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Purification and characterization of a membrane protein (gp45-70) that is a cofactor for cleavage of C3b and C4b
Abstract:
Based on preliminary evidence indicating that a cell-associated protein of U937 (a human monocyte-like cell line) possessed cofactor activity and was not the C3b/C4b receptor, we sought to further characterize this protein. A sequential four-column purification procedure was devised that includes C3(H2O) affinity chromatography to isolate in reasonable yields and purity a cell-associated protein of U937 and several other human cell lines. Based on its pattern and Mr on SDS-PAGE, acidic pI, and ligand specificity, it is identical to a recently described C3(H2O) or C3b-binding membrane glycoprotein of human PBL and cell lines; having no presently identified function, it was termed gp45-70. After purifying this protein, we determined its functional capabilities and compared them to those of the other complement proteins with regulatory activity directed at components comprising the C3 convertases. This protein was the most efficient (50 times that of H) yet-described cofactor for the I-mediated first cleavage of C3b. It also was a cofactor for the first cleavage of C4b, but was not as efficient as C4bp. The second cleavage of C3b and C4b was not efficiently mediated. It had no ability to accelerate decay in the classical or alternative pathway C3 convertases. Based on this unique activity profile and ability to be surface labeled, we have renamed this molecule membrane cofactor protein (MCP). We suggest that this protein plays a major role in preventing autologous complement activation.
Insights
Researchers identified a novel cell-associated protein, now named membrane cofactor protein (MCP), which acts as a potent cofactor for complement system regulation. This protein efficiently cleaves C3b and C4b, crucial for preventing self-tissue damage.
Area of Science:
- Immunology
- Complement System Biology
- Protein Biochemistry
Background:
- Preliminary evidence suggested a U937 cell protein had cofactor activity, distinct from the C3b/C4b receptor.
- A cell-associated protein was identified in U937 and other human cell lines.
Purpose of the Study:
- To further characterize the cell-associated protein from U937 cells.
- To determine the functional capabilities of the purified protein and compare it to known complement regulatory proteins.
Main Methods:
- Sequential four-column purification, including C3(H2O) affinity chromatography.
- Analysis of protein properties: SDS-PAGE, acidic pI, ligand specificity.
- Functional assays comparing cofactor activity for complement component cleavage.
Main Results:
- A protein identical to gp45-70, a C3(H2O)/C3b-binding glycoprotein, was isolated.
- The protein demonstrated highly efficient cofactor activity for the I-mediated first cleavage of C3b (50x more than H).
- It also served as a cofactor for C4b cleavage, though less efficiently than C4bp, and did not accelerate C3 convertase decay.
Conclusions:
- The purified protein, renamed membrane cofactor protein (MCP), exhibits unique complement regulatory functions.
- MCP is the most efficient cofactor described for C3b cleavage.
- MCP likely plays a significant role in preventing autologous complement activation and protecting host tissues.