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Infection and Immunity, November 2008, p. 4876-4882, Vol. 76, No. 11
0019-9567/08/$08.00+0     doi:10.1128/IAI.01717-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Identification and Characterization of the Plasmodium yoelii PyP140/RON4 Protein, an Orthologue of Toxoplasma gondii RON4, Whose Cysteine-Rich Domain Does Not Protect against Lethal Parasite Challenge Infection{triangledown}

David L. Narum,1* Vu Nguyen,1 Yanling Zhang,1 Jacqueline Glen,1 Richard L. Shimp,1 Lynn Lambert,1 Irene T. Ling,2 Karine Reiter,1 Solabomi A. Ogun,2 Carole Long,1 Anthony A. Holder,2 and Raul Herrera1

Malaria Vaccine Development Branch, NIH, Rockville, Maryland 20852,1 Division of Parasitology, National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom2

Received 21 December 2007/ Accepted 3 August 2008

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 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, as observed by immunofluorescence assay (IFA). Here, using MAb 48F8, we have identified the pyp140 gene by screening a P. yoelii {lambda}-Zap cDNA expression library. The pyp140 cDNA covers approximately 90% 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 significant similarity with the Toxoplasma gondii RON4 protein and the Plasmodium falciparum RON4 protein, and the sequence is highly conserved in other Plasmodium species. We produced the cysteine-rich domain of PyP140/RON4 by using the Pichia pastoris expression system and characterized the recombinant protein biochemically and biophysically. 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 immunoblotting 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.

* Corresponding author. Mailing address: Malaria Vaccine Development Branch, NIH, 5640 Fishers Lane, Twinbrook I, Rockville, MD 20852. Phone: (301) 435-2185. Fax: (301) 480-1962. E-mail: dnarum{at}niaid.nih.gov

{triangledown} Published ahead of print on 18 August 2008.

Editor: W. A. Petri, Jr.

Infection and Immunity, November 2008, p. 4876-4882, Vol. 76, No. 11
0019-9567/08/$08.00+0     doi:10.1128/IAI.01717-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Plassmeyer, M. L., Reiter, K., Shimp, R. L. Jr., Kotova, S., Smith, P. D., Hurt, D. E., House, B., Zou, X., Zhang, Y., Hickman, M., Uchime, O., Herrera, R., Nguyen, V., Glen, J., Lebowitz, J., Jin, A. J., Miller, L. H., MacDonald, N. J., Wu, Y., Narum, D. L. (2009). Structure of the Plasmodium falciparum Circumsporozoite Protein, a Leading Malaria Vaccine Candidate. J. Biol. Chem. 284: 26951-26963 [Abstract] [Full Text]  


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