Identification Of The Primary Targets Of Carbamylation In Lens Proteins By Mass Spectrometry And Inactivation Of Glyceraldehydes 3-phosphate Dehydrogenase And Thioltransferase Induced By Carbamylation

PurposeCarbamylation, an important post-translational modification of proteins, inevitably causes conformational changes of lens proteins. It may increase aggregation between crystallin molecules and disrupt the close packing required for transparency thus leading to cataract. The aim of this study was to isolate the primary targets of carbamylation in the lens and identify them by mass spectrometry, and to investigate whether potassium cyanate can inactivate glyceraldehydes 3-phosphate dehydrogenase (GAPDH) and thioltransferase (TTase) in bovine lens. Materials and MethodsSix 2-year-old bovine lenses were obtained from a local abattoir. Four fresh intact bovine lenses were incubated with 100 mM potassium cyanate for 7 and 12 days respectively. The proteins in the water-soluble fractions from the normal control and the cyanate-modified lens proteins were separated by two-dimensional (2-D) gel electrophoresis with identification after silver staining. Protein spots that differed between the normal and carbamylated groups were selected for further analysis using matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The activity of GAPDH and TTase in the water-soluble fraction at 30°C was measured spectrophotometrically.Results1. The 2-D gel results showed that the major lens proteins were in the section of pI 5-8 with relative molecular masses of 20-35 kDa, and changed in the carbamylated fraction like strings of beads indicating modification. The mass spectrometry analysis and a database search identified carbamylated proteins originating fromαA-crystallin,βB2- andγS- (βS)-crystallins, and the first targeted lysine residues are Lys 78, Lys 88 and Lys 99 ofαA-cystallin, Lys 10, Lys 75, Lys 107, Lys 139 and Lys 171 ofβB2-crystallin and Lys 158 ofγS-crystallin.2. Potassium cyanate at concentration of 100 mM inactivated GAPDH in a time-dependent manner. GAPDH activity in control group was very high (341.6±24.7 IU/μg lens protein). All the carbamylated groups offered a statistically significant decrease in activity. After an incubation period of 7 days, the activity of TTase reached 32.1±2.1 (IU/μg lens protein) compared with 38.7±2.7 in the normal control lenses (p=0.02881). The activity of TTase was lower after an incubation of 12 days (p=0.00566). However, there was no statistical difference between the samples incubated with 100 mM KCNO for 7 and 12 days (p=0.19296).Conclusions1.αA-crystallin,βB2- andγS-crystallins may be vulnerable proteins targeted by carbamylation. The accumulated aggregation and loss of chaperone activity may contribute to cataract formation.2. This is the first evidence to show carbamylation is able to inactivate GAPDH and TTase in bovine lenses. This may have implications for the susceptibility of lenticular GAPDH and TTase to carbamylation.