Inhibitory monoclonal antibodies recognise epitopes adjacent to a proteolytic cleavage site on the RAP-1 protein of Plasmodium falciparum

{ "0" : "Institute of Cell and Molecular Biology, Edinburgh, UK" , "1" : "Institute of Cell, Animal and Population Biology, University of Edinburgh, Edinburgh, UK" , "2" : "Max-Planck-Institut für Biochemie, Martinsried bei München, Germany" , "4" : "Malaria" , "5" : "Rhoptry" , "6" : "Inhibitory monoclonal antibody" , "7" : "RAP-1" , "8" : "Epitope mapping" , "9" : "mAb, monoclonal antibody" , "10" : "IPTG, isopropyl-β-thiogalactopyranoside" , "11" : "SDS, sodium dodecyl sulphate" , "12" : "PAGE, polyacrylamide gel electrophoresis" , "13" : "LB, Luria broth"}
Molecular and Biochemical Parasitology (Impact Factor: 1.79). 11/1992; 55(1-2):177-186. DOI: 10.1016/0166-6851(92)90138-A


The low-molecular-weight rhoptry-associated protein (RAP) complex of Plasmodium falciparum consists of at least two gene products, RAP-1 and RAP-2, and has the ability to immunise Saimiri monkeys against experimental P. falciparum infection. Several monoclonal antibodies specifically recognise this complex and in this study we show that purified immunoglobulin derived from these monoclonals is capable of inhibiting parasite growth in vitro. It has previously been shown that RAP-1 initially appears as an 80-kDa protein (p80) in early schizogony and is processed to a 65-kDa protein (p65) in late schizogony. Several of the inhibitory monoclonals recognise both the 80- and 65-kDa proteins by Western blot analysis suggesting that they recognise linear epitopes on RAP-1. We have mapped these epitopes by testing the reactivity of the monoclonals against fragments of the rap-1 gene expressed as β-galactosidase fusion proteins and subsequently against synthetic peptides. All of the epitopes map to a region 10–20 amino acids C-terminal to the proteolytic cleavage site for the processing of p80 to p65 at amino acid 190. We also show that the 65-kDa protein is not present in purified merozoites, suggesting that its generation is associated with merozoite release rather than erythrocyte invasion. These results are discussed with respect to possible inhibitory mechanisms for the monoclonals.

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    • "Interestingly, this protective ability has been associated with the formation of a rhoptry-associated complex consisting of at least three polypeptides of 80 kDa, 65 kDa and 42 kDa [18]. The first two molecules correspond to processed forms of RAP-1, whereas the last one is consistent with the 40–42 kDa mature form of RAP-2 [37]. "
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    ABSTRACT: Invasion of red blood cells (RBCs) by the Plasmodium falciparum malaria merozoite is mediated by parasite surface molecules and proteins contained within apical organelles that are capable of recognizing receptors on the membrane of RBCs. The identification and characterization of these P. falciparum invasion-associated proteins is the first step for unveiling potential new drug and vaccine target molecules to eradicate this deadly disease. Among the exclusive set of malarial vaccine candidates, the members of the rhoptry-associated protein (RAP) family have been associated with the parasite's binding to and invasion of RBCs. Remarkably, the third member of this family (named RAP-3) has been recently detected on the surface of non-infected RBCs exposed to free merozoites, therefore suggesting the participation of this protein during RBC infection. In this study, the sequence of RAP-3 was finely mapped using synthetic peptides in order to identify which are the specific binding regions involved in RAP3-RBC interactions. Two high-activity binding peptides (HABPs) established high affinity interactions with RBC surface molecules of about 27-90 kDa, which were differentially affected by different enzymatic treatments. RAP-1 and RAP-2 HABPs inhibited binding of RAP-3 HABPs to different extents, thus suggesting the recognition of similar binding sites on RBC membrane, as well as ability of RAP-3 HABPs to inhibit P. falciparum infection in vitro. Altogether, these functional analyses of RAP-3 HABPs strongly suggest a potential role for this protein in RBC invasion, and highlight its HABPs as potential targets to develop a fully protective minimal subunit-based malarial vaccine.
    Full-text · Article · Dec 2010 · Peptides
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    • "The role of these rhoptry proteins during RBC invasion and their importance as promissory vaccine candidates have been established by studies reporting the partial inhibition of P. falciparum growth in vitro and in vivo by antibodies targeted against the HMW complex [12] [38], in vitro inhibition of RBC invasion by monoclonal antibodies raised against RAP-1 and RAP-2 [18] [27], lower densities of P. falciparum parasitemia associated to anti-RAMA antibodies [24], and protection of Saimiri boliviensis monkeys against experimental challenge with P. falciparum conferred by immunization with recombinant RAP-1 and RAP-2 [9]. Additionally, the importance of the pfrhoph3 gene in parasite growth and the implication of some RBL proteins in different pathways of RBC invasion have been described [19] [44]. "
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    ABSTRACT: Rhoptries are specialized secretory organelles found in all members of the genus Plasmodium whose proteins have been considered as promising vaccine candidates due to their involvement in cell invasion and the formation of the parasitophorous vacuole (PV). The Plasmodium falciparum Pf34 protein was recently identified as a rhoptry-neck protein located in detergent-resistant microdomains (DRMs) that is expressed in mature intraerythrocytic parasite stages, but its biological function is still unknown. Receptor-ligand assays carried out in this study found that peptides 36,051 ((101)DKKFSESLKAHMDHLKILNN(120)Y), 36,053 ((141)KKYIIKEIQNNKYLNKEKKS(160)), 36,055 ((181)WLESVNNIEEKSNILKNIKS(200)Y) and 36,056 ((201)QLLNNIASLNHTLSEEIKNI(220)Y), located in the central portion of Pf34, were found to establish protease-sensitive interactions of high affinity and specificity with receptors on the surface of red blood cell (RBCs). In vitro assays showed that Pf34 high activity binding peptides (HABPs) inhibit invasion of RBCs by P. falciparum merozoites, therefore suggesting that Pf34 could act as an adhesin during invasion and supporting the inclusion of Pf34 HABPs in further studies to develop antimalarial control methods.
    Full-text · Article · Nov 2010 · Peptides
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    • "Some of the previously identified and characterized rhoptry proteins have been found to actively participate in red blood cell invasion due either to their ability to bind to red blood cells or because monoclonal or polyclonal antibodies raised against them can inhibit in vitro invasion of target cells [23]. Plasmodium falciparum rhoptry-associated protein 1 is localized at the rhoptry base and it is thought that it is implicated in invasion due to the above [11]; more importantly, it has been found that its inoculation confers protection in non-human primates [13]. PfRAP1 is a non-polymorphic protein [9] and its antigenicity has been widely studied in human communities exposed to P. falciparum [24] [25] [26] [27] [28]. "
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    ABSTRACT: Rhoptries are cellular organelles localized at the apical pole of apicomplexan parasites. Their content is rich in lipids and proteins that are released during target cell invasion. Plasmodium falciparum rhoptry-associated protein 1 (RAP1) has been the most widely studied among this parasite species' rhoptry proteins and is considered to be a good anti-malarial vaccine candidate since it displays little polymorphism and induces antibodies in infected humans. Monoclonal antibodies directed against RAP1 are also able to inhibit target cell invasion in vitro and protection against P. falciparum experimental challenge is induced when non-human primates are immunized with this protein expressed in its recombinant form. This study describes identifying and characterizing RAP1 in Plasmodium vivax, the most widespread parasite species causing malaria in humans, producing more than 80 million infections yearly, mainly in Asia and Latin America. This new protein is encoded by a two-exon gene, is proteolytically processed in a similar manner to its falciparum homologue and, as observed by microscopy, the immunofluorescence pattern displayed is suggestive of its rhoptry localization. Further studies evaluating P. vivax RAP1 protective efficacy in non-human primates should be carried out taking into account the relevance that its P. falciparum homologue has as an anti-malarial vaccine candidate.
    Full-text · Article · Apr 2006 · Biochemical and Biophysical Research Communications
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