Biochemical, Biophysical, and Functional Characterization of Bacterially Expressed and Refolded Receptor Binding Domain ofPlasmodium vivax Duffy-binding Protein

Invasion of erythrocytes by malaria parasites is mediated by specific molecular interactions. Plasmodium vivax is completely dependent on interaction with the Duffy blood group antigen to invade human erythrocytes. The P. vivaxDuffy-binding protein, which binds the Duffy antigen during invasion, belongs to a family of erythrocyte-binding proteins that also includesPlasmodium falciparum sialic acid binding protein andPlasmodium knowlesi Duffy binding protein. The receptor binding domains of these proteins lie in a conserved, N-terminal, cysteine-rich region, region II, found in each of these proteins. Here, we have expressed P. vivax region II (PvRII), the P. vivax Duffy binding domain, in Escherichia coli. Recombinant PvRII is incorrectly folded and accumulates in inclusion bodies. We have developed methods to refold and purify recombinant PvRII in its functional conformation. Biochemical, biophysical, and functional characterization confirms that recombinant PvRII is pure, homogeneous, and functionally active in that it binds Duffy-positive human erythrocytes with specificity. Refolded PvRII is highly immunogenic and elicits high titer antibodies that can inhibit binding of P. vivax Duffy-binding protein to erythrocytes, providing support for its development as a vaccine candidate forP. vivax malaria. Development of methods to produce functionally active recombinant PvRII is an important step for structural studies as well as vaccine development. Invasion of erythrocytes by malaria parasites is mediated by specific molecular interactions. Plasmodium vivax is completely dependent on interaction with the Duffy blood group antigen to invade human erythrocytes. The P. vivaxDuffy-binding protein, which binds the Duffy antigen during invasion, belongs to a family of erythrocyte-binding proteins that also includesPlasmodium falciparum sialic acid binding protein andPlasmodium knowlesi Duffy binding protein. The receptor binding domains of these proteins lie in a conserved, N-terminal, cysteine-rich region, region II, found in each of these proteins. Here, we have expressed P. vivax region II (PvRII), the P. vivax Duffy binding domain, in Escherichia coli. Recombinant PvRII is incorrectly folded and accumulates in inclusion bodies. We have developed methods to refold and purify recombinant PvRII in its functional conformation. Biochemical, biophysical, and functional characterization confirms that recombinant PvRII is pure, homogeneous, and functionally active in that it binds Duffy-positive human erythrocytes with specificity. Refolded PvRII is highly immunogenic and elicits high titer antibodies that can inhibit binding of P. vivax Duffy-binding protein to erythrocytes, providing support for its development as a vaccine candidate forP. vivax malaria. Development of methods to produce functionally active recombinant PvRII is an important step for structural studies as well as vaccine development. P. vivax region II Duffy binding-like P. falciparum erythrocyte membrane protein-1 intercellular adhesion molecule 1 guanidine hydrochloride dithiothreitol circular dichroism polyacrylamide gel electrophoresis enzyme-linked immunosorbent assay VCAM, vascular cell adhesion molecule The invasion of erythrocytes by malaria parasites is mediated by specific molecular interactions between host receptors and parasite ligands (1Chitnis C.E. Sinnis P. Miller L.H. Perlmann P. Wahlgren M. Malaria: Molecular and Clinical Aspects. Harwood Academic Publishers Gmbh, London1999: 249-285Crossref Google Scholar). Plasmodium vivax and the related simian malaria parasite Plasmodium knowlesi require interaction with the Duffy blood group antigen to invade human erythrocytes (2Miller L.H. Mason S.J. Clyde D.F. McGinnis M.H. N. Eng. J. Med. 1976; 295: 302-304Crossref PubMed Scopus (957) Google Scholar, 3Miller L.H. Mason S.J. Dvorak J.A. McGinnis M.H. Rothman I.K. Science. 1975; 189: 561-563Crossref PubMed Scopus (459) Google Scholar). P. knowlesi can also invade rhesus erythrocytes using alternative Duffy-independent receptors (4Haynes J.D. Dalton J.P. Klotz F.W. McGinniss M.H. Hadley T.J. Hudson D.E. Miller L.H. J. Exp. Med. 1988; 167: 1873-1881Crossref PubMed Scopus (127) Google Scholar). P. falciparum commonly uses sialic acid residues of glycophorin A as invasion receptors (5Pasvol G. Wainscoat J.S. Weatherall D.J. Nature. 1982; 297: 64-66Crossref PubMed Scopus (153) Google Scholar, 6Breuer W.V. Ginsburg H. Cabantchik Z.I. Bioch. Biophys. 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Cell. 1990; 63: 141-153Abstract Full Text PDF PubMed Scopus (253) Google Scholar, 17Sim B.K.L. Orlandi P.A. Haynes J.D. Klotz F.W. Carter J.M. Camus D. Zegans M.E. Chulay J.D. J. Cell Biol. 1990; 111: 1877-1884Crossref PubMed Scopus (199) Google Scholar, 18Fang X. Kaslow D.C. Adams J.H. Miller L.H. Mol. Biochem. Parasitol. 1991; 44: 125-132Crossref PubMed Scopus (136) Google Scholar). These parasite ligands share similar features and belong to a family of erythrocyte-binding proteins (19Adams J.H. Sim B.K.L. Dolan S.A. Fang X. Kaslow D.C. Miller L.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7085-7089Crossref PubMed Scopus (406) Google Scholar). The extracellular domain of each erythrocyte-binding protein contains two conserved cysteine-rich regions, regions II and VI, at the amino and carboxyl ends, respectively. P. falciparum EBA-175 contains a tandem duplication (F1 and F2) of the N-terminal, conserved, cysteine-rich region. The functional receptor binding domain of each erythrocyte-binding protein lies in region II (20Chitnis C.E. Miller L.H. J. Exp. Med. 1994; 180: 497-506Crossref PubMed Scopus (341) Google Scholar, 21Sim B.K.L. Chitnis C.E. Wasniowska K. Hadley T.J. Miller L.H. Science. 1994; 264: 1941-1944Crossref PubMed Scopus (492) Google Scholar). In the case of EBA-175, region F2 was found to have receptor binding activity (21Sim B.K.L. Chitnis C.E. Wasniowska K. Hadley T.J. Miller L.H. Science. 1994; 264: 1941-1944Crossref PubMed Scopus (492) Google Scholar).P. vivax region II (PvRII)1 specifically binds the human Duffy antigen, and P. falciparum region F2 specifically binds sialic acid residues of glycophorin A. Region II of the P. knowlesi Duffy-binding protein binds both human and rhesus Duffy antigens. P. knowlesi β region II binds sialic acid residues on rhesus erythrocytes, and P. knowlesiγ region II binds as yet unidentified receptors on rhesus erythrocytes (20Chitnis C.E. Miller L.H. J. Exp. Med. 1994; 180: 497-506Crossref PubMed Scopus (341) Google Scholar, 22Ranjan A. Chitnis C.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14067-14072Crossref PubMed Scopus (103) Google Scholar). These conserved, receptor binding domains are referred to as Duffy binding-like (DBL) domains after the binding domain of P. vivax Duffy-binding protein. DBL domains are also found in the extracellular region of the P. falciparum erythrocyte membrane protein-1 (PfEMP-1) family of proteins that are expressed on the surface of P. falciparum-infected trophozoites and schizonts (23Baruch D.I. Pasloske B.L. Singh H.B. Xiahui B. Ma X.C. Feldman M. Howard R.J. Cell. 1995; 82: 77-88Abstract Full Text PDF PubMed Scopus (888) Google Scholar, 24Smith J.D. Chitnis C.E. Craig A.G. Roberts D.J. Hudson-Taylor D.E. Peterson D.S. Pinches B. Newbold C.I. Miller L.H. Cell. 1995; 82: 101-110Abstract Full Text PDF PubMed Scopus (824) Google Scholar, 25Su X. Heatwole V. Wertheimer S.P. Guinet F. Herrfeldt J.A. Peterson D.S. Wellems T. Cell. 1995; 82: 89-100Abstract Full Text PDF PubMed Scopus (1000) Google Scholar). Some members of the PfEMP-1 family bind endothelial receptors such as CD36, ICAM-1, vascular cell adhesion molecule, E-selectin, CD31, chondroitan sulfate A, and hyaluronic acid to mediate cytoadherence, which is responsible for sequestration of P. falciparumtrophozoites and schizonts in the deep vasculature of various host organs (26Ockenhouse C.F. Tandon N.N. Magowan C. Jamieson G.A. Chulay J.D. Science. 1989; 243: 1469-1471Crossref PubMed Scopus (258) Google Scholar, 27Berendt A.R. Simmons D.L. Tansey J. Newbold C.I. Marsh K. Nature. 1989; 341: 51-57Crossref Scopus (574) Google Scholar, 28Ockenhouse C.F. Tegoshi T. Maeno Y. Benjamin C. Ho M. Kan K.E. Thway Y. Win K. Aikawa M. Lobb R.R. J. Exp. Med. 1992; 176: 1183-1189Crossref PubMed Scopus (325) Google Scholar, 29Rogerson S.J. Chaiyaroj S.C. Ng K. Reeder J.C. Brown G.V. J. Exp. 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Med. 2000; 6: 86-90Crossref PubMed Scopus (272) Google Scholar, 33Fried M. Duffy P. Science. 1996; 272: 1502-1504Crossref PubMed Scopus (946) Google Scholar). DBL domains derived from PfEMP-1 have been shown to bind ICAM-1, chondroitan sulfate A, CD31, and uninfected erythrocytes (34Smith J.D. Craig A.G. Kriek N. Hudson-Taylor D. Kyes S. Fagan T. Pinches R. Baruch D.I. Newbold C.I. Miller L.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1766-1771Crossref PubMed Scopus (192) Google Scholar, 35Buffet P.A. Gamain B. Scheidig C. Baruch D. Smith J.D. Hernandez-Rivas R. Pouvelle B. Oishi S. Fujii N. Fusai T. Parzy D. Miller L.H. Gysin J. Scherf A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12743-12748Crossref PubMed Scopus (217) Google Scholar, 36Reeder J.C. Cowman A.F. Davern K.M. Beeson J.G. Thompson J.K. Rogerson S.J. Brown G.V. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5198-5202Crossref PubMed Scopus (204) Google Scholar, 37Rowe A.J. Moulds J.M. Newbold C.I. Miller L.H. Nature. 1997; 398: 292-295Crossref Scopus (479) Google Scholar, 38Chen Q. Barragan A. Fernandez V. Sundstrom A. Schlichtherle M. Sahlen A. Carlson J. Datta S. Wahlgren M. J. Exp. Med. 1998; 187: 15-23Crossref PubMed Scopus (244) Google Scholar, 39Chen Q. Heddini A. Barragan A. Fernandez V. Wahlgren M. J. Exp. Med. 2000; PubMed Scopus (183) Google Scholar). DBL domains are by Plasmodium to bind host receptors to mediate erythrocyte invasion and cytoadherence, two that are to malaria is important to the structural of the interaction of DBL domains with host the development of that inhibit invasion binding DBL domains are also vaccine antibodies such functional domains interaction with erythrocytes endothelial Here, we methods to produce of folded recombinant the binding domain of P. vivaxDuffy-binding protein, which contains amino Recombinant PvRII been expressed in Escherichia from inclusion and in its conformation. We that PvRII is highly immunogenic and elicits high titer antibodies that can inhibit the binding of P. vivaxDuffy-binding protein to erythrocytes, providing support for the development of recombinant PvRII as a vaccine candidate for P. vivax malaria. of functional DBL domains is for and PvRII vivax Duffy-binding to at the was by using and and a the P. vivaxDuffy-binding protein as X. Kaslow D.C. Adams J.H. Miller L.H. Mol. Biochem. Parasitol. 1991; 44: 125-132Crossref PubMed Scopus (136) Google Scholar). The was with and and as a of the in to was with and for of recombinant was with and at was with the at a of and at to of of PvRII was by to the at a of 1 to for at by in in and by by of at in guanidine hydrochloride and with dithiothreitol for at of by using with recombinant PvRII was from inclusion by using a acid as by the inclusion on a acid with The was with at and protein was using a at and at The of the protein was to with PvRII was by in 1 1 and that the protein was was to at for with the of the was for 1 to with by of by the protein was on an with The protein was with a of to PvRII and PvRII was by gel using a gel was Refolded PvRII was on a The for was developed using A acid in and acid in The was with A and and a of A and in was using an protein using of PvRII was for the of PvRII was by using a by of protein was using to the of Biochem. Biophys. 82: PubMed Scopus Google Scholar). The of the assay was using known of be at a of The of in PvRII was in the of to on a of PvRII in as the of in the region from to using a with a of and the 1 of PvRII for was by the protein assay using as a of the was using the of G. R. R. Eng. 1992; PubMed Scopus Google Scholar). in was at for to and in Duffy of erythrocytes by methods using two and Duffy erythrocytes, not with Duffy-positive erythrocytes in the erythrocyte binding the Refolded PvRII was with Duffy-positive and human erythrocytes at for 1 to The was and to erythrocytes. protein was from the erythrocytes with by and by using a acid derived from PvRII of P. vivax Duffy-binding of PvRII as using by and by using a to the of was by PvRII with a PvRII at a of for 1 on and with protein at for 1 The by and with 1 and with proteins by by and by using a of PvRII can be by Refolded PvRII was with of Duffy-positive human erythrocytes to active PvRII with an of human erythrocytes was as a PvRII after was by and as in from the of the for and and in to the in the of for the and of of of and D. with of recombinant PvRII in and by the The on with of PvRII in by the was with to the to on and and the for for of recombinant PvRII using an of with of PvRII and with in for at with of in of with a with and of was to each well and for at The was developed with as the and as the The was by the of and the at was in each well using an as well as at similar the of that with was as (20Chitnis C.E. Miller L.H. J. Exp. Med. 1994; 180: 497-506Crossref PubMed Scopus (341) Google Scholar). in at using with of to the was to PvRII on the surface of (20Chitnis C.E. Miller L.H. J. Exp. Med. 1994; 180: 497-506Crossref PubMed Scopus (341) Google it contains PvRII to the at the and the region and domain of at the in a (20Chitnis C.E. Miller L.H. J. Exp. Med. 1994; 180: 497-506Crossref PubMed Scopus (341) Google Scholar, G.H. D.L. D. R.J. J. 1988; PubMed Google Scholar). The and of the protein to the surface of of PvRII on the cell surface was by as using a in the protein (20Chitnis C.E. Miller L.H. J. Exp. Med. 1994; 180: 497-506Crossref PubMed Scopus (341) Google Scholar, G.H. D.L. D. R.J. J. 1988; PubMed Google Scholar). Binding of erythrocytes to PvRII on the surface was using an erythrocyte binding as (20Chitnis C.E. Miller L.H. J. Exp. Med. 1994; 180: 497-506Crossref PubMed Scopus (341) Google Scholar). of a of Duffy-positive human erythrocytes was to of in to for at erythrocytes by with binding in the of of and as The of with of erythrocytes was in at a of Recombinant PvRII accumulates in inclusion as a expressed in coli. PvRII was in by and by and during to such as of the recombinant protein after of and of and to of PvRII after Refolded PvRII was by and gel as PvRII was by and by 1 of that the of recombinant PvRII is of PvRII are Recombinant PvRII with the of Refolded and PvRII was using a of biophysical, and functional of recombinant PvRII the which is to the is The molecular of recombinant PvRII by is The of PvRII with a are is and the of recombinant The of PvRII by gel on a is with an molecular of that PvRII not not Refolded PvRII on after with that are in the protein 1 The of PvRII was by a that can be to of the protein on in surface Refolded PvRII as a by on a that the is of PvRII with in an in by 1 the of in the protein in PvRII was using the of Biochem. Biophys. 82: PubMed Scopus Google to the of the protein. be at a of in in recombinant PvRII at a of that PvRII contains that of are was to the of The of PvRII at and of the by the of G. R. R. Eng. 1992; PubMed Scopus Google the of for and The of structural from the in the and on of PvRII are of PvRII with in of at and of erythrocyte binding assay was to PvRII is Recombinant PvRII was with Duffy-positive and human erythrocytes to with protein by protein was with by and by a acid derived from PvRII was to Refolded PvRII binds Duffy-positive human erythrocytes not human erythrocytes Refolded PvRII binds erythrocytes with the vivax Duffy-binding protein, that it is folded in its functional conformation. with a high molecular protein that is to cell the of for of protein. with PvRII also with protein not of PvRII is functionally active and of PvRII with erythrocytes by of PvRII by and was to incorrectly folded of PvRII is by Duffy-positive and human erythrocytes to of PvRII was Duffy erythrocytes for the erythrocytes to that of of PvRII is and of PvRII was the to protein after that at of PvRII is by and is Refolded PvRII in was to for of with PvRII by from two of and that PvRII is highly The of to binding of PvRII to erythrocytes was in an erythrocyte binding with to PvRII with the and domain of at the amino and carboxyl ends, to the P. vivax domain to the cell surface (20Chitnis C.E. Miller L.H. J. Exp. Med. 1994; 180: 497-506Crossref PubMed Scopus (341) Google Scholar). in the protein of PvRII on the cell surface (20Chitnis C.E. Miller L.H. J. Exp. Med. 1994; 180: 497-506Crossref PubMed Scopus (341) Google Scholar, G.H. D.L. D. R.J. J. 1988; PubMed Google Scholar). in the of binding after with Duffy-positive human erythrocytes in the of from a with of from a with The of with of erythrocytes was in at a of of of similar are shown in PvRII completely binding of erythrocytes to PvRII to a of These that PvRII can be to high titer antibodies that are of binding of P. vivax Duffy-binding protein to of erythrocyte binding to PvRII expressed on the surface of with of in of with of blood in at a of The of found in two is not from of similar are of with of blood in at a of The of found in two is not from of similar are in a The functional binding domains of erythrocyte-binding proteins to region II, the conserved, N-terminal, cysteine-rich regions that are also referred to as DBL domains (20Chitnis C.E. Miller L.H. J. Exp. Med. 1994; 180: 497-506Crossref PubMed Scopus (341) Google B.K.L. Chitnis C.E. Wasniowska K. Hadley T.J. Miller L.H. Science. 1994; 264: 1941-1944Crossref PubMed Scopus (492) Google Scholar). domains of PfEMP-1 that mediate as well as binding to endothelial receptors such as ICAM-1, chondroitan sulfate A, and have also been to DBL domains (34Smith J.D. Craig A.G. Kriek N. Hudson-Taylor D. Kyes S. Fagan T. Pinches R. Baruch D.I. Newbold C.I. Miller L.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1766-1771Crossref PubMed Scopus (192) Google Scholar, 35Buffet P.A. Gamain B. Scheidig C. Baruch D. Smith J.D. Hernandez-Rivas R. Pouvelle B. Oishi S. Fujii N. Fusai T. Parzy D. Miller L.H. Gysin J. Scherf A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12743-12748Crossref PubMed Scopus (217) Google Scholar, 36Reeder J.C. Cowman A.F. Davern K.M. Beeson J.G. Thompson J.K. Rogerson S.J. Brown G.V. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5198-5202Crossref PubMed Scopus (204) Google Scholar, 37Rowe A.J. Moulds J.M. Newbold C.I. Miller L.H. Nature. 1997; 398: 292-295Crossref Scopus (479) Google Scholar, 38Chen Q. Barragan A. Fernandez V. Sundstrom A. Schlichtherle M. Sahlen A. Carlson J. Datta S. Wahlgren M. J. Exp. Med. 1998; 187: 15-23Crossref PubMed Scopus (244) Google Scholar, 39Chen Q. Heddini A. Barragan A. Fernandez V. Wahlgren M. J. Exp. Med. 2000; PubMed Scopus (183) Google Scholar). DBL domains in two important erythrocyte invasion and the structural for the interaction of DBL domains with host it is to the of DBL domains and these functional We have shown that binding residues to a acid between the and of PvRII A. Chitnis C.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14067-14072Crossref PubMed Scopus (103) Google Scholar). Here, we methods to produce of recombinant PvRII in its functional and biophysical, and functional characterization of the recombinant Recombinant PvRII was expressed in from inclusion in by the of and by and gel The was shown to be pure, and PvRII is as by of The of PvRII by gel is with an molecular of that PvRII not PvRII as a by that it contains a of PvRII binds Duffy-positive human erythrocytes not human erythrocytes, that it is folded in its functional conformation. PvRII is after of PvRII with Duffy-positive human erythrocytes. that the for PvRII is and of PvRII was the to PvRII after with Duffy-positive human erythrocytes that of PvRII is The of PvRII the of as been for DBL domains J.D. G. Gamain B. Baruch D.I. Miller L.H. Mol. Biochem. Parasitol. 2000; PubMed Scopus Google Scholar). to be DBL domains also high for of PvRII are in the by the The binding domain of P. vivax Duffy-binding protein, is a vaccine candidate antibodies functional domain erythrocyte binding and invasion PvRII PvRII to of by that PvRII is highly from completely inhibit erythrocyte binding to PvRII on the surface to a of These that recombinant PvRII can high titer binding providing support for the development of recombinant PvRII as a vaccine for P. vivax malaria. to the in it is not to antibodies PvRII for of erythrocyte invasion by P. the binding domain of P. falciparum EBA-175 have been shown to inhibit erythrocyte invasion by P. falciparum in D.L. Haynes J.D. S. K. H. Sim Infect. Immun. 2000; PubMed Scopus Google Scholar). C. S. and C. antibodies PvRII can be to erythrocyte invasion by P. in binding antibodies PvRII after to P. vivax P. T. Adams J.H. Infect. Immun. 2000; PubMed Scopus Google Scholar). binding antibodies are of human from in completely binding of erythrocytes to PvRII to a of P. T. Adams J.H. Infect. Immun. 2000; PubMed Scopus Google Scholar). Here, we have shown that PvRII inhibit erythrocyte binding to PvRII to a of to be the of high titer antibodies that inhibit binding of P. vivaxDuffy-binding protein to erythrocytes vivax malaria. Recombinant PvRII been expressed in its functional as a protein in using S. D.E. C.F. Mol. Biochem. Parasitol. 2000; PubMed Scopus Google Scholar). Recombinant proteins expressed as proteins in are commonly which is often a for to PvRII in its functional using have been T. P. Barnwell J.W. A.R. F. Kaslow D.C. Adams J.H. Infect. Immun. 2000; Google Scholar, S. D.E. C.F. Mol. Biochem. Parasitol. 2000; PubMed Scopus Google Scholar). is the of methods developed for and of functional PvRII in a These methods be to DBL domains derived from erythrocyte-binding proteins and from extracellular regions of we have similar methods to and refold P. falciparum region the binding domain of P. falciparum Refolded P. falciparum region F2 specifically binds sialic acid residues of human glycophorin The of methods for of folded DBL domains is an important step for structural studies as well as for malaria vaccine development on these functional receptor binding We and of for providing and for for and for P. of for and and for of