01whole.pdf (12.31 MB)
Human UV filters, UV light and oxidative damage in age related nuclear cataract: a thesis submitted in fulfilment of the requirements for the award of the degree of Doctor of Philosophy from Macquarie University
thesisposted on 2022-03-28, 16:54 authored by Nicholas Adam Gad
Purpose: The lenses of humans contain a group of small protective molecules that are known as UV filters. The main UV filters present in human lenses are3-hydroxykynurenine O-β-D-glucoside (3OHKG), kynurenine (Kyn),3-hydroxykynurenine (3OHK) and 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid-O-β-D-glucoside (AHBG). These UV filters protect the lens from UV damage and it is believed that there may be other novel related species present in the lens that act analogously. The UV filters are also implicated in the progressive modification of lens proteins that occur with aging, and the development of age related nuclear cataract in older humans. The first aim of this thesis was to detect, quantify and elucidate the structures of novel 3-hydroxykynurenine glucoside-derived UV filter metabolites present in the human lens. The second aim of this thesis was to investigate bovine lens proteins modified through covalent binding with UV filters and determine if these modified UV filter metabolites could be photosensitisers of oxidative damage. The final aim was to describe the detection and identification of amino acid residues involved in the covalent binding of human lens UV filters and metabolites to bovine lens proteins. Methods: Three novel UV filter compounds 4-(2-amino-3-hydroxyphenyl)-2-hydroxy-4-oxobutanoic acid O-β-D-glucoside (3OHKG-W), 3-hydroxykynurenine O-β-Dglucosideyellow (3OHKG-Y) and 2-amino-3-hydroxyacetophenone O-β-D-glucoside (AHAG), along with Kyn, 3OHK, AHBG and 3OHKG, were detected and quantified by LC-MS/MS in 24 human lenses of different ages, of which 22 were normal and two had age related nuclear cataract. The structures of these compounds were confirmed through total synthesis and spectral analysis. The synthesised analogues (3OHKG-Y, 3OHKG-W, AHAG), together with a previously identified analogue (3OHKG-D) and the major UV filters (3OHKG, Kyn,3OHK), were incubated with bovine lens proteins (BLP) at pH 7.4, to mimic physiological conditions, and at pH 9.5 (which accelerates deamination and encourages binding to protein residues). The modified lens proteins were exposed to UV light under aerobic and anaerobic conditions and in the presence and absence of free UV filters for varying periods of time. The UV light irradiated BLP were then separated through SDS-PAGE to observe protein binding, aggregation and any other observable changes. Finally, the amino acid adducts produced from this incubation were identified by spectral analysis and identities confirmed by total synthesis. Their concentrations in the modified proteins were then quantified following enzymatic hydrolysis of the modified lens proteins with Pronase. Results: As reported in previous studies, 3OHKG concentrations decreased with age in the lens nuclei, whereas the levels of three novel species, 3OHKG-Y, 3OHKG-W and AHAG increased, though to different extents. The deaminated metabolite of 3OHKG (3OHKG-D) was also detected, though in very low concentrations, and only in some of the lenses. In contrast, the concentrations of all the UV filters measured present in the cortex of the lens remained constant with age. Upon irradiation of the modified BLP, different changes were observed between bound UV filters having both an o-aminophenol and α,β-unsaturatedcarbonyl moieties (3OHK) and those with only an α,β-unsaturated carbonyl moiety (3OHKG, Kyn, 3OHKG-D). Metabolites with only an o-aminophenol (AHB) or without an α,β-unsaturated carbonyl moiety (3OHKG-Y, 3OHKG-W or AHAG) no did not experience any visible changes. Evidence of cross-linking and nondisulphide binding was observed in the BLP modified with Kyn, 3OHK, 3OHKG and 3OHKG-D. Irradiation (of at least 120 min) resulted in a significant loss of soluble protein, suggesting aggregation of modified BLP, with the greatest extent of aggregation detected with samples modified at pH 9.5. There was evidence of UV filter binding to BLP under both physiologica land basic conditions. The major lens UV filter, 3OHKG, was found to react with lens proteins at cysteine and histidine residues, with the greatest level of binding on Cys at pH 9.5. Low levels of binding were detected at the Lys residues at pH9.5. These were also observed at pH 7.4, however, only at close to the detection limit. Comparative studies with Kyn and 3OHK similarly resulted in lens proteins modified at Cys residues. The extent of modification was found to be significantly higher at pH 9.5 in all cases. LC-MS/MS analysis of tryptic digests showed modifications at both α- and β-, but not γ-crystallins. By contrast, UV filter compounds that cannot form α,β-unsaturated carbonyl compounds (i.e. cannot deaminate) did not produce covalent modification of lens proteins via side-chain interaction. 3OHKG, Kyn and 3OHK modified lens proteins were found to be coloured and fluorescent, resembling those from aged and age related nuclear cataractous lenses. There was also strong evidence that the structure of the protein in itself inhibits binding to internally (protected) Cys residues. Conclusion: Three novel 3OHKG derived metabolites have been detected in extracts from normal and cataractous human lenses. In the subsequent binding studies of these metabolites (and other UV filter compounds), it was confirmed that binding can only occur between BLP and a UV filter with α,β-unsaturated carbonyl moieties.Covalent binding is a prerequisite to photo-oxidative damage occurring upon irradiation with UV light. No evidence of binding or protein damage was observed in samples treated with AHB, 3OHKG-Y, 3OHKG-W or AHAG upon UV irradiation compared to irradiated untreated BLP. Even though AHB could covalently bind to proteins via auto-oxidation of the o-aminophenol, this was not observed in this study even though AHB displayed evidence of non-covalent binding (at pH 9.5only). Protein modification/treatment was also examined with the UV filter metabolites 3OHKG-W and AHAG, however no modified amino acid residues were observed. There was a very low level of modification with 3OHKG-Y, of similar proportions in both pHs, suggesting some level of spontaneous binding. This generally suggests that, other than 3OHKG-D, 3OHKG derived metabolites are relatively stable and unlikely to react with lens proteins.