Article

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: 2.24). 11/1992; 55(1-2):177-186. DOI: 10.1016/0166-6851(92)90138-A

ABSTRACT 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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    • "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.
    Biochemical and Biophysical Research Communications 04/2006; 341(4):1053-8. DOI:10.1016/j.bbrc.2006.01.061 · 2.28 Impact Factor
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    • "RAP1 together with RAP2 have been viewed as important vaccine candidates as they can partially protect Saimiri monkeys from challenge with P. falciparum (Perrin et al. 1985; Ridley et al. 1990a). Furthermore, mAbs specific for RAP1 can inhibit invasion into erythrocytes (Schofield et al. 1986; Harnyuttanakorn et al. 1992; Howard et al. 1998). The ability to disrupt RAP1 and as a consequence the RAP1–RAP2–RAP3 complex demonstrates that it is not essential for invasion and growth of P. falciparum in human erythrocytes. "
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    ABSTRACT: Malaria is a major human health problem and is responsible for over 2 million deaths per year. It is caused by a number of species of the genus Plasmodium, and Plasmodium falciparum is the causative agent of the most lethal form. Consequently, the development of a vaccine against this parasite is a priority. There are a number of stages of the parasite life cycle that are being targeted for the development of vaccines. Important candidate antigens include proteins on the surface of the asexual merozoite stage, the form that invades the host erythrocyte. The development of methods to manipulate the genome of Plasmodium species has enabled the construction of gain-of-function and loss-of-function mutants and provided new strategies to analyse the role of parasite proteins. This has provided new information on the role of merozoite antigens in erythrocyte invasion and also allows new approaches to address their potential as vaccine candidates.
    Philosophical Transactions of The Royal Society B Biological Sciences 02/2002; 357(1417):25-33. DOI:10.1098/rstb.2001.1010 · 6.31 Impact Factor
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    • "for normal parasite growth and invasion in vitro. This is a surprising result as previous investigators have shown that RAP1 and RAP2 can protect Saimiri monkeys from challenge with P.falciparum (Perrin et al., 1985; Ridley et al., 1990a) and that monoclonal antibodies speci®c for RAP1 can inhibit invasion into erythrocytes (Scho®eld et al., 1986; Harnyuttanakorn et al., 1992; Howard et al., 1998a). These data suggested that members of the low molecular weight complex might be essential for parasite survival and, by their conserved nature, be useful as vaccine antigens (Howard, 1992; Howard and Peterson, 1996). "
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    ABSTRACT: Rhoptry associated protein 1 (RAP1) and 2 (RAP2), together with a poorly described third protein RAP3, form the low molecular weight complex within the rhoptries of Plasmodium falciparum. These proteins are thought to play a role in erythrocyte invasion by the extracellular merozoite and are important vaccine candidates. We used gene-targeting technology in P.falciparum blood-stage parasites to disrupt the RAP1 gene, producing parasites that express severely truncated forms of RAP1. Immunoprecipitation experiments suggest that truncated RAP1 species did not complex with RAP2 and RAP3. Consistent with this were the distinct subcellular localizations of RAP1 and 2 in disrupted RAP1 parasites, where RAP2 does not traffic to the rhoptries but is instead located in a compartment that appears related to the lumen of the endoplasmic reticulum. These results suggest that RAP1 is required to localize RAP2 to the rhoptries, supporting the hypothesis that rhoptry biogenesis is dependent in part on the secretory pathway in the parasite. The observation that apparently host-protective merozoite antigens are not essential for efficient erythrocyte invasion has important implications for vaccine design.
    The EMBO Journal 07/2000; 19(11):2435-43. DOI:10.1093/emboj/19.11.2435 · 10.75 Impact Factor
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