Functional evaluation of complement factor I variants by immunoassays and SDS-PAGE

Area of Science:

• Immunology
• Molecular Biology
• Genetics

Background:

• Factor I (FI) is a critical regulator of the complement system, controlling C3b degradation.
• FI mutations are linked to diseases such as age-related macular degeneration and atypical hemolytic uremic syndrome.

Purpose of the Study:

• To evaluate the functional impact of eight recombinant Factor I genetic variants.
• To assess the co-factor activity of FI with Factor H and soluble CR1.
• To characterize the protease activity of FI variants using diverse analytical methods.

Main Methods:

• SDS-PAGE to analyze C3b degradation.
• ELISA to quantify fluid-phase iC3b generation.
• A novel Luminex bead-based assay for surface-bound C3b degradation.
• Assessment of FI variants with Factor H and soluble CR1 co-factors.

Main Results:

• Mutations in the FIMAC and SP domains of FI significantly reduced protease activity.
• Mutations in the LDLRA2 domain showed no profound functional changes.
• Assays correlated well, but combining methods and co-factors was necessary to detect subtle activity differences in variants Ile55Phe and Gly261Asp.

Conclusions:

• Specific FI domain mutations impair protease activity, affecting complement regulation.
• A multi-method approach combining co-factors is essential for comprehensive assessment of FI variant function.
• Findings offer insights into diagnostic tools for evaluating the impact of FI mutations.