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Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells
Published on: January 29, 2014
Identification of complement receptor type 1-related proteins on primate erythrocytes
M W Nickells1, V B Subramanian, L Clemenza
1Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110.
Insights
This study characterized primate immune adherence receptors (CR1), finding varied sizes and higher expression on red blood cells compared to humans. These receptors bind C3b and C4b, offering insights into CR1 evolution.
Area of Science:
- Immunology
- Primate immunology
- Molecular biology
Background:
- The immune adherence receptor, complement receptor 1 (CR1), plays a crucial role in the immune system.
- Understanding CR1 structure and function across species is vital for comparative immunology.
Purpose of the Study:
- To characterize the structure and function of the immune adherence receptor (CR1, CD35, C3b/C4b receptor) in various primate species.
- To investigate variations in CR1 size, expression levels, and ligand-binding properties.
Main Methods:
- Western blotting and immunoprecipitation to determine protein molecular weights.
- Enzyme-linked immunosorbent assay (ELISA) for protein quantification.
- Affinity chromatography to assess binding to C3b and C4b.
Main Results:
- Primate CR1 on erythrocytes (E) showed diverse molecular weights (55-75 kDa and 130-165 kDa), distinct from human CR1.
- Erythrocytes of most primates expressed significantly higher CR1 levels than human erythrocytes.
- Functional analysis revealed three distinct C3b/C4b binding patterns among primate CR1 variants.
Conclusions:
- Primate CR1 exhibits significant structural and quantitative diversity compared to human CR1.
- These variations suggest evolutionary adaptations in immune adherence mechanisms among primates.
- The study provides a foundation for exploring structure-function relationships of CR1 across primate species.
Abstract:
The purpose of this study was to characterize the structure and function of the immune adherence receptor (CR1, CD35, C3b/C4b receptor) of primates. Western blotting, immunoprecipitation, ELISA, and affinity chromatography with homologous C3b and C4b were utilized. The major cross-reactive E membrane protein of ten species of primates tested was lower in m.w. than was human CR1 and fell into two size groups of 55 to 75 and 130 to 165 kDa. There was 10- to 100-fold more CR1 per primate E than human E. Five species also expressed lesser quantities of a protein similar in m.w. (approximately 200 kDa) to human CR1. In contrast to E, the major cross-reactive protein on PBMC was similar in size to human CR1. Four species also expressed lesser amounts of a lower m.w. protein on their PBMC of the same M(r) as that found on their E. Affinity chromatography demonstrated that the approximately 200-kDa form, if present, was recovered with a similar efficiency to that of human CR1. Three patterns of binding, however, were identified among the lower m.w. proteins: 1) C3b > or = C4b; 2) C4b > C3b; and C3b only or predominantly. The fact that these E proteins cross-react with Ab to human CR1, bind homologous C3b and, in most cases, C4b, and for some species represent the only such protein expressed on their E identifies them as immune adherence receptors. The 70-kDa CR1 of the chimpanzee E seems to arise by alternative splicing of the mRNA encoding the 200-kDa protein. These data raise interesting questions relative to the evolution of CR1 in primates and provide a basis for analysis of structure-function relationships among these size forms of CR1.
