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Published on: May 22, 2020
Activation of human C1r: Western blot analysis reveals slow and dose-dependent activation
Insights
Spontaneous activation of C1r (the first component of the classical complement pathway) is slow and concentration-dependent. Intermolecular activation, not intramolecular autoactivation, is suggested by findings involving serine protease inhibitors.
Area of Science:
- Biochemistry
- Immunology
- Complement System
Background:
- The complement system is crucial for innate and adaptive immunity.
- C1r is a serine protease and a key component of the classical complement pathway.
- Understanding C1r activation mechanisms is vital for immune response regulation.
Purpose of the Study:
- To investigate the spontaneous activation of C1r in the presence of EDTA.
- To elucidate the kinetics and influencing factors of C1r autoactivation.
- To differentiate between intramolecular and intermolecular activation models of C1r.
Main Methods:
- Western blot analysis was employed to detect C1r activation.
- Partially purified native C1r was prepared using ultracentrifugation and sucrose density gradient.
- Single radial immunodiffusion was used to assess C1 inhibitor (C1-INH) levels.
Main Results:
- Spontaneous C1r activation was observed to be slow, with less than 10% activation in 30 minutes.
- Activation rate demonstrated a concentration-dependent relationship.
- Activated C1r was found to enhance the spontaneous activation rate.
- Serine protease inhibitors significantly suppressed C1r activation for up to 1 hour.
Conclusions:
- The observed kinetics and influencing factors are inconsistent with an intramolecular autoactivation model.
- Results suggest that intermolecular activation, potentially by contaminating proteases or existing activated C1r, is the likely mechanism.
- Further research is needed to fully characterize the intermolecular activation pathway of C1r.
Abstract:
Spontaneous activation of C1r in the presence of EDTA was examined by a Western blot. Partially purified native C1r was prepared by ultracentrifugation of fresh serum in 10 to 30% sucrose gradient; final concentration of C1r was one-sixth of the original serum. C1(-)-INH was not detectable by a single radial immunodiffusion (less than 0.5% of serum). The results demonstrated that 1) the rate of spontaneous activation of C1r was slow (less than 10% in 30 min); 2) it was concentration-dependent; 3) it was enhanced by activated C1r; and 4) it was almost completely suppressed by serine protease inhibitors up to 1 h. These results were inconsistent with an intramolecular autoactivation model of C1r in the fluid phase and suggested intermolecular activation by contaminating protease or activated C1r.
