Activation of C1r by proteolytic cleavage

Area of Science:

• Biochemistry
• Immunology
• Complement System

Background:

• The complement system is a critical part of innate immunity.
• Activation of the complement cascade involves a series of enzymatic steps.
• C1r is a serine protease within the C1 complex, but its activation mechanism was not fully understood.

Purpose of the Study:

• To elucidate the mechanism of C1r activation and its role in initiating the classical complement pathway.
• To investigate the structural and functional changes in C1r upon activation.
• To identify factors influencing C1r activation and inhibition.

Main Methods:

• Enzymatic assays to measure C1r's ability to cleave and activate C1s.
• Biochemical analysis, including SDS-PAGE, to study the cleavage of C1r subunits.
• Electrophoresis to compare the properties of C1r and activated C1r.
• Amino acid composition analysis.
• Inhibition studies using various agents and conditions.

Main Results:

• C1r activation requires incubation at 37°C in the absence of calcium, followed by addition of C1s.
• Activation is associated with the cleavage of C1r's 95,000 dalton chains into disulfide-linked 60,000 and 35,000 dalton subunits.
• Activated C1r (C1r) cleaves and activates proenzyme C1s.
• Trypsin cleaves C1r but does not activate it.
• Calcium, polyanethol sulfonate, C1 inactivator, and DFP inhibit C1r activation.
• C1 inactivator forms a complex with C1r on its light chain.
• Proteolytic cleavage of C1r occurs upon addition of aggregated IgG to C1q, C1r, and C1s in the presence of calcium.
• Liquoid inhibits C1r cleavage.

Conclusions:

• C1r is converted from an inactive precursor to an active enzyme (C1r) through proteolytic cleavage, analogous to other complement enzymes.
• This activation process is a biologically significant event in the initiation of the C1 complex.
• The activation and function of C1r are tightly regulated by calcium and specific inhibitors like C1 inactivator.