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* Institute of Genetics, Queens Medical Centre, University of Nottingham, Nottingham, England, United Kingdom;
Institute of Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland, United Kingdom;
Institute for Pathology and Centre for Immune Regulation and
Department of Molecular Biosciences, University of Oslo, Oslo, Norway; and
¶ Department of Rheumatology, Hospital for Joint Diseases, New York University Medical Center, New York, NY 10003
The binding of nonspecific human IgM to the surface of infected erythrocytes is important in rosetting, a major virulence factor in the pathogenesis of severe malaria due to Plasmodium falciparum, and IgM binding has also been implicated in placental malaria. Herein we have identified the IgM-binding parasite ligand from a virulent P. falciparum strain as PfEMP1 (TM284var1 variant), and localized the region within this PfEMP1 variant that binds IgM (DBL4β domain). We have used this parasite IgM-binding protein to investigate the interaction with human IgM. Interaction studies with domain-swapped Abs, IgM mutants, and anti-IgM mAbs showed that PfEMP1 binds to the Fc portion of the human IgM H chain and requires the IgM Cµ4 domain. Polymerization of IgM was shown to be crucial for the interaction because PfEMP1 binding did not occur with mutant monomeric IgM molecules. These results with PfEMP1 protein have physiological relevance because infected erythrocytes from strain TM284 and four other IgM-binding P. falciparum strains showed analogous results to those seen with the DBL4β domain. Detailed investigation of the PfEMP1 binding site on IgM showed that some of the critical amino acids in the IgM Cµ4 domain are equivalent to those regions of IgG and IgA recognized by Fc-binding proteins from bacteria, suggesting that this region of Ig molecules may be of major functional significance in host-microbe interactions. We have therefore shown that PfEMP1 is an Fc-binding protein of malaria parasites specific for polymeric human IgM, and that it shows functional similarities with Fc-binding proteins from pathogenic bacteria.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by a Medical Research Council Career Establishment Award (G0300145) and a European Union Marie Curie Excellence Grant, Antibody Immunotherapy for Malaria (MEXT-CT-2003-509670) to R.J.P., A.R., J.-P.S., and J.A.R. were supported by a Wellcome Trust Senior Fellowship (grant no. 067431).
J.A.R. and R.J.P. share senior authorship. A.G. expressed recombinant DBL domains in bacteria, performed ELISAs, IFAs, and rosetting analysis, purified and characterized Ig domain swaps and mAbs, and generated IgM point mutants. J.-P.S. identified, cloned, and characterized the TM284var1 gene and identified DBL4β as the IgM-binding ligand. R.S.M. helped with size-exclusion chromatography and domain swap culture and purification. A.R. cultured live rosetting parasite isolates and investigated IgM binding by IFAs. I.B.R., R.B., F.-E.J., and I.S. provided IgM domain-swapped expression plasmids, free secretory component, and important discussion. P.K.M. provided a panel of anti-IgM mAbs and contributed to discussion. J.A.R. and R.J.P. conceived and designed the overall study, provided laboratory facilities, and wrote the manuscript together.
2 A.G. and J.-P.S. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Richard J. Pleass, University of Nottingham, Institute of Genetics, Queen’s Medical Centre, Nottingham NG7 2UH, England, U.K. E-mail address: richard.pleass{at}nottingham.ac.uk or Dr. Alexandra Rowe, Institute of Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, West Mains Road, Edinburgh EH9 3JT, Scotland, U.K. E-mail address: alex.rowe{at}ed.ac.uk
4 Abbreviations used in this paper: pIgR, polymeric Ig receptor; CIDR, cysteine-rich interdomain region; DAB, diaminobenzidine; DAPI, 4',6-diamidino-2-phenylindole; DBL, Duffy binding-like; IFA, immunofluorescent assay; NIP, 3-iodo-4-hydroxy-5-nitrophenacetyl; PfEMP1, Plasmodium falciparum erythrocyte membrane protein 1.
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