Gamma-crystallin and free thiols promote N^ε-acetyllysine formation in alpha-crystallin

Abstract

Purpose : Lysine acetylation is a significant post-translational modification in eye lens crystallins. It has been observed that certain lysine residues are favored over others in crystallins for the formation of N^ε-acetyllysine (AcK). We investigated the role of free thiols in such site-specific AcK formation in human α-crystallin.

Methods : Recombinant human αA-crystallin (αAC; 3 mg/mL) and αB-crystallin (αBC; 3 mg/mL) were incubated with acetyl-CoA (AcCoA; 500 µM) for 3 days at 100 mM sodium phosphate buffer, pH 7.4 and 37^oC, and the AcK levels were measured by Western blotting, ELISA, and LC-MS/MS. Cysteine-rich γS-crystallin (γSC; 1 mg/mL) was incubated with histidine-tagged αAC or αBC (3 mg/mL) and AcCoA (500 µM) at pH 7.4 and 37^oC. Histidine-tagged αAC or αBC and γSC were separated using a Ni-sepharose column, and the AcK levels were measured in α-crystallin, as above. A mixture of αBC (3 mg/mL) and AcCoA (500 µM) was incubated with reduced glutathione (GSH; 500 µM) or N-acetyl-lysine (NAC; 500 µM) for 3 days at 37^oC and the AcK content was measured. The AcK levels were compared in reductively alkylated αAC (1 mg/mL) incubated with AcCoA (500 µM) for 3 days and AcCoA-incubated αAC. Three mutants of αBC (K92C, V169C, and E99C) were generated to introduce a cysteine residue within ~5Å of a lysine residue, incubated with AcCoA (500 µM) for 3 days, and the AcK levels in those were measured and compared with the wild type counterparts.

Results : αAC (which contains two cysteine residues) showed significantly higher levels of AcK than αBC (which lacks cysteine residues) upon incubation with AcCoA. The addition of γSC led to enhancement in AcK formation in AcCoA-incubated αBC. Free thiols, GSH and NAC enhanced AcK levels in AcCoA-incubated αBC. Reductive alkylation of cysteine residues significantly reduced AcCoA-mediated AcK formation in αAC. The introduction of cysteine residues within ~5Å of lysine residues resulted in a substantial increase in AcK levels compared to the wild type αBC.

Conclusions : Our findings suggest that AcK formation in α-crystallin is promoted by the proximal cysteine residues and protein-free thiols through a nucleophilic substitution mechanism. These mechanisms could be utilized to increase AcK levels in α-crystallin to reduce lens stiffness associated with presbyopia.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.