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Infect. Immun. doi:10.1128/IAI.01717-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Identification and characterization of the Plasmodium yoelii PyP140/RON4 protein, an orthologue of Toxoplasma gondii RON4; the cysteine-rich domain does not protect against lethal parasite challenge infection

Malaria Vaccine Development Branch, NIH, Rockville, MD 20852; and Division of Parasitology, National Institute for Medical Research, Mill Hill, London, NW7 1AA, U.K.

^* To whom correspondence should be addressed. Email: dnarum{at}niaid.nih.gov.

	Abstract

Previously we identified a Plasmodium yoelii YM 140 kDa merozoite protein, designated PyP140, which formed a complex with Apical Membrane Antigen 1 (AMA1). Furthermore, we had produced a nonprotective monoclonal antibody (mAb), 48F8 that immunoprecipitated metabolically labeled PyP140 and localized the protein to the merozoite's apical end and less frequently to the merozoite surface by immunofluorescence (IFA). Here, using mAb 48F8 we have identified the pyp140 gene by screening a P. yoelii -ZAP cDNA expression library. The pyp140 cDNA covers approximately ninety percent of the putative open reading frame (ORF) of PY02159 from the P. yoelii NL genome sequencing project. Analysis of the complete gene identified the presence of two introns. The ORF encodes a 102,407 Da protein with an amino terminal signal sequence, a series of three unique types of repeats and a cysteine rich region. The binding site of mAb 48F8 was also identified. A Blast search with the deduced amino acid sequence shows a significant similarity with the Toxoplasma gondii RON4 protein, P. falciparum RON4 protein and the sequence is highly conserved in other Plasmodium species. We produced the cysteine rich domain of PyP140/RON4 using the Pichia pastoris expression system and characterized biochemically and biophysically the recombinant protein. BALB/c mice immunized with the protein formulated in oil-in-water adjuvants produced antibodies that recognize parasitized erythrocytes by IFA and native PyP140/RON4 by immunoblot but failed to protect against a lethal P. yoelii YM infection. Our results show that PyP140/RON4 is located within the rhoptries or micronemes. It may associate in part with AMA1, but the conserved cysteine-rich domain does not appear to elicit inhibitory antibodies, a finding that is supported by the marked sequence conservation in this protein within Plasmodium spp, suggesting that it is not under immune pressure.