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

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 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 -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.

The moving junction of apicomplexan parasites: a key structure for invasion

“…The RONs/AMA1 in vitro interaction has been confirmed for Plasmodium (Alexander et al, 2006;Narum et al, 2008;Cao et al, 2009;Collins et al, 2009) and the presence of both PfRON4 and PfAMA1 at the MJ has been recently demonstrated (Riglar et al, 2011). Most of the MJ complex proteins are conserved among Apicomplexa (Table 1).…”

Total and Putative Surface Proteomics of Malaria Parasite Salivary Gland Sporozoites

“…SC-789), 1 g/ml final concentration; rabbit anti-HA (Santa Cruz Biotechnology, catalog no. sc-805) diluted 1:400; mouse anti-PyCSP (clone 2F6) (25); mouse anti-PyRON4 (clone 48F8) (26); Alexa Fluor-conjugated secondary antibodies specific to mouse or rabbit IgG (Alexa Fluor 488 and 594 (Invitrogen), respectively)) and 4Ј,6-diamidino-2-phenylindole (DAPI), which labels A-T rich regions of DNA. Samples were covered with VectaShield (Vector Laboratories, Burlingame, CA) and sealed under a coverglass slip.…”

“…5). Partial co-localization with PyRON4, a known rhoptry neck protein involved in parasite invasion, indicated that PY01024 also was localized to the neck of the rhoptries (26,62). Interestingly, this protein has to date not been implicated in sporozoite host cell infection, and thus further characterization of its role is certainly warranted.…”

Identification and Characterization of the Rhoptry Neck Protein 2 in Babesia divergens and B. microti

“…The tight connection between the parasite and the host cell needed for the MJ occurs when RON2 physically binds to the hydrophobic groove of AMA1. In Plasmodium, RON4 and RON5 also constitute part of the AMA-RON complex of the MJ (49)(50)(51)(52)(53), with RON8 being added in Toxoplasma (40,(54)(55)(56)(57)(58).…”