Multi-crystallin complexes exist in the water-soluble high molecular weight protein fractions of aging normal and cataractous human lenses

Abstract

The purpose of the study was to identify non-covalently held complexes that exist in the water-soluble high molecular weight (WS-HMW) protein fractions of normal human lenses of 20-year-old and 60- to 70-year-old, and in the age-matched 60- to 70-year-old cataractous lenses. The WS protein fractions were prepared from five pooled normal lenses of 20-year-old donors or five pooled lenses of 60- to 70-year-old donors or four pooled cataractous lenses (with nuclear opacity) of 60- to 70-year-old donors. Each WS protein fraction was subjected to size-exclusion chromatography using an Agarose A 5m column to recover the void volume WS-HMW protein fraction. A method known as blue-native polyacrylamide gel electrophoresis (BN-PAGE), which allows the isolation of large multi-protein complexes (MPCs) in their native state for further characterization, was used to separate such complexes from individual WS-HMW protein fractions. The protein species that existed as a complex were excised from a gel and trypsin-digested, and the amino acid sequences of the tryptic fragments analyzed by electrospray tandem mass spectrometry (ES-MS/MS). After the second-dimensional sodium dodecyl sulfate–PAGE during BN-PAGE, protein complexes containing a total of 16, 12, and 24 species with M r between 10 and 90 kDa were identified in the HMW protein fractions of normal lenses of 20-year-old, 60- to 70-year-old and cataractous lenses of 60- to 70-year-old donors, respectively. Based on the amino acid sequences of tryptic peptides of individual protein species in the complexes by the ES-MS/MS method, the presence of α-, β-, and γ-crystallin species along with beaded filament proteins (filensin and phakinin) was observed in the 20-year-old normal lenses. The 60- to 70-year-old normal lenses contained filensin and aldehyde dehydrogenase in addition to the above crystallins. Similarly, the age-matched cataractous lenses also contained the above crystallins and aldehyde dehydrogenase but lacked beaded filament proteins. Protein complexes, held mostly via non-covalent bonding, were seen in the WS-HMW proteins of 20-year-old normal, 60- to 70-year-old normal, and 60- to 70-year-old cataractous lenses. The complexes in the normal lenses were made of α-, β-, and γ-crystallin species, beaded filament proteins (filensin and/or phakinin), and aldehyde dehydrogenase. The complexes in the age-matched cataractous lenses also contained these crystallins, and aldehyde dehydrogenase, but not the beaded filament proteins. Further, the crystallin fragments were greater in number in the cataractous lenses compared to the age-matched normal lenses. During multi-angle light scattering (MALS), the HMW proteins from cataractous lenses exhibited species with lower molecular weight range than age-matched normal lenses. The HMW protein preparations from both normal and cataractous lenses showed spherical structures on electron microscopic analysis.