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The relationship between channel size and the number of C9 molecules in the C5b-9 complex
Insights
The complement C5b-9 channel
Area of Science:
- Biochemistry
- Immunology
- Membrane Biology
Background:
- The complement system is crucial for innate and adaptive immunity.
- The terminal complement complex (T5b-9) forms pores in cell membranes.
- Previous studies suggested C5b-9 channel composition varies with marker size.
Purpose of the Study:
- To investigate how C9 stoichiometry affects the size of the C5b-9 channel.
- To determine if increasing C9 molecules enlarge the functional pore.
- To assess C5b-9 channel formation with larger molecules like ribonuclease A.
Main Methods:
- Dose-response analyses using resealed erythrocyte ghosts.
- Utilizing markers of increasing molecular diameter: sucrose, inulin, and ribonuclease A.
- Sieving experiments with C5b-7 carrying ghosts and markers.
Main Results:
- C5b-9 channel formation shows one-hit kinetics for C5b6.
- Increasing C9 input leads to larger functional channels, accommodating larger molecules.
- Channels allowing inulin (3 nm) require 2-3 C9 units, while ribonuclease A (3.8 nm) requires 4 C9 units.
Conclusions:
- The number of C9 molecules incorporated directly correlates with the functional size of the C5b-9 channel.
- This provides a mechanism for regulating complement-mediated cell lysis based on pore size.
- The study clarifies the dynamic assembly and functional consequences of the terminal complement complex.
Abstract:
We have recently shown by dose-response analyses with resealed erythrocyte ghosts that the channel formed by complement is a monomer of C5b-9 of the composition C5b61C71C81C9n, in which n = 1 for channels permitting passage of sucrose (0.9 nm molecular diameter) and n = 2 for channels allowing transit of inulin (3 nm molecular diameter) (1). We have now continued these experiments and expanded them by including ribonuclease A (molecular diameter, 3.8 nm) as a marker to assess whether additional C9 molecules enlarge the functional C5b-9 channel. Our results show that formation of C5b-9 channels displays one-hit characteristics with respect to C5b6 when tested by transmembrane passage of inulin or ribonuclease A. By contrast, analysis of dose-response curves of C9 indicate that n = 2-3 for channels allowing transit of inulin and n = 4 for channels allowing transit of ribonuclease A. We have also performed sieving experiments with ghosts carrying C5b-7 and containing two small markers, inositol and sucrose. Dose-response curves for C8 were performed in the presence of excess C9 to ensure conversion of all C5b-8 to C5b-9 channels. The results indicate that small channels (approximately 0.8 nm effective diameter) are not formed at high C9 multiplicity, thus confirming the results obtained with the larger markers, i.e., increase of C9 input leads to formation of larger channels.