The Chondroitin Sulfate A-binding Site of the VAR2CSA Protein Involves Multiple N-terminal Domains
2011; Elsevier BV; Volume: 286; Issue: 18 Linguagem: Inglês
10.1074/jbc.m110.191510
ISSN1083-351X
AutoresMadeleine Dahlbäck, Lars Jørgensen, Morten A. Nielsen, Thomas Mandel Clausen, Sisse Bolm Ditlev, Mafalda Resende, Vera V. Pinto, David E. Arnot, Thor G. Theander, Ali Salanti,
Tópico(s)HIV Research and Treatment
ResumoMalaria during pregnancy is a major health problem for African women. The disease is caused by Plasmodium falciparum malaria parasites, which accumulate in the placenta by adhering to chondroitin sulfate A (CSA). The interaction between infected erythrocytes and the placental receptor is mediated by a parasite expressed protein named VAR2CSA. A vaccine protecting pregnant women against placental malaria should induce antibodies inhibiting the interaction between VAR2CSA and CSA. Much effort has been put into defining the part of the 350 kDa VAR2CSA protein that is responsible for binding. It has been shown that full-length recombinant VAR2CSA binds specifically to CSA with high affinity, however to date no sub-fragment of VAR2CSA has been shown to interact with CSA with similar affinity or specificity. In this study, we used a biosensor technology to examine the binding properties of a panel of truncated VAR2CSA proteins. The experiments indicate that the core of the CSA-binding site is situated in three domains, DBL2X-CIDRPAM and a flanking domain, located in the N-terminal part of VAR2CSA. Furthermore, recombinant VAR2CSA subfragments containing this region elicit antibodies with high parasite adhesion blocking activity in animal immunization experiments. Malaria during pregnancy is a major health problem for African women. The disease is caused by Plasmodium falciparum malaria parasites, which accumulate in the placenta by adhering to chondroitin sulfate A (CSA). The interaction between infected erythrocytes and the placental receptor is mediated by a parasite expressed protein named VAR2CSA. A vaccine protecting pregnant women against placental malaria should induce antibodies inhibiting the interaction between VAR2CSA and CSA. Much effort has been put into defining the part of the 350 kDa VAR2CSA protein that is responsible for binding. It has been shown that full-length recombinant VAR2CSA binds specifically to CSA with high affinity, however to date no sub-fragment of VAR2CSA has been shown to interact with CSA with similar affinity or specificity. In this study, we used a biosensor technology to examine the binding properties of a panel of truncated VAR2CSA proteins. The experiments indicate that the core of the CSA-binding site is situated in three domains, DBL2X-CIDRPAM and a flanking domain, located in the N-terminal part of VAR2CSA. Furthermore, recombinant VAR2CSA subfragments containing this region elicit antibodies with high parasite adhesion blocking activity in animal immunization experiments. IntroductionCarbohydrates, or glycans, are highly diverse in structure and biological function and are expressed by virtually all mammalian cells. Many microorganisms, i.e. viruses, bacteria, and protozoa, have evolved mechanisms to establish infection by interacting with glycans on the host cell surface (1Varki A. Cummings R.D. Esko J.D. Freeze H.H. Stanley P. Bertozzi C.R. Hart G.W. Etzler M.E. 2nd Ed. Essentials of Glycobiology. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2009Google Scholar), and the Plasmodium parasites causing malaria are no exception. Of the different Plasmodium species that can infect humans, Plasmodium falciparum is by far the most virulent form and was the cause of 89% of all deaths due to malaria infection in the African region in 2008 (the World Malaria Report 2009, WHO).A number of important potential P. falciparum vaccine targets have been defined as glycan-binding proteins or lectins, e.g. circumsporozoite protein, which interacts with highly sulfated heparan sulfate proteoglycans (HSPG) 3The abbreviations used are: HSPG, heparan sulfate proteoglycan; CSA, chondroitin sulfate A; PfEMP1, P. falciparum erythrocyte membrane protein 1; CIDR, cysteine-rich interdomain region; CSPG, chondroitin sulfate proteoglycan; DBL, Duffy binding-like domain; FCM, flow cytometry; FV2, full-length ectodomain of the VAR2CSA protein without N-terminal segment; GLURP, glutamate-rich protein; ID, interdomain; IE, P. falciparum-infected erythrocyte; MSP-2, merozoite surface protein 2; NTS, N-terminal segment; PAM, pregnancy-associated malaria; PM, placental malaria. during sporozoite invasion of the liver, and the EBA-175 surface antigen that mediates merozoite invasion of erythrocytes through the interaction with sialic acid on glycophorin A (2Brown A. Higgins M.K. Curr. Opin. Struct. Biol. 2010; 20: 560-566Crossref PubMed Scopus (15) Google Scholar). In addition to these proteins, VAR2CSA, a unique member of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family has been characterized as a lectin with binding preference for a low-sulfated form of chondroitin sulfate A (CSA) anchored to proteoglycans (CSPG) in placental tissue (3Salanti A. Staalsoe T. Lavstsen T. Jensen A.T. Sowa M.P. Arnot D.E. Hviid L. Theander T.G. Mol. Microbiol. 2003; 49: 179-191Crossref PubMed Scopus (559) Google Scholar, 4Achur R.N. Valiyaveettil M. Alkhalil A. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40344-40356Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 5Alkhalil A. Achur R.N. Valiyaveettil M. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40357-40364Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 6Khunrae P. Dahlbäck M. Nielsen M.A. Andersen G. Ditlev S.B. Resende M. Pinto V.V. Theander T.G. Higgins M.K. Salanti A. J. Mol. Biol. 2010; 397: 826-834Crossref PubMed Scopus (92) Google Scholar, 7Srivastava A. Gangnard S. Round A. Dechavanne S. Juillerat A. Raynal B. Faure G. Baron B. Ramboarina S. Singh S.K. Belrhali H. England P. Lewit-Bentley A. Scherf A. Bentley G.A. Gamain B. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 4884-4889Crossref PubMed Scopus (121) Google Scholar). VAR2CSA, is a large multidomain antigen expressed on the surface of P. falciparum-infected erythrocytes (IEs) where it mediates sequestration of the IEs to the placenta (8Salanti A. Dahlbäck M. Turner L. Nielsen M.A. Barfod L. Magistrado P. Jensen A.T. Lavstsen T. Ofori M.F. Marsh K. Hviid L. Theander T.G. J. Exp. Med. 2004; 200: 1197-1203Crossref PubMed Scopus (434) Google Scholar). This placental sequestration causes placental malaria (PM), a major cause of mother and offspring morbidity such as severe maternal anemia, low birth weight, and stillbirth (9Brabin B.J. Romagosa C. Abdelgalil S. Menéndez C. Verhoeff F.H. McGready R. Fletcher K.A. Owens S. d'Alessandro U. Nosten F. Fischer P.R. Ordi J. Placenta. 2004; 25: 359-378Crossref PubMed Scopus (279) Google Scholar).Pregnant women and their unborn children are the victims of this severe disease, and it should be feasible to protect them by a vaccine. This is based on the fact that immunity to PM in malaria-endemic regions is acquired as a function of parity (10Fried M. Nosten F. Brockman A. Brabin B.J. Duffy P.E. Nature. 1998; 395: 851-852Crossref PubMed Scopus (453) Google Scholar, 11Ricke C.H. Staalsoe T. Koram K. Akanmori B.D. Riley E.M. Theander T.G. Hviid L. J. Immunol. 2000; 165: 3309-3316Crossref PubMed Scopus (247) Google Scholar, 12Staalsoe T. Megnekou R. Fievét N. Ricke C.H. Zornig H.D. Leke R. Taylor D.W. Deloron P. Hviid L. J. Infect. Dis. 2001; 184: 618-626Crossref PubMed Scopus (143) Google Scholar) and the main antigen-mediating sequestration, VAR2CSA, has been found in all P. falciparum genomes studied. When looking at PfEMP1 sequence variation VAR2CSA appears as an unusually conserved PfEMP1 protein with as high as 75–83% amino acid identity between variants (13Bockhorst J. Lu F. Janes J.H. Keebler J. Gamain B. Awadalla P. Su X.Z. Samudrala R. Jojic N. Smith J.D. Mol. Biochem. Parasitol. 2007; 155: 103-112Crossref PubMed Scopus (99) Google Scholar). The large molecular weight of VAR2CSA makes production of full-length recombinant protein for vaccine use difficult. The ideal scenario for vaccine development is to define a region of the VAR2CSA, which induces antibodies that can abrogate placental adhesion of the full repertoire of genetically different P. falciparum parasites. The consequences of vaccinating to induce VAR2CSA-specific antibodies that are opsonising but not able to block parasite sequestration are not known but could potentially worsen the parasite-induced inflammation in the placenta.Considering the size and the complex structure it is essential to define smaller regions of VAR2CSA that can be included in a vaccine, the obvious approach being to define the glycan-binding site. However, this has not been straightforward and the molecular mechanism underlying the interaction between VAR2CSA and CSA remains unresolved. This is in part due to the difficulties in analyzing protein-glycan binding in vitro, which easily gives rise to false positive identification of nonspecific glycan-binding proteins because of the large impact of low affinity charge-charge interactions (14Dahlbäck M. Nielsen M.A. Salanti A. Trends Parasitol. 2010; 5: 230-235Abstract Full Text Full Text PDF Scopus (24) Google Scholar).To date, vaccine development has relied on the production of a large panel of various recombinant VAR2CSA-derived protein fragments and analysis of their capacity to induce antibodies in animals that can block adhesion of IEs to CSA in vitro. This trial and error approach has generated several potential vaccine antigens (15Nielsen M.A. Pinto V.V. Resende M. Dahlbäck M. Ditlev S.B. Theander T.G. Salanti A. Infect. Immun. 2009; 77: 2482-2487Crossref PubMed Scopus (81) Google Scholar, 16Salanti A. Resende M. Ditlev S.B. Pinto V.V. Dahlbäck M. Andersen G. Manczak T. Theander T.G. Nielsen M.A. Malar. J. 2010; 9: 11Crossref PubMed Scopus (31) Google Scholar, 17Fernandez P. Kviebig N.K. Dechavanne S. Lépolard C. Gysin J. Scherf A. Gamain B. Malar. J. 2008; 7: 170Crossref PubMed Scopus (35) Google Scholar) although the capacity of these proteins to specifically bind CSA is questionable (18Resende M. Ditlev S.B. Nielsen M.A. Bodevin S. Bruun S. Pinto V.V. Clausen H. Turner L. Theander T.G. Salanti A. Dahlbäck M. Int. J. Parasitol. 2009; 39: 1195-1204Crossref PubMed Scopus (39) Google Scholar). Nevertheless, understanding the mode of CSA binding of VAR2CSA is likely to be essential for the optimal design of a vaccine against PM.An important breakthrough in this respect was recently made when the full-length ectodomain of VAR2CSA was successfully expressed as a recombinant protein and for the first time high affinity binding, within the nanomolar range, was attained when binding this complete ectodomain of VAR2CSA to CSA (6Khunrae P. Dahlbäck M. Nielsen M.A. Andersen G. Ditlev S.B. Resende M. Pinto V.V. Theander T.G. Higgins M.K. Salanti A. J. Mol. Biol. 2010; 397: 826-834Crossref PubMed Scopus (92) Google Scholar, 7Srivastava A. Gangnard S. Round A. Dechavanne S. Juillerat A. Raynal B. Faure G. Baron B. Ramboarina S. Singh S.K. Belrhali H. England P. Lewit-Bentley A. Scherf A. Bentley G.A. Gamain B. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 4884-4889Crossref PubMed Scopus (121) Google Scholar). Based on a few truncated proteins and a compact low-resolution structure of VAR2CSA, Srivastava et al. (7Srivastava A. Gangnard S. Round A. Dechavanne S. Juillerat A. Raynal B. Faure G. Baron B. Ramboarina S. Singh S.K. Belrhali H. England P. Lewit-Bentley A. Scherf A. Bentley G.A. Gamain B. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 4884-4889Crossref PubMed Scopus (121) Google Scholar) proposed that the entire ectodomain is required for a specific high affinity CSA interaction. This would explain the reported observations that no single recombinant VAR2CSA domain binds with high affinity to CSA (18Resende M. Ditlev S.B. Nielsen M.A. Bodevin S. Bruun S. Pinto V.V. Clausen H. Turner L. Theander T.G. Salanti A. Dahlbäck M. Int. J. Parasitol. 2009; 39: 1195-1204Crossref PubMed Scopus (39) Google Scholar, 19Khunrae P. Philip J.M. Bull D.R. Higgins M.K. J. Mol. Biol. 2009; 393: 202-213Crossref PubMed Scopus (52) Google Scholar).In this study we have systematically analyzed recombinant fragments of VAR2CSA for affinity to CSA and defined two overlapping protein constructs that possess the same binding properties as the full-length VAR2CSA ectodomain. Specific binding activity locates to the four N-terminal domains, DBL1X-ID1, DBL2X, CIDRPAM, and DBL3X. A major part of the interaction seems to be conferred by proteins encompassing the DBL2X-CIDRPAM region, however an additional domain is required to obtain high affinity binding. Importantly animal-induced antibodies against a recombinant protein encoding the entire binding region effectively block adhesion of IEs to CSA.DISCUSSIONSpecific accumulation of P. falciparum-infected erythrocytes in the developing placenta causes the severe malaria syndrome named placental malaria. The main placental receptor for parasite binding are the CSA chains of CSPG (22Fried M. Duffy P.E. Science. 1996; 272: 1502-1504Crossref PubMed Scopus (930) Google Scholar).The unique sulfation pattern of the placental CSA (4Achur R.N. Valiyaveettil M. Alkhalil A. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40344-40356Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 5Alkhalil A. Achur R.N. Valiyaveettil M. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40357-40364Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) probably explains the exclusively placental trophism. The VAR2CSA protein is the major parasite-encoded antigen associated with placental parasite adhesion and is the leading PM vaccine candidate. Several different regions of VAR2CSA have been proposed as responsible for the binding to CSA (23Gamain B. Trimnell A.R. Scheidig C. Scherf A. Miller L.H. Smith J.D. J. Infect. Dis. 2005; 191: 1010-1013Crossref PubMed Scopus (129) Google Scholar, 24Avril M. Gamain B. Lépolard C. Viaud N. Scherf A. Gysin J. Microbes Infect. 2006; 8: 2863-2871Crossref PubMed Scopus (47) Google Scholar), and it was recently suggested that the entire ectodomain is required for a high affinity interaction (7Srivastava A. Gangnard S. Round A. Dechavanne S. Juillerat A. Raynal B. Faure G. Baron B. Ramboarina S. Singh S.K. Belrhali H. England P. Lewit-Bentley A. Scherf A. Bentley G.A. Gamain B. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 4884-4889Crossref PubMed Scopus (121) Google Scholar). In this study, we aimed to define the exact minimum CSA-binding region of the VAR2CSA protein.Proteins of the PfEMP1 family consist of several highly cysteine-rich domains, which form a complex structure held together by a number of intramolecular disulfide bridges. Because of the many disulfide bonds, the production of these molecules as correctly folded protein has been, and remains, a challenge. VAR2CSA is the only PfEMP1 molecule in which the entire extracellular region has been successfully produced as a recombinant protein, and this has only been done in an insect cell expression system (6Khunrae P. Dahlbäck M. Nielsen M.A. Andersen G. Ditlev S.B. Resende M. Pinto V.V. Theander T.G. Higgins M.K. Salanti A. J. Mol. Biol. 2010; 397: 826-834Crossref PubMed Scopus (92) Google Scholar) and in HEK293 cells (7Srivastava A. Gangnard S. Round A. Dechavanne S. Juillerat A. Raynal B. Faure G. Baron B. Ramboarina S. Singh S.K. Belrhali H. England P. Lewit-Bentley A. Scherf A. Bentley G.A. Gamain B. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 4884-4889Crossref PubMed Scopus (121) Google Scholar). In this study, all VAR2CSA proteins were expressed in baculovirus-infected insect cells and successful formation of intramolecular disulfide bridges was confirmed by reduced and non-reduced SDS-PAGE.Our protein-glycan binding results provide compelling evidence that the receptor binding site of VAR2CSA from the FCR3 parasite lies within the N-terminal region, i.e. DBL1X-ID1-DBL2X-CIDRPAM-DBL3X, within which the binding core consists of DBL2X-CIDRPAM (summarized in Table 2). The DBL2X-CIDRPAM protein does not possess all the elements required for a specific CSA interaction of high affinity, but when this core assembles with either the DBL1X domain in the N-terminal end or the DBL3X domain in the C-terminal end, the CSA binding strength is increased to the level of the full-length protein and the specificity seems to be retained. It is possible that these flanking domains have a stabilizing effect on the binding core without directly participating in the CSA binding. Alternatively, DBL1X and DBL3X exhibit residues that are directly involved in the CSA binding, which is enhanced by the presence of these domains. We have previously shown that the DBL2X single domain does bind to some extend to CSA in a solid phase binding assay but with low specificity (18Resende M. Ditlev S.B. Nielsen M.A. Bodevin S. Bruun S. Pinto V.V. Clausen H. Turner L. Theander T.G. Salanti A. Dahlbäck M. Int. J. Parasitol. 2009; 39: 1195-1204Crossref PubMed Scopus (39) Google Scholar). This is supported by the present data showing that the affinity of the DBL2X protein to CSA is low.The CSA binding properties of the DBL3X domain has been extensively studied (19Khunrae P. Philip J.M. Bull D.R. Higgins M.K. J. Mol. Biol. 2009; 393: 202-213Crossref PubMed Scopus (52) Google Scholar, 25Higgins M.K. J. Biol. Chem. 2008; 283: 21842-21846Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 26Singh K. Gittis A.G. Nguyen P. Gowda D.C. Miller L.H. Garboczi D.N. Nat. Struct. Mol. Biol. 2008; 15: 932-938Crossref PubMed Scopus (84) Google Scholar, 27Singh K. Gitti R.K. Diouf A. Zhou H. Gowda D.C. Miura K. Ostazeski S.A. Fairhurst R.M. Garboczi D.N. Long C.A. J. Biol. Chem. 2010; 285: 24855-24862Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), but these results have not conformed to the expected specificity and affinity of the interaction between VAR2CSA and CSA. Although the DBL3X domain alone binds with weak affinity and not specifically to CSA, this domain has been considered to be part of the true CSA-binding site. Thus, it was an unexpected discovery that DBL3X is not necessary for achieving specific high affinity binding in vitro.Many of the tested proteins also showed some affinity to HSPG. However when looking at the raw data in the sensorgrams it is apparent that the peak response is much lower than the CSPG binding, indicating that only a subfraction of the proteins binds to HSPG. This prompts the use of homogenous proteins as well as direct comparative analyses as shown here.On the basis of our results it seems likely that all four N-terminal domains (DBL1X-ID1-DBL2X-CIDRPAM-DBL3X) form a binding cleft or pocket in the native VAR2CSA protein, which enables specific CSA interactions of high affinity. This kind of multivalent binding is rather typical for protein-glycan interactions, although there are exceptions. Monovalent protein-glycan binding tends to be of relatively low affinity (micro-molar range) and high avidity is therefore often required for the protein-glycan interaction to achieve specificity and function in vivo (1Varki A. Cummings R.D. Esko J.D. Freeze H.H. Stanley P. Bertozzi C.R. Hart G.W. Etzler M.E. 2nd Ed. Essentials of Glycobiology. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2009Google Scholar).The full-length ectodomain of VAR2CSA from the 3D7 parasite binds with similar affinity to CSA as the FCR3 variant analyzed here (7Srivastava A. Gangnard S. Round A. Dechavanne S. Juillerat A. Raynal B. Faure G. Baron B. Ramboarina S. Singh S.K. Belrhali H. England P. Lewit-Bentley A. Scherf A. Bentley G.A. Gamain B. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 4884-4889Crossref PubMed Scopus (121) Google Scholar). It has been shown that the overall structure of VAR2CSA from 3D7 is quite compact, which supports the idea that the binding site resides in multiple rather than single domains (7Srivastava A. Gangnard S. Round A. Dechavanne S. Juillerat A. Raynal B. Faure G. Baron B. Ramboarina S. Singh S.K. Belrhali H. England P. Lewit-Bentley A. Scherf A. Bentley G.A. Gamain B. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 4884-4889Crossref PubMed Scopus (121) Google Scholar). From a broader perspective, it is interesting that the receptor binding region of VAR2CSA from the FCR3 isolate lies within the N-terminal part of the protein, which corresponds to the conserved domain composition of other PfEMP1 molecules (28Su X.Z. Heatwole V.M. Wertheimer S.P. Guinet F. Herrfeldt J.A. Peterson D.S. Ravetch J.A. Wellems T.E. Cell. 1995; 82: 89-100Abstract Full Text PDF PubMed Scopus (990) Google Scholar). Several domains within this N-terminal region have been implicated in receptor binding activities, e.g. CD36 binding of CIDR1 domains (29Smith J.D. Kyes S. Craig A.G. Fagan T. Hudson-Taylor D. Miller L.H. Baruch D.I. Newbold C.I. Mol. Biochem. Parasitol. 1998; 97: 133-148Crossref PubMed Scopus (94) Google Scholar, 30Chen Q. Heddini A. Barragan A. Fernandez V. Pearce S.F. Wahlgren M. J. Exp. Med. 2000; 192: 1-10Crossref PubMed Scopus (181) Google Scholar, 31Robinson B.A. Welch T.L. Smith J.D. Mol. Microbiol. 2003; 47: 1265-1278Crossref PubMed Scopus (162) Google Scholar), ICAM-1 binding of DBLβ-C2 domains (32Smith J.D. Subramanian G. Gamain B. Baruch D.I. Miller L.H. Mol. Biochem. Parasitol. 2000; 110: 293-310Crossref PubMed Scopus (217) Google Scholar, 33Howell D.P. Levin E.A. Springer A.L. Kraemer S.M. Phippard D.J. Schief W.R. Smith J.D. Mol. Microbiol. 2008; 67: 78-87Crossref PubMed Scopus (66) Google Scholar, 34Oleinikov A.V. Amos E. Frye I.T. Rossnagle E. Mutabingwa T.K. Fried M. Duffy P.E. PLoS. Pathog. 2009; 5: e1000386Crossref PubMed Scopus (57) Google Scholar) and binding of the DBL1α domain to HSPG (35Vogt A.M. Barragan A. Chen Q. Kironde F. Spillmann D. Wahlgren M. Blood. 2003; 101: 2405-2411Crossref PubMed Scopus (108) Google Scholar).It is tempting to speculate that all PfEMP1 molecules share a fundamentally similar mode of binding such that the receptor/ligand contact zone is always in the semi-conserved N-terminal part, although the number of domains necessary for high affinity and specificity may vary. However, considerably more structural knowledge about the diverse ectodomains and binding sites of PfEMP1 proteins are needed to substantiate this proposal.The inhibitory function of the VAR2CSA-specific antibodies generated for this study support the location of the CSA-binding site in the N-terminal part since antibodies induced against the NTS-DBL3X protein efficiently abrogate parasite adhesion. The NTS-DBL3X construct binds specifically and with nanomolar affinity to CSPG, which suggests that it displays an essentially fully functional receptor binding site and that epitopes within and around this site are the targets of the inhibitory antibodies. By contrast, the individual domains DBL2X and DBL3X both bind poorly to CSPG, probably because many residues necessary for a strong interaction are not present in a single domain. This would explain why the antigen epitopes necessary for inducing adhesion-blocking antibodies, which are probably conformation dependent and contributed by separate domains, are absent from these constructs. Supporting this view, pooled antibodies raised against individual domains (DBL1X, DBL2X, and DBL3X) were not able to inhibit the interaction between VAR2CSA and CSPG. One additional possible explanation for the lack of adhesion inhibition demonstrated by pooled IgG could be the absence of antibodies specific for the CIDRPAM domain, which seems to be an essential region of the CSA-binding site.Significantly, our current leading PM vaccine candidate, which is based on the DBL4ϵ domain including a small interdomain region (ID4) from the FCR3 parasite, is not required for a specific CSA interaction of high affinity in vitro, although this protein induces inhibitory antibodies that effectively block parasite adhesion to CSA (15Nielsen M.A. Pinto V.V. Resende M. Dahlbäck M. Ditlev S.B. Theander T.G. Salanti A. Infect. Immun. 2009; 77: 2482-2487Crossref PubMed Scopus (81) Google Scholar). The mechanism behind the binding inhibition induced by immunization with this domain is not known, but it could be explained by steric hindrance since the DBL4ϵ domain has been shown by small angle x-ray scattering (SAXS) analysis to be in close proximity to the CSA binding structure of VAR2CSA. 4S. Christoffersen, A. E. Langkilde, L. Turner, T. Lavstsen, B. Berisha, K. E. Jensen, M. A. Nielsen T. G. Theander, S. Larsen, and A. Salanti, manuscript in preparation. These DBL4ϵ-ID4 antibodies have recently proven to be capable of inhibiting the CSA binding of a panel of P. falciparum parasites isolated from the placenta of delivering mothers in both Tanzania and Benin (36Magistrado P.A. Minja D. Doritchamou J. Ndam N.T. John D. Schmiegelow C. Massougbodji A. Dahlbäck M. Ditlev S.B. Pinto V.V. Resende M. Lusingu J. Theander T.G. Salanti A. Nielsen M.A. Vaccine. 2011; 29: 437-443Crossref PubMed Scopus (38) Google Scholar). Cross-inhibition is an essential property of any candidate for PM vaccine development and by targeting the exact CSA-binding site it might be possible to induce an even more potent immune response than has been achieved with the DBL4ϵ-ID4 construct. In addition, such a directed antibody response against the actual receptor binding site would be much more difficult for the parasite to evade by mutation, following introduction of an inhibitory PM vaccine. Possibly the DBL4 domain could be fused to the DBL2X-CIDRPAM to make a stable chimeric protein, both presenting the minimal binding region as well as the DBL4 epitopes that has been shown to induce inhibitory antibodies. IntroductionCarbohydrates, or glycans, are highly diverse in structure and biological function and are expressed by virtually all mammalian cells. Many microorganisms, i.e. viruses, bacteria, and protozoa, have evolved mechanisms to establish infection by interacting with glycans on the host cell surface (1Varki A. Cummings R.D. Esko J.D. Freeze H.H. Stanley P. Bertozzi C.R. Hart G.W. Etzler M.E. 2nd Ed. Essentials of Glycobiology. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2009Google Scholar), and the Plasmodium parasites causing malaria are no exception. Of the different Plasmodium species that can infect humans, Plasmodium falciparum is by far the most virulent form and was the cause of 89% of all deaths due to malaria infection in the African region in 2008 (the World Malaria Report 2009, WHO).A number of important potential P. falciparum vaccine targets have been defined as glycan-binding proteins or lectins, e.g. circumsporozoite protein, which interacts with highly sulfated heparan sulfate proteoglycans (HSPG) 3The abbreviations used are: HSPG, heparan sulfate proteoglycan; CSA, chondroitin sulfate A; PfEMP1, P. falciparum erythrocyte membrane protein 1; CIDR, cysteine-rich interdomain region; CSPG, chondroitin sulfate proteoglycan; DBL, Duffy binding-like domain; FCM, flow cytometry; FV2, full-length ectodomain of the VAR2CSA protein without N-terminal segment; GLURP, glutamate-rich protein; ID, interdomain; IE, P. falciparum-infected erythrocyte; MSP-2, merozoite surface protein 2; NTS, N-terminal segment; PAM, pregnancy-associated malaria; PM, placental malaria. during sporozoite invasion of the liver, and the EBA-175 surface antigen that mediates merozoite invasion of erythrocytes through the interaction with sialic acid on glycophorin A (2Brown A. Higgins M.K. Curr. Opin. Struct. Biol. 2010; 20: 560-566Crossref PubMed Scopus (15) Google Scholar). In addition to these proteins, VAR2CSA, a unique member of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family has been characterized as a lectin with binding preference for a low-sulfated form of chondroitin sulfate A (CSA) anchored to proteoglycans (CSPG) in placental tissue (3Salanti A. Staalsoe T. Lavstsen T. Jensen A.T. Sowa M.P. Arnot D.E. Hviid L. Theander T.G. Mol. Microbiol. 2003; 49: 179-191Crossref PubMed Scopus (559) Google Scholar, 4Achur R.N. Valiyaveettil M. Alkhalil A. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40344-40356Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 5Alkhalil A. Achur R.N. Valiyaveettil M. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40357-40364Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 6Khunrae P. Dahlbäck M. Nielsen M.A. Andersen G. Ditlev S.B. Resende M. Pinto V.V. Theander T.G. Higgins M.K. Salanti A. J. Mol. Biol. 2010; 397: 826-834Crossref PubMed Scopus (92) Google Scholar, 7Srivastava A. Gangnard S. Round A. Dechavanne S. Juillerat A. Raynal B. Faure G. Baron B. Ramboarina S. Singh S.K. Belrhali H. England P. Lewit-Bentley A. Scherf A. Bentley G.A. Gamain B. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 4884-4889Crossref PubMed Scopus (121) Google Scholar). VAR2CSA, is a large multidomain antigen expressed on the surface of P. falciparum-infected erythrocytes (IEs) where it mediates sequestration of the IEs to the placenta (8Salanti A. Dahlbäck M. Turner L. Nielsen M.A. Barfod L. Magistrado P. Jensen A.T. Lavstsen T. Ofori M.F. Marsh K. Hviid L. Theander T.G. J. Exp. Med. 2004; 200: 1197-1203Crossref PubMed Scopus (434) Google Scholar). This placental sequestration causes placental malaria (PM), a major cause of mother and offspring morbidity such as severe maternal anemia, low birth weight, and stillbirth (9Brabin B.J. Romagosa C. Abdelgalil S. Menéndez C. Verhoeff F.H. McGready R. Fletcher K.A. Owens S. d'Alessandro U. Nosten F. Fischer P.R. Ordi J. Placenta. 2004; 25: 359-378Crossref PubMed Scopus (279) Google Scholar).Pregnant women and their unborn children are the victims of this severe disease, and it should be feasible to protect them by a vaccine. This is based on the fact that immunity to PM in malaria-endemic regions is acquired as a function of parity (10Fried M. Nosten F. Brockman A. Brabin B.J. Duffy P.E. Nature. 1998; 395: 851-852Crossref PubMed Scopus (453) Google Scholar, 11Ricke C.H. Staalsoe T. Koram K. Akanmori B.D. Riley E.M. Theander T.G. Hviid L. J. Immunol. 2000; 165: 3309-3316Crossref PubMed Scopus (247) Google Scholar, 12Staalsoe T. Megnekou R. Fievét N. Ricke C.H. Zornig H.D. Leke R. Taylor D.W. Deloron P. Hviid L. J. Infect. Dis. 2001; 184: 618-626Crossref PubMed Scopus (143) Google Scholar) and the main antigen-mediating sequestration, VAR2CSA, has been found in all P. falciparum genomes studied. When looking at PfEMP1 sequence variation VAR2CSA appears as an unusually conserved PfEMP1 protein with as high as 75–83% amino acid identity between variants (13Bockhorst J. Lu F. Janes J.H. Keebler J. Gamain B. Awadalla P. Su X.Z. Samudrala R. Jojic N. Smith J.D. Mol. Biochem. Parasitol. 2007; 155: 103-112Crossref PubMed Scopus (99) Google Scholar). The large molecular weight of VAR2CSA makes production of full-length recombinant protein for vaccine use difficult. The ideal scenario for vaccine development is to define a region of the VAR2CSA, which induces antibodies that can abrogate placental adhesion of the full repertoire of genetically different P. falciparum parasites. The consequences of vaccinating to induce VAR2CSA-specific antibodies that are opsonising but not able to block parasite sequestration are not known but could potentially worsen the parasite-induced inflammation in the placenta.Considering the size and the complex structure it is essential to define smaller regions of VAR2CSA that can be included in a vaccine, the obvious approach being to define the glycan-binding site. However, this has not been straightforward and the molecular mechanism underlying the interaction between VAR2CSA and CSA remains unresolved. This is in part due to the difficulties in analyzing protein-glycan binding in vitro, which easily gives rise to false positive identification of nonspecific glycan-binding proteins because of the large impact of low affinity charge-charge interactions (14Dahlbäck M. Nielsen M.A. Salanti A. Trends Parasitol. 2010; 5: 230-235Abstract Full Text Full Text PDF Scopus (24) Google Scholar).To date, vaccine development has relied on the production of a large panel of various recombinant VAR2CSA-derived protein fragments and analysis of their capacity to induce antibodies in animals that can block adhesion of IEs to CSA in vitro. This trial and error approach has generated several potential vaccine antigens (15Nielsen M.A. Pinto V.V. Resende M. Dahlbäck M. Ditlev S.B. Theander T.G. Salanti A. Infect. Immun. 2009; 77: 2482-2487Crossref PubMed Scopus (81) Google Scholar, 16Salanti A. Resende M. Ditlev S.B. Pinto V.V. Dahlbäck M. Andersen G. Manczak T. Theander T.G. Nielsen M.A. Malar. J. 2010; 9: 11Crossref PubMed Scopus (31) Google Scholar, 17Fernandez P. Kviebig N.K. Dechavanne S. Lépolard C. Gysin J. Scherf A. Gamain B. Malar. J. 2008; 7: 170Crossref PubMed Scopus (35) Google Scholar) although the capacity of these proteins to specifically bind CSA is questionable (18Resende M. Ditlev S.B. Nielsen M.A. Bodevin S. Bruun S. Pinto V.V. Clausen H. Turner L. Theander T.G. Salanti A. Dahlbäck M. Int. J. Parasitol. 2009; 39: 1195-1204Crossref PubMed Scopus (39) Google Scholar). Nevertheless, understanding the mode of CSA binding of VAR2CSA is likely to be essential for the optimal design of a vaccine against PM.An important breakthrough in this respect was recently made when the full-length ectodomain of VAR2CSA was successfully expressed as a recombinant protein and for the first time high affinity binding, within the nanomolar range, was attained when binding this complete ectodomain of VAR2CSA to CSA (6Khunrae P. Dahlbäck M. Nielsen M.A. Andersen G. Ditlev S.B. Resende M. Pinto V.V. Theander T.G. Higgins M.K. Salanti A. J. Mol. Biol. 2010; 397: 826-834Crossref PubMed Scopus (92) Google Scholar, 7Srivastava A. Gangnard S. Round A. Dechavanne S. Juillerat A. Raynal B. Faure G. Baron B. Ramboarina S. Singh S.K. Belrhali H. England P. Lewit-Bentley A. Scherf A. Bentley G.A. Gamain B. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 4884-4889Crossref PubMed Scopus (121) Google Scholar). Based on a few truncated proteins and a compact low-resolution structure of VAR2CSA, Srivastava et al. (7Srivastava A. Gangnard S. Round A. Dechavanne S. Juillerat A. Raynal B. Faure G. Baron B. Ramboarina S. Singh S.K. Belrhali H. England P. Lewit-Bentley A. Scherf A. Bentley G.A. Gamain B. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 4884-4889Crossref PubMed Scopus (121) Google Scholar) proposed that the entire ectodomain is required for a specific high affinity CSA interaction. This would explain the reported observations that no single recombinant VAR2CSA domain binds with high affinity to CSA (18Resende M. Ditlev S.B. Nielsen M.A. Bodevin S. Bruun S. Pinto V.V. Clausen H. Turner L. Theander T.G. Salanti A. Dahlbäck M. Int. J. Parasitol. 2009; 39: 1195-1204Crossref PubMed Scopus (39) Google Scholar, 19Khunrae P. Philip J.M. Bull D.R. Higgins M.K. J. Mol. Biol. 2009; 393: 202-213Crossref PubMed Scopus (52) Google Scholar).In this study we have systematically analyzed recombinant fragments of VAR2CSA for affinity to CSA and defined two overlapping protein constructs that possess the same binding properties as the full-length VAR2CSA ectodomain. Specific binding activity locates to the four N-terminal domains, DBL1X-ID1, DBL2X, CIDRPAM, and DBL3X. A major part of the interaction seems to be conferred by proteins encompassing the DBL2X-CIDRPAM region, however an additional domain is required to obtain high affinity binding. Importantly animal-induced antibodies against a recombinant protein encoding the entire binding region effectively block adhesion of IEs to CSA.
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