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Expression of a hybrid complement regulatory protein, membrane cofactor protein decay accelerating factor on Chinese
Insights
A novel hybrid membrane cofactor protein-decay accelerating factor (MCP-DAF) was created to regulate complement system activation. This hybrid protein shows potential for reducing complement-mediated cell damage, particularly through the alternative pathway.
Area of Science:
- Immunology
- Cell Biology
Background:
- Complement system activation is regulated by membrane cofactor protein (MCP) and decay accelerating factor (DAF).
- MCP and DAF have complementary functions in preventing complement-mediated cell damage.
Purpose of the Study:
- To investigate the regulatory activity of a hybrid MCP-DAF molecule.
- To compare the efficacy of hybrid MCP-DAF with MCP, DAF, and co-expressed MCP + DAF.
Main Methods:
- Transfection of Chinese hamster ovary (CHO) cells with cDNA encoding MCP, DAF, or a hybrid MCP-DAF.
- Assessing complement C3 deposition via classical and alternative pathways on sensitized CHO cells.
- Evaluating complement-mediated cytolysis of CHO cells.
Main Results:
- Hybrid MCP-DAF demonstrated significant C3 deposition blocking via the alternative pathway.
- MCP + DAF showed the highest C3 deposition blocking via the classical pathway.
- Cellular protection against cytolysis generally correlated with reduced C3 deposition.
Conclusions:
- Hybrid MCP-DAF offers potent protection against alternative pathway-mediated complement activation and cell damage.
- MCP-DAF may be a valuable tool for mitigating complement-mediated pathologies, especially those involving the alternative pathway.
Abstract:
C activation on the cell surface is supposedly regulated by membrane cofactor protein (MCP) and decay accelerating factor (DAF). These are complementary in function: MCP acts as a cofactor in factor I-mediated C3b and C4b inactivation, thus preventing the assembly of C3 convertases, whereas DAF accelerates spontaneous decay of the assembled C3 convertase. In this report, a hybrid MCP-DAF was expressed on Chinese hamster ovary cells by transfecting cDNA, and its regulatory activity was compared with those of MCP and DAF transfectants and with a transfectant expressing both MCP and DAF (MCP + DAF). The C3 deposition on sensitized CHO cells through activation of the classical pathway was blocked to a different degree with these transfectants, the order being MCP + DAF > DAF > hybrid MCP-DAF > MCP. Likewise, the C3 deposition via the alternative pathway was blocked efficiently in the order hybrid > MCP + DAF > MCP. The C-mediated cytolysis of CHO cells virtually reflected the degree of C3 fragment deposition. The MCP-DAF transfectant acquired additive protective activity against alternative pathway-mediated C3 deposition and cytolysis but was less potent in circumventing classical pathway attack than cells that expressed DAF alone or DAF + MCP. Hybrid MCP-DAF may be useful for alleviating C-mediated cell damage, especially via the alternative pathway.