Non-oxidative modification of lens crystallins by kynurenine: a novel post-translational protein modification with possible relevance to ageing and cataract

In humans, the crystallin proteins of the ocular lens become yellow-coloure d and fluorescent with ageing. With the development of senile nuclear catar act, the crystallins become brown and additional fluorophores are formed. T he mechanism underlying crystallin colouration is not known but may involve interaction with kynurenine-derived UV filter compounds. We have recently identified a sulphur-linked glutathionyl-3-hydroxykynurenine glucoside addu ct in the lens and speculated that kynurenine may also form adducts with GS H and possibly with nucleophilic amino acids of the crystalline (e.g. Cys). Here we show that kynurenine modifies calf lens crystallins non-oxidativel y to yield coloured (365 nm absorbing), fluorescent (Ex 380 nm/Em 450-490 n m) protein adducts. Carboxymethylation and succinylation of crystallins inh ibited kynurenine-mediated modification by approx. 90%, suggesting that Cys , Lys and possibly His residues may be involved. This was confirmed by show ing that kynurenine formed adducts with GSH as well as with poly-His and po ly-lys. NMR studies revealed that the novel poly-Lys-kynurenine covalent li nkage was via the epsilon-amino group of the Lys side chain and the beta C of the kynurenine side chain. Analysis of tryptic peptides of kynurenine-mo dified crystallins revealed that all of the coloured peptides contained eit her His, Cys or an internal Lys residue. We propose a novel mechanism of ky nurenine-mediated crystallin modification which does not require UV light o r oxidative conditions as catalysts. Rather, we suggest that the side chain of kynurenine-derived lens UV filters becomes deaminated to yield an alpha ,beta-unsaturated carbonyl which is highly susceptible to attack by nucleop hilic amino acid residues of the crystallins. The inability of the lens fib re cells to metabolise their constituent proteins results in the accumulati on of coloured/fluorescent crystallins with age. (C) 2000 Elsevier Science B.V. All rights reserved.