Complement component C5: engineering of a mutant that is specifically cleaved by the C4-specific C1s protease

Area of Science:

• Immunology
• Protease biochemistry

Background:

• Complement component C3 can be engineered for C1s protease susceptibility.
• The complement system involves complex proteolytic cascades.

Purpose of the Study:

• To investigate if similar modifications could render complement component C5 susceptible to C1s protease.
• To characterize the protease sensitivity of engineered C5 variants.

Main Methods:

• Site-directed mutagenesis was used to substitute amino acid residues in C5.
• Engineered C5 variants (ASC4) were tested for cleavage by C1s and C5 convertase.
• Protease activity was compared between precursor and mature forms of wild-type and mutant C5.

Main Results:

• Small substitutions in C5 did not confer C1s sensitivity.
• A hybrid C5 protein (ASC4), with both upstream and downstream substitutions, was cleaved by C1s.
• ASC4 showed differential cleavage sensitivity between precursor and mature forms for C1s and C5 convertase.
• Wild-type C5 was cleaved by C5 convertase but not C1s.

Conclusions:

• A mutant C5 molecule (ASC4) can be constructed to be cleaved by both C1s and C5 convertase.
• The recognition sites for C1s and C5 convertase on C5 are likely distinct and spatially separated.
• C5 convertase recognition involves residues distant from the cleavage site.