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Potent inhibition of terminal complement assembly by clusterin: characterization of its impact on C9 polymerization
1Department of Biochemistry, University of Minnesota, St. Paul 55108, USA.
Insights
Clusterin, a complement inhibitor, blocks the assembly of C9 and C5b-7, significantly reducing complement-mediated cell lysis. This interaction protects cells by slowing down the complement cascade, offering a potent defense mechanism.
Area of Science:
- Immunology
- Biochemistry
- Molecular Biology
Background:
- Clusterin (CL) is a heterodimeric apolipoprotein with complement inhibitory functions.
- The terminal complement pathway, involving proteins C5b-9, is crucial for cell lysis.
- Understanding clusterin's precise inhibitory mechanisms is vital for its therapeutic potential.
Purpose of the Study:
- To characterize the interactions between clusterin and assembling terminal complement proteins.
- To elucidate the modes of action and binding sites of clusterin in complement inhibition.
- To quantify the impact of clusterin on C9 assembly and complement-mediated cytolysis.
Main Methods:
- Utilized fluorescein isothiocyanate-labeled C9 (FITC-C9) to monitor C9 assembly via fluorescence changes.
- Performed kinetic analysis of C9 assembly and dissociation rates.
- Assessed clusterin binding affinities to C5b-8 and C5b-9(1) complexes.
Main Results:
- Clusterin inhibited C9 assembly on C5b-8 and C5b-9, and prevented C5b-7 membrane attachment.
- Clusterin binding to C5b-8-(FITC-C9)1 quenched fluorescence, indicating binding to the C9 site.
- Clusterin exhibited higher affinity for C5b-8 and C5b-9(1) complexes than C9 itself.
Conclusions:
- Clusterin acts as a potent inhibitor of terminal complement pathway assembly through multiple mechanisms.
- Even low levels of active clusterin can significantly reduce complement-mediated cytolysis, offering cellular protection.
- Clusterin's inhibitory activity suggests a role in protecting nucleated cells from complement attack.
Abstract:
The interactions of the heterodimeric apolipoprotein and complement inhibitor, clusterin (CL, 80 kDa), with actively assembling terminal complement proteins were characterized. Clusterin inhibited at three sites and by two modes of action. Clusterin inhibited C9 assembly on C5b-8 and C5b-9 and also bound to C5b-7 to prevent membrane attachment. The impact on C5b-9 assembly was the most potent. C9 assembly was monitored by assembly-induced fluorescence changes of C9 labeled with fluorescein isothiocyanate (FITC-C9). Assembly of monomeric FITC-C9 with C5b-8 or C5b-9(1) produced a substantial decrease in fluorescence intensity due to changes in the environment of the probe. Addition of the next subunit of unlabeled C9 produced a further small change. One equivalent of FITC-C9 bound to C5b-8 at low temperatures, but the fluorescence change and addition of more C9 did not occur until the temperaure was increased. Kinetic analysis of the fluorescence change suggested an irreversible, first-order process with an activation energy of 29 kcal/mol (k = 0.12 s(-1) at 25 degrees C). The kinetic properties differed for C9 addition to C5b-9(1) (0.27 s(-1) at 25 degrees C, 21 kcal/mol), indicating that C9 activation occurred at a different or altered site. Clusterin binding to C5b-8-(FITC-C9)1 caused fluorescence quenching similar to that of unlabeled C9, indicating that it bound to the C9 binding site. Clusterin binding to C5b-8 and C5b-9(1) was reversible with affinities that were 2 and 15 times that of C9 for the C5b-8 and C5b-9(1) complexes, respectively. The results suggested that the presence of <10% of the circulating clusterin in its heterodimeric, active form could reduce the rate of complement cytolysis of nucleated cells by 10-fold, and under some conditions by 100-fold or more. This would provide a high level of protection for certain cells and may allow time for action by other inhibitors of complement.