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Elucidation of the disulfide-bonding pattern in the factor I modules of the sixth component (C6) of human complement
S Lengweiler1, J Schaller, R G DiScipio
1Department of Chemistry and Biochemistry, University of Bern, Switzerland.
Insights
Researchers mapped disulfide bridges in complement component C6
Area of Science:
- Biochemistry
- Immunology
- Proteomics
Background:
- Complement component C6 (C6) possesses two tandem factor I modules at its C-terminus.
- Understanding the structure of C6 is crucial for comprehending complement system function.
Purpose of the Study:
- To precisely map the disulfide bridges within the factor I modules of human complement component C6.
- To elucidate the structural organization of C6 and its relationship to other complement proteins.
Main Methods:
- Limited proteolysis of native C6 using trypsin and subtilisin.
- Separation of peptide fragments via reversed-phase high-performance liquid chromatography (RP-HPLC).
- Detection and identification of cystine-containing peptides using fluorescence assays, amino acid analysis, and Edman degradation.
Main Results:
- A specific pattern of disulfide bonds was determined: Cys752-Cys802, Cys763-Cys780, Cys765-Cys816, Cys772-Cys795, Cys841-Cys852, Cys846-Cys898, Cys859-Cys876, Cys861-Cys911, and Cys867-Cys891.
- The identified disulfide bridges provide a detailed structural map of the C6 C-terminal domains.
Conclusions:
- The study successfully localized multiple disulfide bonds within the factor I modules of complement component C6.
- These findings contribute to the structural understanding of C6 and facilitate comparisons with related complement components like C7 and factor I.
Abstract:
Complement component C6 is known to contain two factor I modules in tandem at its C-terminus. To localize the disulfide bridges in those domains, native C6 was cleaved with trypsin, followed by subtilisin. The resulting digests were separated by reversed-phase HPLC, and all of the potential cystine-containing fragments were detected by a fluorescence assay and amino acid composition analyses. Final identification of the disulfide bonds was achieved by Edman degradation of the corresponding peptides. From the data gained a 1-3, 2-9, 4-7, 5-10, 6-8 pattern was determined (Cys752-Cys802, Cys763-Cys780, Cys765-Cys816, Cys772-Cys795, Cys841-Cys852, Cys846-Cys898, Cys859-Cys876, Cys861-Cys911, Cys867-Cys891). These findings are compared with the strongly related complement components C7 and factor I.