Racemization in cataractous lens from diabetic and aging individuals: analysis of Asp 58 residue in αA-crystallin

Abstract

Cataract is the leading cause of visual impairment globally. Racemization of lens proteins may contribute to cataract formation in aging individuals. As a special type of age-related cataract (ARC), diabetic cataract (DC) is characterized by the early onset of cortical opacification and finally developed into a mixed type of cortical and nuclear opacification. We compared racemization of Asp 58 residue, a hotspot position in αA-crystallin, from the cortex and nucleus of diabetic and age-matched senile cataractous lenses, by identifying L-Asp/L-isoAsp/D-Asp/D-isoAsp by mass spectrometry. Compared to nondiabetic cataractous lenses, DC lenses showed a significantly increased cortex/nucleus ratio of D-Asp 58, which originated primarily from an increased percentage of D-Asp 58 in the lens cortex of DC. Moreover, patients diagnosed with diabetes for over 10 years showed a lower cortex/nucleus ratio of D-isoAsp 58 in the lens compared with those who had a shorter duration of diabetes, which originated mainly from an increased percentage of D-isoAsp 58 in the lens nucleus of DC with increasing time of hyperglycemia. Further analysis confirmed decreased protein solubility in diabetic cataractous lenses. The different racemization pattern in DC may be distinguished from ARC and influence its phenotype over the protracted duration of diabetes.

Keywords: aging; aspartyl residue; cataract; crystallin; diabetes; lens; racemization.

Figure 1

(A) Representative LC-MS/MS trace showing the separation of the four Asp isomers of the αA-crystallin tryptic peptide (55–65) TVLDSGISEVR. Peptides containing D-Asp, D-isoAsp, L-Asp, or L-isoAsp at position 58 were synthesized. To measure racemization in αA-crystallin, all forms of the peptide were summed and modifications for each were expressed as a% of the total peak area. (B) Representative graphs showing the separation of the four Asp 58 isomers in αA-crystallin of ARC lenses. (C) Representative graphs showing the separation of the four Asp 58 isomers in αA-crystallin of DC lenses. (D) The percentage of each Asp 58 isomer in αA-crystallin from lenses of patients with ARC and DC. (E) The cortex/nucleus ratio of each Asp 58 isomer in αA-crystallin from cortex and nucleus of ARC and DC lenses after dissection. (F) The percentage of each Asp 58 isomer in αA-crystallin from cortex and nucleus of ARC and DC lenses after dissection.

Figure 2

(A) The cortex/nucleus ratio of each Asp 58 isomer in αA-crystallin of two subgroups of DC lenses according to the duration of diabetes (less than ten years and more than ten years). (B) The percentage of each Asp 58 isomer in αA-crystallin of two subgroups of DC lenses according to the duration of diabetes (less than ten years and more than ten years).

Figure 3

Protein solubility changes in lenses of ARC and DC patients (^*P = 0.005, ARC cortex vs. DC cortex; ^**P = 0.013, DC cortex < 10 years vs. DC cortex > 10 years; ^***P < 0.001, ARC cortex vs, DC cortex > 10 years; ^****P = 0.007, DC nucleus < 10 years vs. DC nucleus > 10 years).

Figure 4

Illustration of the normal L-Asp 58 spontaneously converted to L-isoAsp 58, D-Asp 58, and D-isoAsp 58 in diabetic lenses. (A) The main difference in racemization at Asp residues in the lenses of DC patients was an increased percentage of D-Asp 58 in the cortex, leading to the early stage of cortical opacification in DC. (B) Over the protracted duration of diabetes, the percentage of D-isoAsp 58 in the nucleus increased, leading to the further nuclear opacification on the original basis of cortical opacification in the late stage of DC. L-Asp = L-Asp 58, L-isoAsp = L-isoAsp 58, D-Asp = D-Asp 58, D-isoAsp = D-isoAsp 58.