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Glutathione-catalyzed disulfide-linking of C9 in the membrane attack complex of complement
Insights
Glutathione can covalently crosslink complement C9 proteins within the membrane attack complex (MAC). This crosslinking reveals C9
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- The membrane attack complex (MAC) of complement, a C5b-9 complex, is formed by five proteins linked noncovalently.
- Understanding the structural dynamics and protein interactions within the MAC is crucial for comprehending its function.
Purpose of the Study:
- To investigate the potential for covalent crosslinking of C9 molecules within the MAC.
- To determine if glutathione can induce disulfide linkages between C9 subunits in the C5b-9 complex.
Main Methods:
- Preparation of tetramolecular C5b-8 complexes bound to phospholipid vesicles.
- Incubation of C5b-8 complexes with purified 131I-C9 in the presence of glutathione.
- Analysis of C9 crosslinking and complex formation using radiolabeling and gel electrophoresis.
Main Results:
- Glutathione induced disulfide-linked dimeric C9 formation within the C5b-9 complex.
- Approximately one-third of C9 in a high-C9-binding complex was disulfide-linked.
- In a lower-C9-binding complex, about one-fifth of C9 subunits were cross-linked.
- C9 alone was not significantly cross-linked by glutathione, indicating dependence on the C5b-8 complex.
Conclusions:
- C9 exhibits a unique self-associating property when incorporated into the C5b-8 complex.
- Glutathione-mediated disulfide crosslinking provides insights into C9 polymerization within the MAC.
- These findings highlight a novel aspect of MAC structural assembly and regulation.
Abstract:
The membrane attack complex of complement (the dimeric C5b-9 complex) is a multimolecular assemblage of five proteins (C5b, C6, C7, C8, and C9) which are held together by noncovalent forces. We found that C9 molecules in the complex can be covalently crosslinked (disulfide-linked) by glutathione. In this experiment, the tetramolecular C5b-8 complex bound to phospholipid vesicles was first prepared from purified C5b-6, incubated (37 degrees C, 20 min) with an excess of 131I-C9 in the presence of 1 mM glutathione; an average of 5.3 molecules of C9 per C5b-8 were bound and the C5b-9 complex formed was predominantly a dimeric C5b-9 complex. About one-third of C9 in this C5b-9 complex was found to be in a disulfide-linked dimeric form. The C5b-9 complex, having only an average of 0.9 molecules of C9 per C5b-8, was also prepared in the presence of glutathione; this C5b-9 preparation contained both monomeric and dimeric C5b-9 complexes, and about one-fifth of the C9 subunits was in a cross-linked dimeric form. By contrast, C9 in the absence of the C5b-8 complex was not significantly cross-linked by glutathione. These results indicate that C9 has a unique property to associate with itself upon reaction with the C5b-8 complex.