasite Plasmodium falciparum^*

Franka Teuscher¹, Jonathan Lowther¹, Tina S. Skinner-Adams^¶2, Tobias Spielmann³, Matthew W. A. Dixon⁴, Colin M. Stack⁵, Sheila Donnelly, Artur Mucha^||, Pawe Kafarski^||, Stamatia Vassiliou^**, Donald L. Gardiner⁶, John P. Dalton¹⁷, and Katharine R. Trenholme, Supported by NHMRC program Grant 290208 and a generous donation from Mark Nicholson, Alice Hill, and the Tudor Foundation¹⁸

From the Malaria Biology Laboratory, The Queensland Institute of Medical Research, 300 Herston Rd, Herston, Brisbane, Queensland 4006, Australia, the Institute for the Biotechnology of Infectious Diseases (IBID), University of Technology Sydney, Level 6, Building 4, Thomas and Harris Street, Ultimo, Sydney, New South Wales 2007, Australia, the ^¶University of Queensland, Department of Medicine, Central Clinical Division, Brisbane, Queensland 4072, Australia, the ^||Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wybrzeze Wyspianskiego 27, 50-370 Wroclaw, Poland, and the ^**Laboratory of Organic Chemistry, Department of Chemistry, University of Athens, Panepistimiopolis Zografou, 15771 Athens, Greece

A member of the M18 family of aspartyl aminopeptidases is expressed by all intra-erythrocytic stages of the human malaria parasite Plasmodium falciparum (PfM18AAP), with highest expression levels in rings. Functionally active recombinant enzyme, rPfM18AAP, and native enzyme in cytosolic extracts of malaria parasites are 560-kDa octomers that exhibit optimal activity at neutral pH and require the presence of metal ions to maintain enzymatic activity and stability. Like the human aspartyl aminopeptidase, the exopeptidase activity of PfM18AAP is exclusive to N-terminal acidic amino acids, glutamate and aspartate, making this enzyme of particular interest and suggesting that it may function alongside the malaria cytosolic neutral aminopeptidases in the release of amino acids from host hemoglobin-derived peptides. Whereas immunocytochemical studies using transgenic P. falciparum parasites show that PfM18AAP is expressed in the cytosol, immunoblotting experiments revealed that the enzyme is also trafficked out of the parasite into the surrounding parasitophorous vacuole. Antisense-mediated knockdown of PfM18AAP results in a lethal phenotype as a result of significant intracellular damage and validates this enzyme as a target at which novel antimalarial drugs could be directed. Novel phosphinic derivatives of aspartate and glutamate showed modest inhibition of rPfM18AAP but did not inhibit malaria growth in culture. However, we were able to draw valuable observations concerning the structure-activity relationship of these inhibitors that can be employed in future inhibitor optimization studies.

Received for publication, June 15, 2007 , and in revised form, August 8, 2007.

^* This work was supported by Australian Research Council Discovery Project Grant DPO666128. 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. S1.

¹ These authors contributed equally to this manuscript.

² Recipient of a University of Queensland Postdoctoral Fellowship and a Ramaciotti Development grant.

³ Supported by a fellowship from the Swiss National Science Foundation.

⁴ Supported by an Australia and New Zealand Trustees Ph.D. scholarship.

⁵ Supported by a Basic Research Grant obtained from Enterprise Ireland.

⁶ Supported by National Health and Medical Research Council (NHMRC) program Grant 290208 and a generous donation from the Mark Nicholson, Alice Hill, and the Tudor Foundation.

⁷ Recipient of a New South Wales BioFirst award.

⁸ To whom correspondence should be addressed: Malaria Biology Laboratory, The Queensland Institute of Medical Research, 300 Herston Rd., Herston, Brisbane, Queensland 4006, Australia. Tel.: 61-7-33620432; Fax: 61-7-33620104; E-mail: Katharine.Trenholme{at}qimr.edu.au.

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