Proteomic Analysis of Human Age-related Nuclear Cataracts and Normal Lens Nuclei

Abstract

PURPOSE: To identify proteomic differences between age-related nuclear cataracts (ARNCs) and normal lens nuclei. METHODS: Total solubilized proteins from ARNC lens nuclei with different grades were compared with normal controls by 2-D differential in-gel electrophoresis (2-D DIGE). Proteins with different abundances were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses determined the compositions of high molecular weight (HMW; >200 kDa) aggregates found in ARNC lens nuclei. Western blot analysis was used to verify the changes in αA-crystallin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels. RESULTS: The 2-D differential in-gel electrophoresis results showed that nine proteins were significantly less abundant in lens nuclei from ARNC patients than in control lens nuclei. Six proteins (αA-, βA3-, βA4-, βB1-, and γD-crystallin and putative uncharacterized protein DKFZp434A0627 from the CRYGS family) tended to decrease as the cataract grade increased, while the other three proteins (αB-crystallin, GAPDH, and retinal dehydrogenase 1) did not show such a tendency. SDS-PAGE showed decreased protein levels at ∼20 kDa in ARNC lenses but significantly increased levels at HMW (>200 kDa). Liquid chromatography tandem mass spectrometry analysis showed that the HMW aggregates derived largely from crystallins also contained filensin, phakinin, and carbonyl reductase 1. Of all the components, αA-crystallin accounted for the highest fraction. αA-, αB-, and γD-crystallin and DKFZp434A0627 were more prone to aggregate than other crystallins. CONCLUSIONS: The results show that crystallins, especially αA-crystallin, aggregate irreversibly during ARNC development. Some enzymes (GAPDH, retinal dehydrogenase 1, and carbonyl reductase 1) may be involved in and/or accelerate this process.