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Transmembrane channel formation by complement: functional analysis of the number of C5b6, C7, C8, and C9 molecules
Insights
Complement proteins C5b6, C7, and C8 act as one-hit wonders in forming membrane channels. However, C9
Area of Science:
- Immunology
- Biochemistry
- Membrane Biology
Background:
- Sequential addition of complement proteins C5b6, C7, C8, and C9 to erythrocyte ghosts forms transmembrane channels.
- These channels involve hydrophobic peptide insertion into the cell membrane.
Purpose of the Study:
- To quantify the number of each complement protein required for membrane channel assembly.
- To elucidate the molecular composition of complement-mediated channels using different sized markers.
Main Methods:
- Erythrocyte ghosts were sequentially treated with complement proteins C5b6, C7, C8, and C9.
- Quantitative analysis of sucrose and inulin release from ghosts after channel formation.
- Assessed the relationship between protein dose and marker release to determine protein participation characteristics.
Main Results:
- C5b6, C7, and C8 exhibited one-hit kinetics in channel formation, irrespective of marker size.
- C9 showed one-hit kinetics for sucrose release but multi-hit kinetics for inulin release.
- Channel composition is proposed as a monomer of C5b–9 (C5b61C71C81C9n).
Conclusions:
- The number of C9 molecules (n) varies depending on the channel size and marker transit.
- n=1 for sucrose channels and n=2 for inulin channels, suggesting distinct channel structures.
- Results support a model where complement channels have variable C9 stoichiometry.
Abstract:
Earlier studies have shown that sequential treatment of resealed erythrocyte ghosts with C5b6, C7, C8, and C9 leads to insertion of hydrophobic peptides from these complement proteins into the membrane and assembly of transmembrane channels. The number of molecules of each of the proteins required for assembly of the membrane-associated channel structure was evaluated by measuring the quantitative relationship between the doses of the individual proteins and the release of two trapped markers, sucrose and inulin, from ghosts after channel formation. The incubation period was sufficient to attain equilibrium of marker distribution between the ghosts and the extracellular fluid. Two markers of different size (sucrose and inulin, 0.9 and 3 nm molecular diameter, respectively) were used in order to develop information on the molecular composition of small and large channels, respectively. We found that participation of C5b6, C7, and C8 in channel formation displayed one-hit characteristics, regardless of marker size. By contrast, the participation of C9 was one-hit with respect to the sucrose marker, whereas with respect to the inulin marker the C9 reaction was multi-hit. Our results are compatible with the view that these markers are released through a channel structure in the membrane that is a monomer of C5b--9 of the composition C5b61 C71C81C9n, in which n = 1 for channels permitting passage of sucrose and n = 2 for channels allowing transit of inulin.