June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
αB-Crystallin is the Major Succinylated Protein in Human Lenses
Author Affiliations & Notes
  • SANDIP KUMAR NANDI
    OPHTHALMOLOGY, UNIVERSITY OF COLORADO, SCHOOL OF MEDICINE, Aurora, Colorado, United States
  • Stefan Rakete
    OPHTHALMOLOGY, UNIVERSITY OF COLORADO, SCHOOL OF MEDICINE, Aurora, Colorado, United States
  • Rooban Nahomi
    OPHTHALMOLOGY, UNIVERSITY OF COLORADO, SCHOOL OF MEDICINE, Aurora, Colorado, United States
  • Cole Michel
    PHARMACEUTICAL SCIENCES, UNIVERSITY OF COLORADO, SCHOOL OF PHARMACY, AURORA, Colorado, United States
  • Alexandra Dunbar
    OPHTHALMOLOGY, UNIVERSITY OF COLORADO, SCHOOL OF MEDICINE, Aurora, Colorado, United States
  • Kristofer S Fritz
    PHARMACEUTICAL SCIENCES, UNIVERSITY OF COLORADO, SCHOOL OF PHARMACY, AURORA, Colorado, United States
  • Ram H Nagaraj
    OPHTHALMOLOGY, UNIVERSITY OF COLORADO, SCHOOL OF MEDICINE, Aurora, Colorado, United States
    PHARMACEUTICAL SCIENCES, UNIVERSITY OF COLORADO, SCHOOL OF PHARMACY, AURORA, Colorado, United States
  • Footnotes
    Commercial Relationships   SANDIP NANDI, None; Stefan Rakete, None; Rooban Nahomi, None; Cole Michel, None; Alexandra Dunbar, None; Kristofer Fritz, None; Ram Nagaraj, None
  • Footnotes
    Support  EY022061, EY023286 and a challenge grant from RPB
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 5305. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to Subscribers Only
      Sign In or Create an Account ×
    • Get Citation

      SANDIP KUMAR NANDI, Stefan Rakete, Rooban Nahomi, Cole Michel, Alexandra Dunbar, Kristofer S Fritz, Ram H Nagaraj; αB-Crystallin is the Major Succinylated Protein in Human Lenses. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5305.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
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 Nε-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.

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×