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Lisa Kuttner-Kondo, Viviana Ferreira, Claudio Cortes, Satya Yadav, Joe Hollyfield; Surface plasmon resonance indicates that complement factor H, C3b and native properdin bind to carboxyethylpyrrole-adducted BSA. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4584.
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The initiating events in AMD may include an accumulation of lipid oxidation products, such as the carboxyethylpyrrole (CEP) moiety that is derived from docosahexaenoic acid. While complement activation may be essential for disposal of oxidative waste, it is also implicated in the degenerative disease process. This study investigated the kinetics of complement factor H (CFH; a negative regulator of alternative pathway complement activation), native properdin (P2 dimer, P3 trimer, and P4 tetramer; a positive regulator of the same pathway), and complement C3b binding to CEP-BSA.
SPR experiments were performed using a Biacore 3000. CEP-BSA with pyrrole to protein (P/P) ratio 7 (as measured by the Ehrlich assay) and BSA were immobilized on CM5 chips by amine coupling. The human CFH, C3b, P2, P3, or P4, or recombinant CFH (rCFH) fragments diluted in HBS-P buffer were injected over the chips, and the sensorgrams analyzed. rCFH fragments included complement control protein (CCP) repeat domains 1-3, 5-7, 6-8, 7-9, 17-19 (with 6XHis tag), as well as CCPs 18-20 and 19-20.
CFH, native properdin forms, and C3b bound to CEP-BSA while they didn’t bind to the reference surface where BSA was immobilized. Kinetic rate constants were determined using Biaevaluation software (V4.0.1) by the global-fit method. The data showed affinity values (KD) in the range 10-6 M (C3b) to 10-7 M (P3). CFH appears to have the highest association-rate constant (ka), while the native properdins have the slowest dissociation kinetics (kd). The rCFH fragments CCPs 5-7, 6-8, 18-20 and 19-20 bound to CEP-BSA P/P 7, with affinity values in the range of 1.4 X 10-6 M to 4.4 X 10-6 M. The other recombinants did not bind.
CFH likely binds to CEP moieties through its “heparin-binding” domains, CCPs 18-20 and 6-8. Properdin, a positively-charged multimer, avidly bound to the CEP moieties. Therefore, the negative charges of CEP are probably responsible for attraction through electrostatic interactions. As the CEP-BSA was bound by both positive and negative regulators of the complement pathway, a balance may be necessary for allowing complement activation (promoted by properdin), while regulating the extent of activation (by CFH) to a level that facilitates opsonization and removal of lipid oxidation products, but not tissue damage.
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