CD59, Disulphide-Locked Human C9 and Horse C9 Inhibit Human Membrane Attack Complex Assembly by Similar Mechanisms

Area of Science:

• Immunology
• Complement System
• Molecular Biology

Background:

• The membrane attack complex (MAC), formed by plasma proteins C5b-C9, lyses target cells.
• CD59 regulates MAC formation by preventing C9 polymerization into the lytic pore.
• Engineered C9lock and species-incompatible horse C9 (HoC9) exhibit altered lytic activity.

Purpose of the Study:

• To compare the inhibitory mechanisms of soluble CD59 (sCD59), C9lock, and HoC9 on MAC assembly.
• To elucidate the functional differences between HoC9 and human C9.
• To identify novel strategies for inhibiting MAC formation in pathological conditions.

Main Methods:

• Recombinant expression and affinity purification of MAC inhibitors (sCD59, C9lock, HoC9, HuC9).
• Binding assays (ELISA) to detect intermediate MAC complex binding.
• Haemolytic assays to assess inhibition of MAC-mediated lysis.

Main Results:

• sCD59, C9lock, and HoC9 effectively inhibited human serum-mediated haemolysis via classical and alternative pathways.
• All three inhibitors bound C5b-8 intermediates but not C5b-7, competitively blocking C9-mediated lysis.
• Inhibitors also bound mouse and rat C5b-8, blocking human C9-mediated lysis, indicating conserved binding sites.

Conclusions:

• sCD59, C9lock, and HoC9 share mechanistic similarities in inhibiting MAC assembly by targeting C5b-8.
• Functional differences between HoC9 and HuC9 were clarified, highlighting species compatibility in MAC formation.
• These MAC inhibitors serve as valuable tools for studying MAC assembly and offer potential therapeutic strategies.