Glycation of human lens proteins: Preferential glycation of αA subunits

Abstract

Glycation of crystallins and high molecular weight (HMW) aggregates was followed during aging (16–85 years) and in diabetes (44 and 70 years old). Lens soluble and insoluble fractions were reduced with [ 3H]NaBH 4 and separated by molecular sieve HPLC. The protein content in each HPLC peak was measured by the Lowry method. The tritium incorporation, expressed as cpm mg −1 protein, was taken as a measure of early glycation and specific non-tryptophan fluorescence (E x: 370 nm; E m: 440 nm), expressed as relative fluorescence U mg −1 protein, was taken as a measure of advanced glycation. The youngest lenses analysed were 16 and 17 years old and these provided the baseline values. The results showed that during aging there was about a three-fold increase in tritium incorporation and fluorescence of α-crystallin, while the increases in β and γ were only two-fold from the levels seen in 16- and 17-year-old lenses. On the other hand, both the soluble and insoluble HMW aggregate fractions showed up to five-fold increase in tritium incorporation during aging. The fluorescence was about two-fold higher in the insoluble HMW aggregates as compared to the soluble HMW aggregates in 16- and 17-year-old lenses and both showed an increase of about three-fold during aging. Diabetes resulted in an approximately 10–50% increase in tritium incorporation and non-tryptophan fluorescence of various crystallins and HMW aggregates. Since α-crystallins showed a higher level of glycation than other crystallins, and soluble HMW aggregates appear to have consisted predominantly of α-crystallins, we further determined the level of glycation of α-crystallin subunits. Alpha-crystallins from 50–85-year-old lenses was used to separate the subunits by both reverse-phase HPLC (RP-HPLC) and gel electrophoresis. Determination of the protein bound radioactivity showed that αA was more glycated than αB. Furthermore, the RP-HPLC also separated a modified α peak, in addition to αA and αB, which presumably originated from HMW aggregates. The modified α had an even higher level of early glycation than αA. Interestingly, the fluorescence associated with the modified α was also several fold higher than αA and αB.