A CR1 polymorphism associated with constitutive erythrocyte CR1 levels affects binding to C4b but not C3b

Abstract

The erythrocyte type one complement receptor (E-CR1) mediates erythrocyte binding of complement-opsonized immune complexes (IC), and helps protect against random deposition of circulating IC. Two linked CR1 polymorphisms occur in binding domains, at I643T and Q981H. In Caucasians, the variant alleles (643T, 981H) are associated with low constitutive E-CR1 expression levels. This study was conducted to determine if these polymorphisms affect ligand binding, and if so, represent risk factors for the autoimmune IC disease, systemic lupus erythematosus (SLE). In an ELISA comparing relative ligand binding differences, E-CR1 from individuals homozygous for the variant residues (643TT/981HH) exhibited greater binding to C4b, but not C3b, than homozygous wild-type E-CR1. Analysis of single-binding domain CR1 constructs demonstrated that the 981H residue imparted this enhanced C4b binding. No differences were observed in the 981H allele frequency between Caucasian controls (0.170, n = 100) and SLE patients (0.130, n = 150, P = 0.133), or between African American controls (0.169, n = 71) and SLE patients (0.157, n = 67). In a subset of individuals assessed for CR1 size, excluding from this analysis those expressing at least one B allele revealed a trend for over-representation of the 981H allele in Caucasian controls (0.231 frequency, n = 26) versus SLE patients (0.139, n = 83, P = 0.089), but again no difference between African American controls (0.188, n = 24) and SLE patients (0.191, n = 34). These data suggest that the 981H residue compensates for low constitutive expression of E-CR1 in Caucasians by enhancing C4b binding. This may contribute protection against SLE.

Figure 1

Relative E-CR1 concentrations as determined using different capture and detecting anti-CR1 antibodies.

Figure 2

Single binding domain CR1 constructs, containing SCRs 8–11 for the analysis of the I643T polymorphism, or SCRs 15–18 for the analysis of the Q981H polymorphism.

Figure 3

Ethidium bromide-stained gel following PCR amplification and BstNI digestion for determining G3093T genotype.

Figure 4

Results of ELISA for measuring binding E-CR1 from solubilized erythrocyte membranes from two individuals who were homozygous wild-type (WT; 643II/981QQ) or homozygous variant (Var; 643TT/981HH). Soluble erythrocyte membranes containing CR1 were incubated in parallel in wells coated with J3D3, C3b or C4b, and bound CR1 were detected using biotinylated E11, followed by strepavidin-HRP. A single WT and Var sample were assayed together in each ELISA. For assays comparing WT1 versus Var1 (n = 4, d.f. = 11), C3b was coated at 1·2 µg/ml and C4b was coated at 2·5 µg/ml. For assays comparing WT2 versus Var2 (n = 3, d.f. = 8), C3b was coated at 2·5 µg/ml and C4b was coated at 5 µg/ml. Mean and standard error are shown.

Figure 5

Results of ELISA comparing the binding of partially purified wild-type (WT1) or variant (Var1) E-CR1 or solubilized erythrocyte membranes containing a second wild-type E-CR1 (WT2), to C4b deposited on IgG-coated wells (n = 4, df = (2,18)). Mean and standard error are shown.

Figure 6

Results of ELISA comparing the binding of 643-I and 643-T constructs (n = 2, df = 9), or the 981-Q and 981-H constructs (n = 3, df = 14) to C3b and C4b. Mean and standard error are shown.

Figure 7

Average E-CR1 levels in a subset of race-matched controls and SLE patients. (a)Average E-CR1 levels measured in normal control Caucasians and African Americans grouped by Q981H genotype. For Caucasians, n = 25 QQ, 10 QH, 2 HH. For African Americans, n = 23 QQ, 11 QH. Comparison between E-CR1 levels of controls (same as (a)) and SLE patients, according to Q981H genotype. For Caucasian SLE patients, n = 74 QQ, 19 QH, 1 HH. For African American SLE patients, n = 18 QQ, 9 QH. Mean and standard error are shown. **P < 0·005 for Caucasian QQ versus QH (a) or controls versus SLE patients (b).