Substitution of two amino acids confers C3b binding to the C4b binding site of CR1 (CD35). Analysis based on ligand binding by chimpanzee erythrocyte complement receptor
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V B Subramanian1, L Clemenza, M Krych
1Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.
Insights
Chimpanzee complement receptor type 1 (CR177) binds both C3b and C4b. Specific amino acid substitutions in this CR177 protein enable dual ligand binding and enhanced cofactor activity, mimicking human CR1 function.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Complement receptor type 1 (CR1) plays a crucial role in the innate immune system by binding complement fragments like C3b and C4b.
- Alternative splicing of the CR1 gene can produce variants with distinct functional properties.
- Chimpanzee CR177 (Ch E CR177) is an alternatively spliced CR1 variant with an unknown specific function.
Purpose of the Study:
- To investigate the structural basis for the C3b binding capability of chimpanzee CR177 (Ch E CR177).
- To determine the role of specific amino acid substitutions in Ch E CR177's ligand binding and cofactor activity.
- To compare the functional properties of Ch E CR177 with human CR1 (Hu CR1).
Main Methods:
- Site-directed mutagenesis was used to create truncated human CR1 constructs with specific amino acid substitutions.
- Ligand binding assays were performed to assess the binding of C3b and C4b to the engineered CR1 constructs.
- Cofactor activity assays were utilized to evaluate the functional capacity of the modified CR1 variants.
Main Results:
- Ch E CR177 binds both C4b and C3b, unlike human CR1 which primarily binds C4b in its homologous region.
- Single amino acid substitutions in the putative binding site of human CR1 did not confer C3b binding.
- A double amino acid substitution in the engineered human CR1 construct successfully induced C3b binding and enhanced cofactor activity for C3b, while retaining C4b binding.
Conclusions:
- The dual C3b and C4b binding ability of Ch E CR177 is attributed to specific amino acid substitutions within its complement protein-repeating modules.
- These substitutions enhance cofactor activity for C3b, suggesting a functional analogy to human E CR1 despite size and structural differences.
- The findings provide insights into the molecular mechanisms underlying complement receptor diversity and function.
Abstract:
The chimpanzee (Ch) E complement receptor type 1 (CR177) appears to be an alternatively spliced product of the Ch CR1 gene transcript. Its cDNA-derived amino acid sequence contains complement protein-repeating modules (CP) 1-6, 28, 29, and 30 in tandem and is 98.8% homologous to the corresponding regions of human (Hu) CR1. It differs from the C4b binding site of Hu CR1 only by two amino acids, Tyr for Ser37 in CP 1 and Asp for Gly79 in CP 2. However, in addition to binding C4b, Ch E binds C3b. As homologous substitution of one of these amino acids (Tyr for Ser37) was previously shown to not confer C3b binding, we reasoned that either single substitution of the other amino acid or a combination of the two amino acid changes would be required for C3b binding. To test this, using a truncated form of Hu CR1 that has a binding site only for C4b, we made these additional constructs. Single substitution of either amino acid did not affect the ligand binding or cofactor activity. However, the double substitution induced C3b binding and increased cofactor activity for C3b without changing the C4b-binding property. Of interest, these two amino acids are also found in the homologous positions of CP 9 and 16, which form part of the C3b binding site of Hu CR1. Thus, Ch E CR177, one-third of the size and with only a single ligand binding site, by acquiring key amino acid substitutions, binds C3b and C4b and functions analogous to Hu E CR1.