“Our recent study revealed an age-related truncation affecting essential GSH biosynthesis enzyme, the γ-glutamylcysteine ligase catalytic subunit (GCLC), at aspartate residue 499,” they wrote in the study.

In findings from the study published in Science Advances, the researchers, at Augusta University in Augusta, Georgia, noted the truncated GCLC fragments compete with full-length GCLC in forming a heterocomplex with modifier subunit (GCLM) but show a marked reduction in enzymatic activity.¹

“Crucially, using an aspartate-to-glutamate mutation knock-in (D499E-KI) mouse model that blocks GCLC truncation, we observed a notable relay in ARC formation compared with WT [wild-type] mice: Nearly 50% of D499E-KI mice remained cataract free vs [approximately] 20% of the WT mice at their age of 20 months,” the researchers wrote. “Our findings concerning age-related GCLC truncation might be the key to understanding the profound reduction in lens GSH with age. By halting GCLC truncation, we can rejuvenate lens GSH levels and considerably postpone cataract onset.”

Mark Packer, president, Packer Research Associates in Boulder, Colorado, and a member of the Ophthalmology Times Editorial Advisory Board, noted this new research from Augusta University provides “compelling evidence that impaired enzymatic production of the antioxidant glutathione in the aging human lens contributes to cataract formation, and has shown that restoration of enzymatic function prevents cataracts in a mouse model.” Packer was not involved in the study.

“The underlying mechanism of the decrease in enzymatic function remains to be fully understood,” he said in comments to Ophthalmology Times on the study. “However, this line of research hold promise for cataract prevention in the future. In today’s world, doctors have plenty of reasons to recommend maintaining healthful antioxidant levels through diet.”

Study results

The researchers found that GCLC underwent age-related truncation in mouse and human lenses. GCL is a critical enzyme involved in the biosynthesis of intracellular GSH, acting as the rate-limiting step. GCL is comprised of 2 subunits: the catalytic subunit (GCLC) and the modifier subunit (GCLM).

The researchers found that although GCLC has the catalytic domain, its interaction with GCLM is necessary for optimal enzymatic activity.²

“During our investigation of GCLC protein expression in older mouse lenses, we unexpectedly observed a smaller molecular weight band, approximately 60,000 Da (referred to as G60 in this study),” they wrote. “Intriguingly, G60 exhibited an age-related increase in mouse lenses and reached similar levels to [that of] the full-length GCLC (termed G73 in this study) in aged lenses.”

The researchers noted that they also analyzed collected human lenses with an average postmortem interval of 18 hours and observed a similar age-related increase in G60.¹

“Calculating the ratio of G60:G73, we identified a positive age association (R2, 0.6772) in human lenses,” they wrote. “On the basis of age-related human lens growth and physiological behavior, we categorized lenses into 4 groups: below 20 years (group I) as a period of rapid growth, 21 to 40 years (group II) as the optimal performance period, 41 to 65 years (group III) as a period prone to presbyopia development, and beyond 65 years (66 to 100 years, group IV) associated with higher cataractogenesis.”

Moreover, the researchers pointed out that they witnessed a nearly linear relationship of G60:G73 from groups I to IV. Notably, full-length GCLC (G73) levels were also increased in aged mice and human lenses.

“Through comparisons with GCLC knockout (KO) mouse lenses, we confirmed that this band represents a truncated form of GCLC,” the researchers added.

To examine the condition of the smaller truncated fragment with a size of approximately 13 kDa (termed G13 in this study), the researchers generated an antibody that specifically recognizes G13 using a mouse G13 recombinant fragment.

“With this antibody, we detected an age-related increase in the G13 fragment in mouse lenses, suggesting that the truncated G13 fragment is also stably present in the aged lens,” they wrote. “Unfortunately, our homemade C-terminal antibody did not recognize the human G13 fragment.”

Furthermore, the researchers noted GCL activity continues to decline during aging in human and mouse lenses. They used liquid chromatography and mass spectrometry to detect a remarkable age-related decrease in GCL activity in the human lens. In mice, they detected a decline in GCL activity linked with age.

“There was a clear correlation between lenticular GSH levels and GCL activities,” the researchers wrote. “These findings strongly emphasize the pivotal role of GCL activity in maintaining lens GSH homeostasis.”

As part of the methodology, the researchers noted that all chemicals used were of analytical reagent grade. Milli-Q water was used for the preparation of standards and reagents.

In addition, the researchers noted normal human eye globe span of 5 to 76 years was obtained from the Georgia Eye Bank in Atlanta with an average postmortem time of 18 hours. The lens was then dissected and immediately processed for individual assays^.1

Researchers also noted animal experiments were conducted in compliance with the approved protocols by the Animal Care and Use Committee at Augusta University in Georgia, following the Association for Research in Vision and Ophthalmology guidelines for the ethical use of animals in ophthalmic and vision research.

The researchers concluded that their efforts represent an interesting discovery, identifying an age-related truncation of the GHS biosynthesis enzyme GCLC.

“This cleavage does not lead to a decrease in GCL holoenzyme complex formation, but it does significantly impair GCL enzymatic activity,” they wrote. “By generating KI mice without GCLC cleavage, we demonstrated that blocking GCLC cleavage successfully maintained lens GSH levels even in older mice and [that] it significantly delayed age-related cataract formation. These findings open up a crucial path for developing a therapeutic approach to delay age-related cataractogenesis, potentially offering strategies to mitigate the impact of cataracts associated with aging.”

References

1. Wei Z, Hao C, Radeen KR, Srinivasagan R, Chen JK, Sharma S, McGee-Lawrence ME, Hamrick MW, Monnier VM, Fan X. Prevention of age-related truncation of γ-glutamylcysteine ligase catalytic subunit (GCLC) delays cataract formation. Sci Adv. 2024 Apr 26;10(17):eadl1088. doi: 10.1126/sciadv.adl1088. Epub 2024 Apr 26. PMID: 38669339; PMCID: PMC11051666.

2. Chen Y, Shertzer HG, Schneider SN, Nebert DW, Dalton TP. Glutamate cysteine ligase catalysis: dependence on ATP and modifier subunit for regulation of tissue glutathione levels. J Biol Chem. 2005 Oct 7;280(40):33766-74. doi: 10.1074/jbc.M504604200. Epub 2005 Aug 4. PMID: 16081425.