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Identification and Stoichiometry of Glycosylphosphatidylinositol-anchored Membrane Proteins of the Human Malaria Parasite Plasmodium falciparum*S

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Most proteins that coat the surface of the extracellular forms of the human malaria parasite Plasmodium falciparum are attached to the plasma membrane via glycosylphosphatidylinositol (GPI) anchors. These proteins are exposed to neutralizing antibodies, and several are advanced vaccine candidates. To identify the GPI-anchored proteome of P. falciparum we used a combination of proteomic and computational approaches. Focusing on the clinically relevant blood stage of the life cycle, proteomic analysis of proteins labeled with radioactive glucosamine identified GPI anchoring on 11 proteins (merozoite surface protein (MSP)-1, -2, -4, -5, -10, rhoptry-associated membrane antigen, apical sushi protein, Pf92, Pf38, Pf12, and Pf34). These proteins represent ∼94% of the GPI-anchored schizont/merozoite proteome and constitute by far the largest validated set of GPI-anchored proteins in this organism. Moreover MSP-1 and MSP-2 were present in similar copy number, and we estimated that together these proteins comprise approximately two-thirds of the total membrane-associated surface coat. This is the first time the stoichiometry of MSPs has been examined. We observed that available software performed poorly in predicting GPI anchoring on P. falciparum proteins where such modification had been validated by proteomics. Therefore, we developed a hidden Markov model (GPI-HMM) trained on P. falciparum sequences and used this to rank all proteins encoded in the completed P. falciparum genome according to their likelihood of being GPI-anchored. GPI-HMM predicted GPI modification on all validated proteins, on several known membrane proteins, and on a number of novel, presumably surface, proteins expressed in the blood, insect, and/or pre-erythrocytic stages of the life cycle. Together this work identified 11 and predicted a further 19 GPI-anchored proteins in P. falciparum.

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Published, MCP Papers in Press, April 7, 2006, DOI 10.1074/mcp.M600035-MCP200

*

This work was supported in part by the National Health and Medical Research Council of Australia and the Wellcome Trust (UK). 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.

S

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

§

Both authors contributed equally to this work.

**

International research scholars of the Howard Hughes Medical Institute.