Annotation and characterization of the Plasmodium vivax rhoptry neck protein 4 (Pv RON4)

Abstract

BackgroundThe tight junction (TJ) is one of the most important structures established during merozoite invasion of host cells and a large amount of proteins stored in Toxoplasma and Plasmodium parasites’ apical organelles are involved in forming the TJ. Plasmodium falciparum and Toxoplasma gondii apical membrane antigen 1 (AMA-1) and rhoptry neck proteins (RONs) are the two main TJ components. It has been shown that RON4 plays an essential role during merozoite and sporozoite invasion to target cells. This study has focused on characterizing a novel Plasmodium vivax rhoptry protein, RON4, which is homologous to PfRON4 and PkRON4.MethodsThe ron4 gene was re-annotated in the P. vivax genome using various bioinformatics tools and taking PfRON4 and PkRON4 amino acid sequences as templates. Gene synteny, as well as identity and similarity values between open reading frames (ORFs) belonging to the three species were assessed. The gene transcription of pvron4, and the expression and localization of the encoded protein were also determined in the VCG-1 strain by molecular and immunological studies. Nucleotide and amino acid sequences obtained for pvron4 in VCG-1 were compared to those from strains coming from different geographical areas.ResultsPvRON4 is a 733 amino acid long protein, which is encoded by three exons, having similar transcription and translation patterns to those reported for its homologue, PfRON4. Sequencing PvRON4 from the VCG-1 strain and comparing it to P. vivax strains from different geographical locations has shown two conserved regions separated by a low complexity variable region, possibly acting as a “smokescreen”. PvRON4 contains a predicted signal sequence, a coiled-coil α-helical motif, two tandem repeats and six conserved cysteines towards the carboxy-terminus and is a soluble protein lacking predicted transmembranal domains or a GPI anchor. Indirect immunofluorescence assays have shown that PvRON4 is expressed at the apical end of schizonts and co-localizes at the rhoptry neck with PvRON2.ConclusionsGenomic, transcriptional and expression data reported for PvRON4, as well as its primary structure characteristics suggest that this protein participates in reticulocyte invasion, as has been shown for its homologue PfRON4.

Receptor-ligand and parasite protein-protein interactions inPlasmodium vivax: Analysing rhoptry neck proteins 2 and 4

“…Each protein's main characteristics are shown. Signal sequence (dark blue), transmembrane domains (black), coiled‐coil domains (grey), and cysteine residues conserved amongst Plasmodium vivax strains (dotted red lines) (Arevalo‐Pinzon et al, ; Arevalo‐Pinzon, Curtidor, Abril, & Patarroyo, ). The locations are shown of two Pv RON2 fragments ( Pv RON2‐RI and Pv RON2‐RII) and one Pv RON4 fragment, which were cloned in vectors to be expressed in COS‐7 cells and E scherichia coli .…”

“…A single band was found for each fragment by western blot and Coomassie blue staining, coinciding with the expected molecular weights (Figure a, left). The three recombinant fragments contained conserved cysteine residues amongst the different P. vivax strains (Arevalo‐Pinzon et al, ; Arevalo‐Pinzon, Curtidor, Abril, & Patarroyo, ). Although internal disulphide linkage formation in these fragments is still unknown, previous studies with the RON2sp1 show that disulphide bridge formation between Cys2051‐Cys2063 ( Pv RON2sp1) and Cys2037‐Cys2049 ( Pf RON2sp1) in RON2 carboxy terminal region is important for this peptide's interaction with the AMA‐1 hydrophobic groove (Vulliez‐Le Normand et al, ; Vulliez‐Le Normand et al, ).…”

“…Using P. vivax strains adapted in non-human primates (Pico de Coana et al, 2003) or infected patients' blood (Bozdech et al, 2008) has enabled comparative approaches with other Plasmodium species (Patarroyo, Calderon, & Moreno-Perez, 2012) or using omic sciences (Moreno-Perez, Degano, Ibarrola, Muro, & Patarroyo, 2014;Venkatesh et al, 2016) to identify a significant amount of proteins expressed in P. vivax schizonts (Sch) and Mz (Patarroyo et al, 2012). PvRON2 (Arevalo-Pinzon, , PvRON4 (Arevalo-Pinzon, Curtidor, Abril, & Patarroyo, 2013), and PvRON5 (Arevalo-Pinzon, Bermudez, have been identified recently; these are homologous to those identified in P. falciparum, which are located in the apical extreme of P. vivax Colombia Guaviare 1 (VCG-1) strain Sch. Different biochemical techniques have been used to report a conserved PvRON5 fragment located towards the carboxyl-terminal extreme interacting with RBC, having a preference for CD71 + cells (Arevalo-Pinzon et al, 2015), suggesting that RONs could be participating in host-parasite interactions, similar to that found in P. falciparum.…”

A targeted immunomic approach identifies diagnostic antigens in the human pathogen Babesia microti

“…Studies in other protozoan parasites such as Plasmodium falciparum , P. vivax, and Theileria sp. have identified surface antigens that are critical for cell adhesion and are the primary targets of host immunity . Several of these antigens contain a glycosylphosphatidylinositol (GPI) anchor and are among the most antigenic and abundant proteins expressed during the life cycle of many parasites …”

“…have identified surface antigens that are critical for cell adhesion and are the primary targets of host immunity. [15][16][17] Several of these antigens contain a glycosylphosphatidylinositol (GPI) anchor [17][18][19] and are among the most antigenic and abundant proteins expressed during the life cycle of many parasites. [20][21][22] Identification of the full set of GPI-anchored proteins (GPI-proteome) in an organism can be determined using both bioinformatics and experimental techniques.…”