RESUMO
We describe an approach for the rapid mapping of epitopes within a malaria antigen using a combination of phage display techniques. Phage display of antigen fragments identifies the location of the epitopes, then random peptide libraries displayed on phage are employed to identify accurately amino acids involved in the epitope. Finally, phage display of mutant fragments confirms the role of each residue in the epitope. This approach was applied to the apical membrane antigen-1 (AMA1), which is a leading candidate for inclusion in a vaccine directed against the asexual blood stages of Plasmodium falciparum. As part of the effort both to understand the function of AMA1 in the parasite life cycle and to define the specificity of protective immune responses, a panel of monoclonal antibodies (MAbs) was generated to obtain binding reagents to the various domains within the molecule. There is a pressing need to determine rapidly the regions recognized by these antibodies and the structural requirements required within AMA1 for high affinity binding of the MAbs. Using phage displaying random AMA1 fragments, it was shown that MAb5G8 recognizes a short linear epitope within the pro-domain of AMA1 whereas the epitope recognized by MAb 1F9 is reduction sensitive and resides within a disulphide-bonded 57 amino acid sub-domain of domain-1. Phage displaying random peptide libraries and mutant AMA1 fragments were employed for fine mapping of the MAb5G8 core epitope to a three-residue sequence in the AMA1 prodomain.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Protozoários/imunologia , Mapeamento de Epitopos/métodos , Proteínas de Membrana/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Especificidade de Anticorpos , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Bacteriófagos/genética , Dissulfetos/química , Epitopos/imunologia , Eritrócitos/parasitologia , Proteínas de Membrana/química , Dados de Sequência Molecular , Mutação , Biblioteca de Peptídeos , Peptídeos/genética , Peptídeos/imunologia , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Alinhamento de SequênciaRESUMO
The histidine-rich protein 2 (PfHRP2) of Plasmodium falciparum has been implicated in the detoxification of ferriprotoporphyrin IX (FP) moieties that are produced as by-products of the digestion of haemoglobin. In this work, we have used a spectroscopic analysis to confirm that recombinant PfHRP2 binds FP. A monoclonal antibody that recognises both recombinant and authentic PfHRP2 was used in immunofluorescence microscopy studies. We found that PfHRP2 is mainly located in the erythrocyte cytosol of infected erythrocytes, however, dual labelling studies suggest that the location of a sub-population of the PfHRP2 molecules overlaps with that of the food vacuole-associated protein, P-glycoprotein homologue (Pgh-1). A semi-quantitative analysis of the level of PfHRP2 in infected erythrocytes suggests a concentration of a few micromolar in the food vacuole. Under conditions designed to mimic the parasite food vacuole, we found that 1.2 microM PfHRP2 is sufficient to catalyse the conversion of about 30% of a 100 microM sample of FP to beta-haematin within 24 h. Moreover, PfHRP2 is capable of promoting the H(2)O(2)-induced degradation of FP at pH 5.2. PfHRP2 also efficiently enhances the ability of FP to catalyse the H(2)O(2)-mediated oxidation of the model co-factor, ortho-phenylene diamine (OPD). These data suggest that PfHRP2 may promote the detoxification of FP and reactive oxygen species within the food vacuole. By contrast, PfHRP2 inhibits the destruction of FP by glutathione (GSH) at pH 7.4. This suggests that PfHRP2 is not a catalyst of FP degradation outside the food vacuole.
