Quantitative analysis of C4b dimer binding to distinct sites on the C3b/C4b receptor (CR1).
The complement receptor CR1 (CD35) is a transmembrane protein composed in its extracellular portion of short consensus repeats (SCR 1-30) organized into four long homologous repeats (LHR-A, LHR-B, LHR-C, and LHR-D). Each LHR, except LHR-D, contains a binding site for C3b and/or C4b within its first four SCR. The binding reaction between CR1 and soluble dimers of C4b (C4b2) was analyzed using the native receptor on human erythrocytes and full-length recombinant CR1 expressed in stably transfected Chinese hamster ovary (CHO) cells. CR1 mutants expressed similarly were used to determine the SCR of LHR-A required for C4b2 binding and the potential of C3b binding sites in CR1 to bind C4b2. Erythrocyte CR1, CHO cells expressing full-length recombinant CR1 (ABCD), constructs ACD, and SCR(1-4)D each bound C4b2 with similar affinities (Kd, approximately 4 x 10(-7) M). Construct SCR(1-2)D bound C4b2 with lower affinity (Kd, 1.4 x 10(-6) M) indicating that SCR(1-4) are required for a fully functional C4b2 binding site. Construct SCR(15-18)D, which contains a C3b site, also bound C4b2 with lower affinity (Kd 1.2 x 10(-6) M) than its binding to C3b dimers. Constructs SCR(15-16)D and D did not bind C4b2. Each CR1 construct that bound C4b2 functioned as a cofactor for factor I-mediated cleavage to C4d.
Reilly
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University of New Mexico School of Medicine, Albuquerque 87131.
Makrides
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Ford
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PJ
Marsh
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HC
Mold
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C
D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism*; D015935:Complement C4b / Q000378:metabolism*; D003169:Complement Inactivator Proteins; D006224:Cricetinae; D004912:Erythrocytes / Q000378:metabolism; D006023:Glycoproteins; D006801:Humans; D007700:Kinetics; D017463:Receptors, Complement 3b / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D002352:Carrier Proteins; D003169:Complement Inactivator Proteins; D006023:Glycoproteins; D017463:Receptors, Complement 3b; D011994:Recombinant Proteins; D015935:Complement C4b