title: A library of functional recombinant cell-surface and secreted P. falciparum merozoite proteins.
abstract: Malaria, an infectious disease caused by parasites of the Plasmodium genus, is one of the world's major public health concerns causing up to a million deaths annually, mostly because of P. falciparum infections. All of the clinical symptoms are associated with the blood stage of the disease, an obligate part of the parasite life cycle, when a form of the parasite called the merozoite recognizes and invades host erythrocytes. During erythrocyte invasion, merozoites are directly exposed to the host humoral immune system making the blood stage of the parasite a conceptually attractive therapeutic target. Progress in the functional and molecular characterization of P. falciparum merozoite proteins, however, has been hampered by the technical challenges associated with expressing these proteins in a biochemically active recombinant form. This challenge is particularly acute for extracellular proteins, which are the likely targets of host antibody responses, because they contain structurally critical post-translational modifications that are not added by some recombinant expression systems. Here, we report the development of a method that uses a mammalian expression system to compile a protein resource containing the entire ectodomains of 42 P. falciparum merozoite secreted and cell surface proteins, many of which have not previously been characterized. Importantly, we are able to recapitulate known biochemical activities by showing that recombinant MSP1-MSP7 and P12-P41 directly interact, and that both recombinant EBA175 and EBA140 can bind human erythrocytes in a sialic acid-dependent manner. Finally, we use sera from malaria-exposed immune adults to profile the relative immunoreactivity of the proteins and show that the majority of the antigens contain conformational (heat-labile) epitopes. We envisage that this resource of recombinant proteins will make a valuable contribution toward a molecular understanding of the blood stage of P. falciparum infections and facilitate the comparative screening of antigens as blood-stage vaccine candidates.
authors.affiliation: Cell Surface Signalling laboratory, Wellcome Trust Sanger Institute, Cambridge CB10 1HH, UK;
authors.forename: Cécile
authors.initials: C
authors.lastname: Crosnier
authors.forename: Madushi
authors.initials: M
authors.lastname: Wanaguru
authors.forename: Brian
authors.initials: B
authors.lastname: McDade
authors.forename: Faith H
authors.initials: FH
authors.lastname: Osier
authors.forename: Kevin
authors.initials: K
authors.lastname: Marsh
authors.forename: Julian C
authors.initials: JC
authors.lastname: Rayner
authors.forename: Gavin J
authors.initials: GJ
authors.lastname: Wright
mesh_terms: D000953:Antigens, Protozoan / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D000954:Antigens, Surface / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D003001:Cloning, Molecular; D004912:Erythrocytes / Q000469:parasitology*; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D007106:Immune Sera / Q000737:chemistry; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D020066:Merozoite Surface Protein 1 / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D052940:Merozoites / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D058977:Molecular Sequence Annotation; D019151:Peptide Library; D010963:Plasmodium falciparum / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding; D020543:Proteome / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012794:Sialic Acids / Q000378:metabolism
chemical_list: D000953:Antigens, Protozoan; D000954:Antigens, Surface; D002352:Carrier Proteins; C438380:EBA140 protein, Plasmodium falciparum; D007106:Immune Sera; D008565:Membrane Proteins; D020066:Merozoite Surface Protein 1; D019151:Peptide Library; D020543:Proteome; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D012794:Sialic Acids; C065668:erythrocyte-binding antigen 175, Plasmodium; C439571:merozoite surface protein 7, Plasmodium
references.citation: PLoS Pathog. 2012;8(11):e1003031
references.pmid: 23166499
references.citation: Mol Biochem Parasitol. 2009 Jun;165(2):153-61
references.pmid: 19428662
references.citation: Nat Genet. 2007 Jan;39(1):126-30
references.pmid: 17159981
references.citation: Lancet. 2012 Feb 4;379(9814):413-31
references.pmid: 22305225
references.citation: Vaccine. 2000 Nov 22;19(7-8):816-24
references.pmid: 11115704
references.citation: J Biol Chem. 2003 Jul 11;278(28):25977-81
references.pmid: 12716913
references.citation: Mol Biochem Parasitol. 2001 Sep 28;117(1):83-9
references.pmid: 11551634
references.citation: J Mol Biol. 1999 Sep 24;292(3):741-58
references.pmid: 10497036
references.citation: Mol Cell Proteomics. 2010 Dec;9(12):2654-65
references.pmid: 20802085
references.citation: Structure. 2007 Mar;15(3):267-73
references.pmid: 17355862
references.citation: N Engl J Med. 2012 Dec 13;367(24):2284-95
references.pmid: 23136909
references.citation: Mol Biochem Parasitol. 2000 Dec;111(2):377-90
references.pmid: 11163444
references.citation: PLoS One. 2008 May 14;3(5):e2189
references.pmid: 18478103
references.citation: Mol Biochem Parasitol. 2006 Aug;148(2):144-60
references.pmid: 16644028
references.citation: Mol Biochem Parasitol. 1996 Oct 1;80(2):159-69
references.pmid: 8892293
references.citation: Science. 1994 Jun 24;264(5167):1941-4
references.pmid: 8009226
references.citation: J Biol Chem. 2004 Feb 6;279(6):4648-56
references.pmid: 14613941
references.citation: BMC Biol. 2010 Jun 04;8:76
references.pmid: 20525357
references.citation: Int J Parasitol. 2000 Feb;30(2):113-8
references.pmid: 10704592
references.citation: PLoS One. 2008 Mar 05;3(3):e1732
references.pmid: 18320051
references.citation: Proc Natl Acad Sci U S A. 2008 Mar 18;105(11):4301-5
references.pmid: 18332422
references.citation: Nucleic Acids Res. 2002 Jan 15;30(2):E9
references.pmid: 11788735
references.citation: Eukaryot Cell. 2002 Dec;1(6):915-25
references.pmid: 12477792
references.citation: J Mol Biol. 2004 Jul 16;340(4):783-95
references.pmid: 15223320
references.citation: Genome Biol. 2009;10(9):R99
references.pmid: 19765300
references.citation: Proteomics. 2008 Nov;8(22):4680-94
references.pmid: 18937256
references.citation: PLoS One. 2009;4(4):e5410
references.pmid: 19404387
references.citation: Yeast. 2001 Jan 30;18(2):137-50
references.pmid: 11169756
references.citation: Trends Parasitol. 2008 May;24(5):210-8
references.pmid: 18420458
references.citation: Mol Biotechnol. 2006 Oct;34(2):225-37
references.pmid: 17172668
references.citation: Vaccine. 2000 Jan 31;18(14):1402-11
references.pmid: 10618538
references.citation: Infect Immun. 2008 Apr;76(4):1702-8
references.pmid: 18268027
references.citation: Protein Eng. 1994 Apr;7(4):515-21
references.pmid: 7518084
references.citation: Proc Int Conf Intell Syst Mol Biol. 1998;6:175-82
references.pmid: 9783223
references.citation: Nat Med. 2003 Jan;9(1):87-92
references.pmid: 12469115
references.citation: Mol Cell Proteomics. 2011 Sep;10(9):M111.007948
references.pmid: 21628511
references.citation: Cell. 2006 Feb 24;124(4):755-66
references.pmid: 16497586
references.citation: Infect Immun. 2010 Jan;78(1):486-94
references.pmid: 19884337
references.citation: Infect Immun. 2008 May;76(5):2240-8
references.pmid: 18316390
references.citation: N Engl J Med. 2011 Nov 17;365(20):1863-75
references.pmid: 22007715
references.citation: Nature. 2011 Nov 09;480(7378):534-7
references.pmid: 22080952
references.citation: Protein Expr Purif. 2004 Mar;34(1):87-94
references.pmid: 14766303
references.citation: Nature. 2002 Oct 3;419(6906):498-511
references.pmid: 12368864
references.citation: Genome Res. 2004 Oct;14(10B):2076-82
references.pmid: 15489329
references.citation: Proc Natl Acad Sci U S A. 2010 Mar 16;107(11):4884-9
references.pmid: 20194779
references.citation: Proc Natl Acad Sci U S A. 2010 Apr 13;107(15):6958-63
references.pmid: 20351286
references.citation: PLoS Med. 2007 Nov 13;4(11):e320
references.pmid: 18001147
references.citation: Clin Infect Dis. 2010 Oct 15;51(8):e50-60
references.pmid: 20843207
references.citation: PLoS Med. 2010 Jan 19;7(1):e1000218
references.pmid: 20098724
references.citation: J Biol Chem. 2006 Oct 20;281(42):31517-27
references.pmid: 16940297
references.citation: Mol Biochem Parasitol. 2007 Jan;151(1):100-10
references.pmid: 17125854
references.citation: Exp Parasitol. 2004 Nov-Dec;108(3-4):186-9
references.pmid: 15582517
references.citation: Curr Opin Biotechnol. 2009 Dec;20(6):700-7
references.pmid: 19889531
references.citation: Malar J. 2008 Oct 01;7:197
references.pmid: 18828893
references.citation: Immunol Cell Biol. 2009 Jul;87(5):377-90
references.pmid: 19417768
references.citation: Nat Biotechnol. 2012 Apr 10;30(4):316-7
references.pmid: 22491276
references.citation: PLoS One. 2012;7(7):e41937
references.pmid: 22848665
references.citation: Nat Commun. 2011 Dec 20;2:601
references.pmid: 22186897
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
references.citation: Mol Biochem Parasitol. 2009 Feb;163(2):119-22
references.pmid: 19026694
references.citation: Anal Biochem. 2012 May 1;424(1):45-53
references.pmid: 22342946
references.citation: Int J Parasitol. 2009 Feb;39(3):371-80
references.pmid: 19000690