Copper Reductase Activity and Free Radical Chemistry by Cataract-Associated Human Lens ?-Crystallins

Cataracts are caused by high-molecular-weight aggregates of human eye lens proteins that scatter light, causing lens opacity. Metal ions have emerged as important potential players in the etiology of cataract disease, as human lens gamma-crystallins are susceptible to metal-induced aggregation. Here, the interaction of Cu2+ ions with gamma D-, gamma C-, and gamma S-crystallins, the three most abundant gamma-crystallins in the lens, has been evaluated. Cu2+ ions induced non-amyloid aggregation in all three proteins. Solution turbidimetry, sodium dodecyl sulfate poly(acrylamide) gel electrophoresis (SDS-PAGE), circular dichroism, and differential scanning calorimetry showed that the mechanism for Cu-induced aggregation involves: (i) loss of beta-sheet structure in the N-terminal domain; (ii) decreased thermal and kinetic stability; (iii) formation of metal-bridged species; and (iv) formation of disulfide-bridged dimers. Isothermal titration calorimetry (ITC) revealed distinct Cu2+ binding affinities in the gamma-crystallins. Electron paramagnetic resonance (EPR) revealed two distinct Cu2+ binding sites in each protein. Spin quantitation demonstrated the reduction of gamma-crystallin-bound Cu2+ ions to Cu+ under aerobic conditions, while X-ray absorption spectroscopy (XAS) confirmed the presence of linear or trigonal Cu+ binding sites in gamma-crystallins. Our EPR and XAS studies revealed that gamma-crystallins' Cu2+ reductase activity yields a protein-based free radical that is likely a Tyr-based species in human gamma D-crystallin. This unique free radical chemistry carried out by distinct redox-active Cu sites in human lens gamma-crystallins likely contributes to the mechanism of copper-induced aggregation. In the context of an aging human lens, gamma-crystallins could act not only as structural proteins but also as key players for metal and redox homeostasis.

Automated summary

Cataracts are caused by the aggregation of high-molecular-weight proteins in the human eye lens, which scatter light and cause lens opacity. Metal ions play an important role in the development of cataract disease, as human lens gamma-crystallins are susceptible to metal-induced aggregation.