Doctor of Philosophy
Department of Chemistry
Aquilina, J. Andrew, Reaction of 3-Hydroxykynurenine with lens proteins and synthetic peptides, Doctor of Philosophy thesis, Department of Chemistry, University of Wollongong, 2000. https://ro.uow.edu.au/theses/1169
3-Hydroxykynurenine (30HKyn), present in the human lens as a UV-filter, is a powerful antioxidant which has also been implicated as a carcinogen and neurotoxin. The neurotoxicity results from the oxidation of30HKyn due to the formation of H202 and possibly hydroxyl radicals. Oxidation of o-aminophenols, such as 30HKyn, also results in the formation of highly reactive quinonimines. Thus one possible consequence of 30HKyn oxidation may be the covalent modification of cellular macromolecules. Such a process could contribute to the neurotoxicity and may potentially be important in other tissues, such as the human lens, where 3-hydroxykynurenine functions as a UV filter.
In this study the roles of UV-light, pH, glutathione and oxygen were examined, with the objective of determining how these factors may possibly affect the binding of 30HKyn to lens proteins (crystallins) under the conditions found within the lens itself. The presence of oxygen was found to be an important parameter for determining the extent to which 30HKyn reacts with protein, and when it was totally excluded, little modification was observed. UV-light was not required for activation, but was found to augment the extent of modification and crosslinking, while an elevated pH, which is known to accelerate the rate of 30HKyn oxidation, did not markedly increase the extent of reaction with the crystallins. 30HKyn binding was accompanied by crystallin aggregation, pigmentation, and development of non-tryptophan fluorescence, all of which have been associated with cataract formation.
The inclusion of glutathione, a ubiquitous antioxidant, in reaction mixtures resulted in a delayed onset of crystallin modification. This effect was apparent at concentrations of glutathione greater than 1 mM. When glutathione levels fell below 1 mM, crystallins became modified by 30HKyn. Since lens glutathione concentrations decrease with age, and are known to be lower in the lens nucleus than the cortex, this region appears particularly vulnerable to modification by this UV filter.
Using the model dipeptides glycyllysine (GK) and glycylglycine (GG), it was demonstrated that 3-hydroxykynurenine can bind to protein α-amino groups, and further, that under oxidative conditions, 3-hydroxykynurenine can function to cross-link polypeptide chains. The structures of a cross-linked moiety using the peptide GK and a covalently modified GG have been elucidated. The modified species were both coloured and fluorescent, and involved cyclisation of the N-terminal glycine to form a heteroaromatic ring at the C-2 and C-3 positions of 30HKyn. GK was cross-linked through a benzoxazole moiety whereas GG preferably formed a benzimidazole linked compound. The compounds were named quinilinobenzoxamine (QBA) and 2- diglycylimidazolekynurenine.
The γ-crystallin family of lens proteins possess unblocked N-termini, the terminal residues of which are GK. In a reaction mixture of γ-crystallin and 30HKyn a dimerised γ-crystallin species was observed. LC-MS of a tryptic digest of this product failed to reveal a digest fragment corresponding to QBA. A synthetic peptide corresponding to the nine Nterminal residues of γ-crystallin was found to be cross-linked in the manner of QBA. Tryptic digestion of this product revealed a major product corresponding to a chymotryptic activity which was also present in the γ-crystallin / 30HKyn dimer. As QBA could not be identified in the digests, it was proposed that trypsin may destabilise the benzoxazole moiety, resulting in a structurally altered product, the characteristics of which are not known.
Finally, the major reaction product of the tetrapeptide tuftsin (TKPR) and 30HKyn was examined. This peptide which has threonine as the N-terminal residue, was able to form a dimer via the same mechanism as QBA. This result suggests that 30HKyn can react with any peptide that has a free N-terminus, regardless of the identity of the amino acid (except proline). This finding has identified a host of potentially modified species in the reaction of 30HKyn with the free N-terminal residues of proteins. In the lens where 30HKyn is known to be continually synthesised, this may provide valuable biomarkers in disease states such as senile nuclear cataract.