Isolating the Epstein-Barr virus gp350/220 binding site on complement receptor type 2 (CR2/CD21).
Complement receptor type 2 (CR2/CD21) is essential for the attachment of Epstein-Barr virus (EBV) to the surface of B-lymphocytes in an interaction mediated by the viral envelope glycoprotein 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 residues (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 evidence that both SCR1 and SCR2 make contact with gp350. Specifically, two anti-CR2 monoclonal antibodies, designated as monoclonal antibodies 171 and 1048 whose primary epitopes are located within SCR2, inhibit binding of wild-type CR2 to EBV gp350; with regard to SCR1, both K562 cells expressing an S15P mutation and recombinant S15P CR2 proteins exhibit diminished gp350 binding.
Young
Kendra A
KA
Department of Medicine and Immunology, University of Colorado at Denver and Health Sciences Center, Aurora, Colorado 80045, USA.
Chen
Xiaojiang S
XS
Holers
V Michael
VM
Hannan
Jonathan P
JP
D019943:Amino Acid Substitution; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001402:B-Lymphocytes / Q000737:chemistry / Q000378:metabolism / Q000821:virology; D001665:Binding Sites / Q000502:physiology; D003179:Complement C3b / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015933:Complement C3d / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004854:Herpesvirus 4, Human / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D020014:K562 Cells; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D017464:Receptors, Complement 3d / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053585:Virus Attachment
D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D010446:Peptide Fragments; D017464:Receptors, Complement 3d; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C045477:complement C3d,g; D003179:Complement C3b; D015933:Complement C3d