Abstract:AIM: To attempt to identify the primary targets of carbamylation in bovine lenses incubated under physiological condition. METHODS: Fresh intact bovine lenses were incubated with [14C]-labelled potassium cyanate for seven days. The water-soluble proteins (WSP) of both cortex and nucleus lens were isolated by size-exclusion chromatography on a Sephacryl S-300HR column. The higher radioactive fractions were pooled and freeze-dried, and separated further by loading on an Affinity Blue column to separate some enzymes. In addition, WSP from cortex was separated directly by affinity chromatography. The most reactive fractions with higher radioactivity from [14C]-cyanate were further analyzed by SDS-gels and mass spectrometry. RESULTS: The majority of protein incorporating [14C]-labelled potassium cyanate was in the water-soluble fractions, and much more in the cortex than in the nucleus. Chromatography results demonstrated that the major incorporated [14C]-carbamylated crystallins were fractions corresponding to α-crystallin, β-crystallin and ξ-crystallin in the cortex, but β-crystallin and γ-crystallin in the nucleus. The SDS gels showed that bound fractions of cortex crystallins after Affi-Gel Blue separation were abundant with 20 and 35kDa proteins. However, the bound fractions of nucleus crystallins mainly showed 20kDa proteins. Mass spectrometry analysis of these higher radioactivity fractions and a database search revealed that the proteins were originated from bovine α-crystallin A and B chains and ξ-crystallin in the cortex; βA1 and αB-crystallins with a little γB-crystallin in the nucleus respectively. Further analysis suggested the location of this carbamylation of αB-crystallin in the nucleus to be at Lys 92 and 103. CONCLUSION: α-and ξ-crystallin from cortex can be prefe- rentially targeted by carbamylation during whole lens incubations. Carbamylation of these crystallins at the earlier stage may result in further unfolding and misfolding of lens proteins, leading to aggregation of crystallins and eventually to cataract formation.
