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The ninth component of human complement (C9). Functional activity of the b fragment
Insights
Investigating human complement protein C9
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- The complement system is crucial for innate and adaptive immunity.
- Complement protein C9 is the terminal component of the membrane attack complex (MAC).
- Understanding C9's structure-function relationship is key to its role in cell lysis.
Purpose of the Study:
- To elucidate the domain structure and functional activities of human complement protein C9.
- To differentiate the roles of the NH2-terminal (C9a) and COOH-terminal (C9b) fragments of C9.
Main Methods:
- Cleavage of C9 with alpha-thrombin to obtain C9a and C9b fragments.
- Separation and renaturation of fragments using SDS-PAGE and dialysis.
- Functional assays including ion channel formation in lipid membranes, marker release from liposomes, and erythrocyte lysis.
- Secondary structure prediction analysis.
Main Results:
- The C9b fragment formed ion-conducting channels in lipid membranes with specific conductances and lifetimes, and mediated lipid-specific ion transport (K+ over Na+).
- Both C9a and C9b fragments induced marker release from liposomes and lysed erythrocytes independently of the C5b-8 complex.
- Secondary structure predictions suggested amphipathic alpha-helices in C9b and beta-sheets in C9a.
Conclusions:
- The COOH-terminal C9b fragment likely contains the channel-forming domain.
- The NH2-terminal C9a fragment may possess surface-binding domains.
- These findings suggest distinct functional domains within monomeric C9 involved in membrane attack complex assembly and function.
Abstract:
The domain structure of human complement protein C9 was investigated by determining the functional activities of the NH2-terminal (C9a) and COOH-terminal (C9b) fragments obtained by cleavage of C9 with alpha-thrombin. The two fragments were separated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renatured by dialysis against buffers containing zwitterionic detergents. The C9b fragment produced membranolytic activities in three independent assays. First, it produced single, ion-conducting channels of varying conductances in planar lipid membranes. Most of the channels had an average conductance of 11 picoSiemens and an average lifetime of about 30 s. The channels showed lipid specificity and a 3-fold preference for conducting K+ over Na+. Second, the fragment also caused specific marker release from liposomes which was inhibitable by a C9b-specific monoclonal antibody, and third, it lysed erythrocytes in the absence of a fully assembled C5b-8 complex. The isolated C9a fragment did not produce single channels in planar lipid membranes but was also effective in releasing markers from liposomes and in lysing erythrocytes. Secondary structure predictions indicate the presence of several amphiphilic, "surface-seeking" segments in the primary structure of C9 which are mainly alpha-helices in C9b and beta-sheets in C9a. These results may indicate the presence of surface-binding domains in the NH2-terminal half and channel-forming domains in the COOH-terminal portion of native, monomeric C9.