Cell-bound C4b resists reduction by reducing agents: analysis by chain structure and by hemolytic activity

Area of Science:

• Biochemistry
• Immunology
• Complement System

Background:

• The complement system is a crucial part of innate immunity.
• Complement component 4b (C4b) plays a key role in complement activation pathways.
• Understanding the structural and functional integrity of C4b is essential for comprehending immune responses.

Purpose of the Study:

• To investigate the effect of reducing agents on cell-bound complement component 4b (C4b).
• To determine the role of C4b gamma-chains in its hemolytic activity and function.
• To assess the impact of different reducing agents on C4b stability and interaction with antibodies.

Main Methods:

• Treatment of EA3H-C4hu cells with 2-mercaptoethanol (MSH) and dithiothreitol (DTT).
• Analysis of residual C4b chains using antibody-based detection.
• Assessment of hemolytic activity and generation of SAC4b2a.
• Kinetic studies to evaluate the removal of C4b chains.

Main Results:

• MSH treatment did not alter C4b chains, reactivity with anti-C4 antibody, or SAC4b2a generation.
• DTT treatment removed approximately 70% of C4b gamma-chains and over 95% of C4b activity.
• Alpha' and beta chains of C4b remained unaffected by DTT.
• Kinetic studies revealed heterogeneity in cell-bound C4b, with 30% of gamma-chains resistant to DTT removal.

Conclusions:

• The C4b gamma-chain is essential for C4b's hemolytic activity and its ability to generate SAC4b2a.
• Cell-bound C4b exhibits heterogeneity, with some gamma-chains being more resistant to reduction.
• MSH is not suitable for cleaving C4b gamma-chains, whereas DTT effectively removes most of them, highlighting differential effects of reducing agents on C4b structure and function.