Isolating the Epstein-Barr virus gp350/220 binding site on complement receptor type 2 (CR2/CD21)

Abstract

Isolating the Epstein-Barr Virus gp350/220 Binding Site on Complement Receptor Type 2 (CR2/CD21)* Received for publication,July 31, 2007, and in revised form, October 9, 2007 Published, JBC Papers in Press,October 9, 2007, DOI 10.1074/jbc.M706324200 Kendra A. Young‡, Xiaojiang S. Chen§, V. Michael Holers‡, and Jonathan P. Hannan‡1 From the ‡Department of Medicine and Immunology, University of Colorado at Denver and Health Sciences Center, Aurora, Colorado 80045 and the §Department of Molecular and Computational Biology, University of Southern California, Los Angeles, California 90089 Complement receptor type 2 (CR2/CD21) is essential for the attachment of Epstein-Barr virus (EBV) to the surface of B-lym- phocytes in an interaction mediated by the viral envelope glyco- protein gp350. The heavily glycosylated structure of EBV gp350 has recently been elucidated by x-ray crystallography, and the CR2 binding site on this protein has been characterized. To identify the corresponding gp350 binding site on CR2, we have undertaken a site-directed mutagenesis study targeting regions of CR2 that have previously been implicated in the binding of CR2 to the C3d/C3dg fragments of complement component C3. Wild-type or mutant forms of CR2 were expressed on K562 cells, and the ability of these CR2-expressing cells to bind gp350 was measured using flow cytometry. Mutations directed toward the two N-terminal extracellular domains of CR2 (SCR1-2) reveal that a large contiguous surface of CR2 SCR1-2 is involved in gp350 binding, including a number of positively charged res- idues (Arg-13, (Arg-28, (Arg-36, Lys-41, Lys-57, Lys-67, and Arg-83). These data appear to complement the CR2 binding site on gp350, which is characterized by a preponderance of negative charge. In addition to identifying the importance of charge in the formation of a CR2-gp350 complex, we also provide evi- dence that both SCR1 and SCR2 make contact with gp350. Spe- cifically, two anti-CR2 monoclonal antibodies, desi

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