title: Biochemical analysis of the Plasmodium falciparum erythrocyte-binding antigen-175 (EBA175)-glycophorin-A interaction: implications for vaccine design.
abstract: PfEBA175 has an important role in the invasion of human erythrocytes by Plasmodium falciparum and is therefore considered a high priority blood-stage malaria vaccine candidate. PfEBA175 mediates adhesion to erythrocytes through binding of the Duffy-binding-like (DBL) domains in its extracellular domain to Neu5Acα2-3Gal displayed on the O-linked glycans of glycophorin-A (GYPA). Because of the difficulties in expressing active full-length (FL) P. falciparum proteins in a recombinant form, previous analyses of the PfEBA175-GYPA interaction have largely focused on the DBL domains alone, and therefore they have not been performed in the context of the native protein sequence. Here, we express the entire ectodomain of PfEBA175 (PfEBA175 FL) in soluble form, allowing us to compare the biochemical and immunological properties with a fragment containing only the tandem DBL domains ("region II," PfEBA175 RII). Recombinant PfEBA175 FL bound human erythrocytes in a trypsin and neuraminidase-sensitive manner and recognized Neu5Acα2-3Gal-containing glycans, confirming its biochemical activity. A quantitative binding analysis showed that PfEBA175 FL interacted with native GYPA with a KD ∼0.26 μM and is capable of self-association. By comparison, the RII fragment alone bound GYPA with a lower affinity demonstrating that regions outside of the DBL domains are important for interactions with GYPA; antibodies directed to these other regions also contributed to the inhibition of parasite invasion. These data demonstrate the importance of PfEBA175 regions other than the DBL domains in the interaction with GYPA and merit their inclusion in an EBA175-based vaccine.
authors.affiliation: From the Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH,; the Malaria Programme, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA.
authors.forename: Madushi
authors.initials: M
authors.lastname: Wanaguru
authors.affiliation: From the Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH,; the Malaria Programme, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA.
authors.forename: Cécile
authors.initials: C
authors.lastname: Crosnier
authors.affiliation: the Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom.
authors.forename: Steven
authors.initials: S
authors.lastname: Johnson
authors.affiliation: the Malaria Programme, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA.
authors.forename: Julian C
authors.initials: JC
authors.lastname: Rayner
authors.affiliation: From the Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH,; the Malaria Programme, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA. Electronic address: gw2@sanger.ac.uk.
authors.forename: Gavin J
authors.initials: GJ
authors.lastname: Wright
mesh_terms: D000953:Antigens, Protozoan / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D004912:Erythrocytes / Q000276:immunology* / Q000378:metabolism / Q000469:parasitology; D006021:Glycophorins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D017780:Malaria Vaccines / Q000235:genetics / Q000276:immunology*; D016778:Malaria, Falciparum / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000517:prevention & control; D010963:Plasmodium falciparum / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins
chemical_list: D000953:Antigens, Protozoan; C409972:GYPA protein, human; D006021:Glycophorins; D017780:Malaria Vaccines; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C065668:erythrocyte-binding antigen 175, Plasmodium
keywords: Carbohydrate-binding Protein; Cell Invasion; Cell Surface Receptor; Infectious Diseases; Malaria; Protein-Protein Interactions; Surface Plasmon Resonance (SPR)
references.citation: Cell. 2005 Jul 29;122(2):183-93
references.pmid: 16051144
references.citation: Mol Biochem Parasitol. 2002 Aug 7;123(1):23-33
references.pmid: 12165386
references.citation: BMC Biol. 2010 Jun 04;8:76
references.pmid: 20525357
references.citation: Trends Parasitol. 2011 May;27(5):222-9
references.pmid: 21354860
references.citation: PLoS Pathog. 2012;8(11):e1003031
references.pmid: 23166499
references.citation: Mol Biosyst. 2009 Dec;5(12):1405-12
references.pmid: 19593473
references.citation: J Clin Invest. 2010 Dec;120(12):4168-78
references.pmid: 21123952
references.citation: PLoS Med. 2008 Jul 22;5(7):e142
references.pmid: 18651785
references.citation: Nat Struct Mol Biol. 2011 Jul 10;18(8):908-14
references.pmid: 21743458
references.citation: Parasitology. 2000 Mar;120 ( Pt 3):225-35
references.pmid: 10759080
references.citation: Nature. 2010 Sep 23;467(7314):420-5
references.pmid: 20864995
references.citation: Science. 1994 Jun 24;264(5167):1941-4
references.pmid: 8009226
references.citation: Cytometry A. 2010 Nov;77(11):1067-74
references.pmid: 20872885
references.citation: Infect Immun. 1998 Sep;66(9):4203-7
references.pmid: 9712768
references.citation: Am J Phys Anthropol. 1998 Oct;107(2):187-98
references.pmid: 9786333
references.citation: Proc Natl Acad Sci U S A. 2005 Sep 6;102(36):12819-24
references.pmid: 16126901
references.citation: Nucleic Acids Res. 2002 Jan 15;30(2):E9
references.pmid: 11788735
references.citation: J Cell Biol. 2006 Sep 25;174(7):1023-33
references.pmid: 17000879
references.citation: Immunity. 2000 Aug;13(2):233-42
references.pmid: 10981966
references.citation: Nature. 2011 Nov 09;480(7378):534-7
references.pmid: 22080952
references.citation: Infect Immun. 2011 Mar;79(3):1107-17
references.pmid: 21149582
references.citation: Infect Immun. 2005 Oct;73(10):6530-6
references.pmid: 16177327
references.citation: Clin Infect Dis. 2010 Oct 15;51(8):e50-60
references.pmid: 20843207
references.citation: Clin Vaccine Immunol. 2010 Oct;17(10):1552-9
references.pmid: 20702657
references.citation: Science. 1997 Apr 4;276(5309):131-3
references.pmid: 9082985
references.citation: Mol Biochem Parasitol. 1990 May;40(2):285-94
references.pmid: 2194125
references.citation: Lancet. 2012 Feb 4;379(9814):413-31
references.pmid: 22305225
references.citation: J Exp Med. 1993 Nov 1;178(5):1497-505
references.pmid: 8228803
references.citation: Nature. 2002 Feb 7;415(6872):673-9
references.pmid: 11832955
references.citation: Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):7085-9
references.pmid: 1496004
references.citation: Clin Microbiol Infect. 2011 Nov;17(11):1600-7
references.pmid: 21883665
references.citation: Expert Rev Vaccines. 2011 Dec;10(12):1697-708
references.pmid: 22085173
references.citation: Vaccine. 2006 Jul 26;24(31-32):5839-51
references.pmid: 16735084
references.citation: PLoS One. 2011 Apr 14;6(4):e18393
references.pmid: 21533224
references.citation: Proc Natl Acad Sci U S A. 2011 May 3;108(18):7553-8
references.pmid: 21502513
references.citation: Science. 1985 Nov 1;230(4725):553-6
references.pmid: 3901257
references.citation: J Cell Biol. 1992 Feb;116(4):901-9
references.pmid: 1310320
references.citation: Malar J. 2008 Oct 01;7:197
references.pmid: 18828893
references.citation: Proc Natl Acad Sci U S A. 1975 Aug;72(8):2964-8
references.pmid: 1059087
references.citation: Genome Res. 2008 Apr;18(4):622-30
references.pmid: 18296487
references.citation: Vaccine. 2013 Jan 2;31(2):373-9
references.pmid: 23146673