αB-Crystallin is the Major Succinylated Protein in Human Lenses

Abstract

Purpose : Human lens proteins are chemically modified throughout life and some modifications accumulate because of the negligible protein turnover rate. Succinylation of lysine residues is one such modification. Here, we have investigated the effect of age on succinylation of lens proteins and the consequences of succinylation on the structure and chaperone activity of αB-crystallin (αB).

Methods : Water-soluble and insoluble protein fractions of human lenses (donor ages 20 to 73 years) were prepared and analyzed for N^ε-succinyllysine (SuccK) by western blotting. SuccK quantification in enzyme digested lens samples was done using UPLC-MS/MS. Succinylation of recombinant αB was performed using succinyl CoA. Mass spectrometric studies were performed to identify SuccK in proteins. Chaperone activity of αB was determined using two client proteins, insulin and citrate synthase. Far UV-CD, tryptophan fluorescence and DLS studies were performed to investigate the effect of succinylation on the secondary, tertiary and quaternary structure of αB. Surface hydrophobicity of αB was determined spectrofluorimetrically by probing with bis-ANS.

Results : All major crystallins of the human lens contain SuccK modification. However, western blotting and immunoprecipitation studies showed that αB to be predominantly succinylated. The SuccK quantification by LC-MS/MS showed a range between 1.2 and 14.3 pmol/mg lens protein. The aged lenses (age>60 years) showed slightly lower levels than young lenses (age<30 years). Immunohistochemical analyses showed SuccK throughout the lens. Mass spectrometric analyses showed SuccK at K70, K90, K92, K166, K174 and K175 in human lens αB. In vitro succinylation of recombinant human αB to the level observed in human lenses resulted in succinylation of K72, K92, K150 and K166 and improved the chaperone activity without major changes in either the secondary or tertiary structure.

Conclusions : Our study demonstrated that succinylation of human lens proteins occurs early in life and continues to be present through old age, and αB-crystallin to be the dominant succinylated protein. Succinylation improved the chaperone activity of αB without altering its structure, which suggest that it is an adoptive gain of function modification to protect lens during aging.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.