Quantitative analysis of C4b dimer binding to distinct sites on the C3b/C4b receptor (CR1)

Area of Science:

• Immunology
• Molecular Biology
• Biochemistry

Background:

• Complement receptor 1 (CR1/CD35) is a transmembrane protein with extracellular short consensus repeats (SCRs).
• CR1's structure includes four long homologous repeats (LHRs), each containing binding sites for complement fragments C3b and C4b.

Purpose of the Study:

• To analyze the binding of CR1 to soluble C4b2 dimers.
• To identify the specific SCRs within LHR-A responsible for C4b2 binding.
• To investigate if C3b binding sites on CR1 can also bind C4b2.

Main Methods:

• Utilized native CR1 on human erythrocytes and recombinant full-length CR1 expressed in Chinese hamster ovary (CHO) cells.
• Employed CR1 mutants to map C4b2 binding sites.
• Measured binding affinities (Kd) of CR1 constructs to C4b2.

Main Results:

• CR1, full-length CR1 (ABCD), ACD, and SCR(1-4)D constructs bound C4b2 with similar high affinity (Kd ≈ 4 x 10⁻⁷ M).
• SCR(1-2)D showed lower affinity (Kd = 1.4 x 10⁻⁶ M), indicating SCRs 1-4 are essential for optimal C4b2 binding.
• SCR(15-18)D, a C3b binding site, exhibited lower affinity for C4b2 (Kd = 1.2 x 10⁻⁶ M).
• CR1 constructs binding C4b2 also acted as cofactors for factor I-mediated C4d cleavage.

Conclusions:

• The N-terminal SCRs (1-4) of CR1 are critical for high-affinity C4b2 binding.
• While C3b binding sites can weakly bind C4b2, the primary binding site is distinct and located within the first four SCRs.
• CR1's ability to bind C4b2 and facilitate its cleavage is essential for complement regulation.