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Functional role of the linker between the complement control protein modules of complement protease C1s
Isabelle Bally1, Véronique Rossi, Nicole M Thielens
1Laboratoire d'Enzymologie Moléculaire, Institut de Biologie Structurale Jean-Pierre Ebel, Grenoble, France.
Journal of Immunology (Baltimore, Md. : 1950)
|September 24, 2005
Summary
The linker connecting C1s protease modules is crucial for cleaving C4 but not C2. Mutations in this linker affect C4 cleavage, impacting the complement system
Area of Science:
- Biochemistry
- Immunology
- Molecular Biology
Background:
- C1s is a serine protease essential for the classical complement pathway, cleaving C4 and C2.
- Its catalytic domain includes two CCP modules linked by a flexible four-residue segment (Gln340-Pro-Val-Asp343).
Purpose of the Study:
- To investigate the functional significance of the linker connecting the CCP1 and CCP2 modules of human C1s.
- To determine the linker's role in C1 complex assembly, C1s activation, and substrate cleavage (C4 and C2).
Main Methods:
- Site-directed mutagenesis of the C1s linker region (residues 340-343).
- Baculovirus-mediated expression and functional characterization of C1s mutants.
- Assays for C1 complex assembly, C1s activation by C1r, and C4/C2 cleavage activity.
Main Results:
- Mutations in the C1s linker did not affect C1 assembly or C1s activation by C1r.
- The Q340E mutation rendered C1s resistant to activation.
- Most linker mutations significantly reduced C4-cleaving activity, while C2 cleavage remained largely unaffected.
- Flexibility of the linker is critical for efficient C4 cleavage, likely by optimizing substrate positioning.
Conclusions:
- The C1s CCP1-CCP2 linker's flexibility is dispensable for C1 assembly and C1s activation.
- The linker plays a critical role in C4 cleavage by facilitating optimal substrate interaction with the C1s active site.
- These findings elucidate the structural determinants of C1s function in the complement cascade.