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Functional and structural domains of the sixth component (C6) of human complement
Y Nakano1, M Hashimoto, N H Choi
1School of Pharmaceutical Sciences, Showa University, Tokyo, Japan.
Chemical & Pharmaceutical Bulletin
|February 1, 1991
Summary
Serine protease inhibitors like diisopropyl fluorophosphate (DFP) inactivate C6 complement protein. Proteolytic digestion identified functional domains, revealing C6 binds C5 within short consensus repeats, even after DFP modification.
Area of Science:
- Biochemistry
- Immunology
- Complement System
Background:
- C6 is a complement protein involved in cell lysis.
- Serine protease inhibitors like DFP and PMSF are known to affect C6 activity.
- Understanding C6's functional domains is crucial for its role in the complement cascade.
Purpose of the Study:
- To reinvestigate the effects of DFP and PMSF on C6 hemolytic activity.
- To elucidate the functional and structural domains of C6 using proteolytic digestion.
- To identify the specific binding site of C6 with C5.
Main Methods:
- Inactivation of C6 using serine protease inhibitors (DFP, PMSF).
- Limited proteolytic digestion of C6 with various proteases (trypsin, plasmin, lysyl endopeptidase, V8 protease).
- Analysis of C6 fragments using SDS-PAGE and Western blotting.
- Binding assays of C6 fragments with C5 using sucrose density ultracentrifugation.
Main Results:
- DFP and PMSF inactivated C6 in a dose-dependent manner (1-10 mM).
- Staphylococcus aureus V8 protease yielded disulfide bond-independent fragments.
- [3H]DFP labeling identified a 38 kDa fragment as the primary reactive site.
- C6 fragments (53, 38, 35, and 27.5 kDa) interacted with C5, regardless of DFP treatment.
Conclusions:
- C6 retains its ability to interact with C5 even after modification by DFP.
- The binding domain of C6 with C5 is located within the two short consensus repeats.
- Proteolytic mapping provides insights into the functional domains of C6 involved in C5 interaction.