Sialic acid-dependent binding of baculovirus-expressed recombinant antigens from Plasmodium falciparum EBA-175 to Glycophorin A.
The Plasmodium falciparum Erythrocyte Binding Antigen-175, EBA-175, is a soluble merozoite stage parasite protein which binds to glycophorin A surface receptors on human erythrocytes. We have expressed two conserved cysteine-rich regions, region II and region VI, of this protein as soluble His-tagged polypeptides in insect cell culture, and have tested their function in erythrocyte and glycophorin A binding assays. Recombinant region II polypeptides comprised of the F2 sub-domain or the entire region II (F1 and F2 sub-domains together) bound to erythrocytes and to purified glycophorin A in a manner similar to the binding of native P. falciparum EBA-175 to human red cells. Removal of sialic acid residues from the red cell surface totally abolished recombinant region II binding, while trypsin treatment of the erythrocyte surface reduced but did not eliminate recombinant region II binding. Synthetic peptides from three discontinuous regions of the F2 sub-domain of region II inhibited human erythrocyte cell binding and glycophorin A receptor recognition. Immune sera raised against EBA-175 recombinant proteins recognized native P. falciparum-derived EBA-175, and sera from malaria-immune adults recognized recombinant antigens attesting to both the antigenicity and immunogenicity of proteins. These results suggest that the functionally-active recombinant region II domain of EBA-175 may be an attractive candidate for inclusion in multi-component asexual blood stage vaccines.
Department of Immunology, Walter Reed Army Institute of Research, Silver Spring, MD, USA. chris.ockenhouse@na.amedd.army.mil
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Ockenhouse
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Barbosa
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Blackall
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Murphy
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Kashala
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Dutta
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Lanar
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Daugherty
D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000096:biosynthesis* / Q000235:genetics; D016367:Baculoviridae / Q000235:genetics; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism; D005822:Genetic Vectors; D006021:Glycophorins / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D009439:Neuraminidase / Q000494:pharmacology; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D010455:Peptides / Q000138:chemical synthesis / Q000235:genetics / Q000494:pharmacology; D010963:Plasmodium falciparum / Q000276:immunology*; D011485:Protein Binding; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D012680:Sensitivity and Specificity; D014357:Trypsin / Q000494:pharmacology
D000953:Antigens, Protozoan; D002352:Carrier Proteins; D006021:Glycophorins; D007106:Immune Sera; D010446:Peptide Fragments; D010455:Peptides; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C065668:erythrocyte-binding antigen 175, Plasmodium; D009439:Neuraminidase; D014357:Trypsin; D019158:N-Acetylneuraminic Acid