Abstract
The sub-retinal pigment epithelial deposits that are a hallmark of age-related macular degeneration contain both C3b and millimolar levels of zinc. C3 is the central protein of complement, whereas C3u is formed by the spontaneous hydrolysis of the thioester bridge in C3. During activation, C3 is cleaved to form active C3b, then C3b is inactivated by Factor I and Factor H to form the C3c and C3d fragments. The interaction of zinc with C3 was quantified using analytical ultracentrifugation and x-ray scattering. C3, C3u, and C3b associated strongly in >100 μM zinc, whereas C3c and C3d showed weak association. With zinc, C3 forms soluble oligomers, whereas C3u and C3b precipitate. We conclude that the C3, C3u, and C3b association with zinc depended on the relative positions of C3d and C3c in each protein. Computational predictions showed that putative weak zinc binding sites with different capacities exist in all five proteins, in agreement with experiments. Factor H forms large oligomers in >10 μM zinc. In contrast to C3b or Factor H alone, the solubility of the central C3b-Factor H complex was much reduced at 60 μM zinc and even more so at >100 μM zinc. The removal of the C3b-Factor H complex by zinc explains the reduced C3u/C3b inactivation rates by zinc. Zinc-induced precipitation may contribute to the initial development of sub-retinal pigment epithelial deposits in the retina as well as reducing the progression to advanced age-related macular degeneration in higher risk patients.
Highlights
Sub-retinal pigment epithelial deposits contain complement proteins and bioavailable zinc
We have shown that Bruch’s membrane contains bioavailable zinc, especially in samples that contain substantial sRPEds, evidenced by their labeling in the presence of selective fluorescence sensors that only bind to bioavailable zinc [67]
When pathophysiological amounts of Ͼ100 M zinc are present together with high levels of localized inflammation, our results show that zinc will precipitate and remove the C3b-FH complex and free C3b
Summary
Sub-retinal pigment epithelial deposits contain complement proteins and bioavailable zinc. The sub-retinal pigment epithelial deposits that are a hallmark of age-related macular degeneration contain both C3b and millimolar levels of zinc. Zinc-induced precipitation may contribute to the initial development of sub-retinal pigment epithelial deposits in the retina as well as reducing the progression to advanced agerelated macular degeneration in higher risk patients. The formation of convertase enzyme complexes (such as those originating from complement Factor B) cleaves the ANA/C3a domain from C3 to form C3b. To determine the extent to which surface-bound zinc causes each of C3, C3u, C3b, C3c, and C3d to self-associate, we employed analytical ultracentrifugation and synchrotron x-ray scattering [36] combined with metal binding-site predictions for these proteins [37]. Molecular mechanisms are suggested for the initial formation of sRPEds that lead to AMD as well as an explanation for the role of zinc in reducing the occurrence of developing advanced AMD in high risk patients [38, 39]
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