Molecular & Biochemical Parasitology 137 (2004) 55–64 Functional interdependence of the DBL domain and c2 region for binding of the Plasmodium falciparum variant antigen to ICAM-1  Amy L. Springer a,b , Leia M. Smith a , Donald Q. Mackay a , Siri O. Nelson a , Joseph D. Smith a,b,∗ a Seattle Biomedical Research Institute, 307 Westlake Avenue N., Suite 500, Seattle, WA 98109-5219, USA b Department of Pathobiology, University of Washington, Seattle, WA 98195, USA Received 11 December 2003; received in revised form 12 December 2003; accepted 28 March 2004 Available online 25 May 2004 Abstract Cytoadherence of Plasmodium falciparum-infected erythrocytes is associated with severe malaria and is primarily mediated through binding of the variant surface antigen P. falciparum erythrocyte membrane protein 1 (PfEMP1) to specific host ligands. Infected erythrocyte binding to Intercellular Adhesion Molecule 1 (ICAM-1) has been implicated as having a role in cerebral malaria, a major cause of death from P. falciparum infection. We have examined ICAM-1-binding PfEMP1 proteins in the cytoadhesive P. falciparum strain IT4/25/5 in order to extend our understanding of binding. For A4tres, the ICAM-1 binding region was previously shown to reside within contiguous DBL2β and c2 domains. We determined the gene sequence encoding IT-ICAM var, and showed that ICAM-1 binding in this protein also maps to DBL2βc2 domains that have 48% amino acid identity to A4tres. By truncation and chimera analysis, most of the DBL2β and the first half of the c2 region were required for A4tres binding to ICAM-1, suggesting this tandem should be considered a structural-functional combination for ICAM-1 binding. Of interest, a chimera formed between two different ICAM-1 binding domains did not bind ICAM-1, suggesting a functional interdependence between DBL2β and c2 from the same protein. As gene recombination and gene conversion are important mechanisms for generating diversity in the PfEMP1 protein family, this finding implies an extra level of constraint on the functional evolution of binding traits. Knowledge about the PfEMP1::ICAM-1 interaction may allow the development of interventions to prevent binding and disease. © 2004 Elsevier B.V. All rights reserved. Keywords: Malaria; Plasmodium; Cytoadherence; ICAM-1; var; PfEMP1 1. Introduction Adhesion of Plasmodium falciparum-infected erythro- cytes is strongly implicated in the pathogenesis of severe malaria infections. Infected erythrocyte (IE) binding to en- dothelium allows parasites to avoid spleen-dependent killing mechanisms and contributes to systemic and organ-specific disease complications [1]. Cytoadherence is mediated pri- marily by the P. falciparum erythrocyte membrane protein 1 family (PfEMP1), a family of variant antigens expressed Abbreviations: CIDR, cysteine-rich interdomain region; DBL, Duffy binding-like; IE, infected erythroctye; ICAM-1, Intercellular Adhesion Molecule 1; PfEMP1, Plasmodium falciparum erythrocyte membrane protein 1  Note: The nucleotide sequence reported in this paper is available in the GenBank database with the accession number: AY578326. ∗ Corresponding author. Tel.: +1 206 256 7384; fax: +1 206 256 7229. E-mail address: joe.smith@sbri.org (J.D. Smith). on the infected erythrocyte surface [2], encoded by var genes. Numerous host receptors have been identified as specific ligands for infected erythrocyte cytoadherence, including CD36 [3], ICAM-1 [4], and chondroitin sulfate A [5–7]. The variable binding properties of PfEMP1 pro- teins to different host receptors is believed to influence the specificity of infected erythrocytes for different endothelial microvasculature and disease manifestation. Each parasite genotype contains approximately 60 var genes encoding different PfEMP1 proteins [8–10], although only one protein appears to be expressed at a time [11,12]. The encoded proteins vary in their adhesive properties as well as in the number and sequence of two distinct extra- cellular domains: Duffy-binding like (DBL) domains and Cysteine-rich interdomain regions (CIDR) [8]. The PfEMP1 binding regions for different host receptors have begun to be mapped to specific DBL and CIDR domains [13–22]. These adhesive domains can be grouped into different se- 0166-6851/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2004.03.019