title: Potent inhibition of terminal complement assembly by clusterin: characterization of its impact on C9 polymerization.
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.
authors.affiliation: Department of Biochemistry, University of Minnesota, St. Paul 55108, USA.
authors.forename: J F
authors.initials: JF
authors.lastname: McDonald
authors.forename: G L
authors.initials: GL
authors.lastname: Nelsestuen
mesh_terms: D051152:Clusterin; D003186:Complement C9 / Q000737:chemistry / Q000378:metabolism*; D003169:Complement Inactivator Proteins / Q000378:metabolism / Q000494:pharmacology*; D015938:Complement Membrane Attack Complex / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006023:Glycoproteins / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008027:Light; D018832:Molecular Chaperones; D011108:Polymers; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
chemical_list: C495242:CLU protein, human; D051152:Clusterin; D003186:Complement C9; D003169:Complement Inactivator Proteins; D015938:Complement Membrane Attack Complex; D005456:Fluorescent Dyes; D006023:Glycoproteins; D018832:Molecular Chaperones; D011108:Polymers; D016650:Fluorescein-5-isothiocyanate