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: 2.73). 11/1992; 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.

  • [Show abstract] [Hide abstract]
    ABSTRACT: A Babesia bovis merozoite protein, Bb-1, was localized by immunoelectron microscopy to an apical organelle known as the spherical body. This unique structure appears to be analogous to dense granules of other apicomplexan protozoa. Similar to previously described dense granule proteins of Plasmodium spp., Bb-1 is secreted during or just after invasion of host erythrocytes and becomes associated with the cytoplasmic face of the infected cell. The amino terminal sequence of Bb-1 contains a predicted signal peptide and is similar to the amino terminus of another spherical body protein (BvVA1225) which is also translocated to the erythrocyte membrane. Importantly, these two spherical body proteins are the major components of a protective fraction of B. bovis antigen. There is marked conservation of Bb-1 amino acid sequences and B-lymphocyte epitopes among geographic strains. However, a divergent Bb-1 allele (Bv80) in Australia strains encodes six regions of amino acid polymorphism, including a region of tetrapeptide repeats in the C-terminal half of the polypeptide. Two of the polymorphic regions map to previously defined Th1 epitopes on Bb-1.
    Molecular and Biochemical Parasitology 01/1995; 69(2):149-159. · 2.73 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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 hypo-thesis that rhoptry bio-genesis 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. Keywords: malaria/molecular parasitology/targeted gene disruption/vaccine antigens
    The EMBO Journal 06/2000; 19(11):2435-2443. · 9.82 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Malaria is a major global threat, that results in more than 2 million deaths each year. The treatment of malaria is becoming extremely difficult due to the emergence of drug-resistant parasites, the absence of an effective vaccine, and the spread of insecticide-resistant vectors. Thus, malarial therapy needs new chemotherapeutic approaches leading to the search for new drug targets. Here, we discuss different approaches to identifying novel antimalarial drug targets. We have also given due attention to the existing validated targets with a view to develop novel, rationally designed lead molecules. Some of the important parasite proteins are claimed to be the targets; however, further in vitro or in vivo structure-function studies of such proteins are crucial to validate these proteins as suitable targets. The interactome analysis among apicoplast, mitochondrion and genomic DNA will also be useful in identifying vital pathways or proteins regulating critical pathways for parasite growth and survival, and could be attractive targets. Molecules responsible for parasite invasion to host erythrocytes and ion channels of infected erythrocytes, essential for intra-erythrocyte survival and stage progression of parasites are also becoming attractive targets. This review will discuss and highlight the current understanding regarding the potential antimalarial drug targets, which could be utilized to develop novel antimalarials.
    Expert Review of Clinical Pharmacology 09/2009; 2(5):469-89.