The glutathione biosynthesis enzyme subunit GCLC undergoes age- and diabetes-related fragmentation and inactivation: relevance to age-related cataractogenesis

Abstract

Purpose : Oxidation has been documented as the major risk factor in age-related cataract formation. Glutathione (GSH), the most abundant lens antioxidant, is continuously decreasing in the aging lens. The critical role of GSH in maintaining lens redox status and transparency is very well recognized. Yet, 70 years after the first report of GSH presence in the human lens, the underlying mechanisms of impaired GSH biosynthesis and profound reduction of lens GSH levels in aged human lens are still poorly understood. Below we report the unexpected discovery that GSH biosynthesis enzyme undergoes age-related fragmentation and inactivation.

Methods : The posttranslational processing of γ-glutamyl cysteine ligase, catalytic subunit (GCLC) in human and mouse lenses at different age was determined by immunoblot and mass spectrometry (MS). The cleavage site was predicted by in-silico computational analysis and was confirmed by MS and amino acids mutagenesis approach. The GCLC activity was determined by LC/MS. The protein interaction between GCLC and GCLM was determined by two-hybrid system as well as size exclusion fractionation of the lens protein extract. The stability and functions of truncated GCLC fragments were also determined in FHL124 and GCLC^-/- MEF cell culture.

Results : We have found an age associated C-terminal 13kD truncation and accumulation of N-terminal 60kD fragment (GCLC⁶⁰) in both human and mouse lenses. The GCLC⁶⁰ accumulation reaches almost equal level relative to full length in mouse at age of 12mos and human at age over 65. The computational analysis and MS determination indicates aspartate 499 (D499) is the caspase-3 like cleavage site, and this was further confirmed by D499A and D499E mutation. Over 8-fold reduction of the enzymatic activity was found in truncated form (GCLC⁶⁰) vs. full length GCLC. The interaction between truncated form (GCLC60) and GCLM is largely maintained based on size exclusion fractionation analysis.

Conclusions : Holoenzyme formation between GCLC and GCLM is essential for GSH biosynthesis in vivo. The competitive binding between GCLC⁶⁰ and GCLC with GCLM will impair GSH biosynthesis. This unexpected discovery may well have been the most important mechanism of age-related loss of the lens GSH. We therefore hypothesize that truncation of GCLC is a key event in the pathogenesis of age-related cataractogenesis.

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