Asp Isomerization Increases Aggregation of Α-Crystallin and Decreases Its Chaperone Activity in Human Lens of Various Ages.

alpha-Crystallin, comprising 40-50 subunits of alpha A- and alpha B-subunits, is a long-lived major soluble chaperone protein in lens. During aging, alpha-crystallin forms aggregates of high molecular weight (HMW) protein and eventually becomes water-insoluble (WI). Isomerization of Asp in alpha-crystallin has been proposed as a trigger of protein aggregation, ultimately leading to cataract formation. Here, we have investigated the relationship between protein aggregation and Asp isomerization of alpha A-crystallin by a series of analyses of the soluble alpha-crystallin, HMW and WI fractions from human lens samples of different ages (10-76 years). Analytical ultracentrifugation showed that the HMW fraction had a peak sedimentation coefficient of 40 S and a wide distribution of values (10-450 S) for lens of all ages, whereas the alpha-crystallin had a much smaller peak sedimentation coefficient (10-20 S) and was less heterogeneous, regardless of lens age. Measurement of the ratio of isomers (L alpha-, L beta-, D alpha-, D beta-) at Asp58, Asp91/92 and Asp151 in alpha A-crystallin by liquid chromatography-mass spectrometry showed that the proportion of isomers at all three sites increased in order of aggregation level (alpha-crystallin < HMW < WI fractions). Among the abnormal isomers of Asp58 and Asp151, D beta-isomers were predominant with a very few exceptions. Notably, the chaperone activity of HMW protein was minimal for lens of all ages, whereas that of alpha-crystallin decreased with increasing lens age. Thus, abnormal aggregation caused by Asp isomerization might contribute to the loss of chaperone activity of alpha-crystallin in aged human lens.