3-hydroxykynurenine in the human lens

The human lens contains three kynurenine UV filters, 3- hydroxykynurenine O-β-Dglucoside (3OHKynG), kynurenine (Kyn) and 3-hydroxykynurenine (3OHKyn), and it absorbs UV light in the 300-400 nm region due to their presence. UV filters may also prevent UV-induced photodamage to the retina and lens. After middle age, the UV filters, 3OHKynG and Kyn become bound progressively to proteins in the centre of our lenses. This feature is, in part, responsible for normal age-dependant human lens colouration. To provide proof that 3OHKyn is bound to normal human lenses, model studies were undertaken. Cysteine (Cys), histidine (His) and lysine (Lys) residues in lens proteins had been previously shown to bind to UV filters in vivo, therefore adducts of these amino acids and 3OHKyn were synthesised and characterised by mass spectrometry, fluorescence, UV-visible and NMR spectroscopy in Chapter 2. The stability properties of each of the 3OHKyn amino acid adducts were also determined, with incubations performed at pH 4.0 and pH 7.2. 3OHKyn-t-Boc-His was identified as the most stable of the three adducts. 3OHKyn-t-Boc-Lys and 3OHKyn-Cys both decomposed at pH 7.2 forming numerous oxidation products. The stability of each adduct to acid hydrolysis was also examined. In Chapter 3, calf lens protein was incubated with 3OHKyn, and acid hydrolysis showed that Cys was the primary site of modification when the incubation was undertaken at pH 7.2. However, when the incubation was undertaken at a higher pH (for example, pH 9.5), 3OHKyn readily modified Cys, His and Lys residues. Previously acid hydrolysis of human lens protein had identified Kyn attachment to the proteins. However, acid hydrolysis was not an appropriate method for detecting 3OHKyn attached to human lens proteins because 3OHKynG is also bound to human lens proteins. Therefore, a new assay was developed, and it was found that 3OHKyn does indeed bind to human lens proteins in an age-dependant manner. The assay also provides data for 3OHKynG and Kyn attachment to human lens proteins. In Chapter 3, α-crystallin was also incubated with 3OHKyn under low oxygen tension, and the findings from this study showed that 3OHKyn modified the Cys residue in αAcrystallin. In addition, oxidation of methionine and tryptophan was observed. Agerelated nuclear cataract is associated with colouration, insolubilisation and extensive oxidation of Cys and methionine residues. It appears that 3OHKyn in the lens may promote the oxidation and modifications of proteins, and may contribute to oxidative stress in the human lens. In Chapter 4, the aim was to examine if 3OHKyn could act as a crosslinker of cataract lens proteins. 3OHKyn is known to readily oxidise and yield highly reactive species. It was therefore proposed that 3OHKyn bound to lens proteins could promote crosslinking, insolubilisation and colouration of lens proteins following formation of oxidised species. 3OHKyn amino acid adducts were incubated with excess amino acids, and the resulting products examined. These compounds may be analogous to those that would form in a cataract lens. In addition, 3OHKyn-modified protein was incubated and the products were examined by SDS-PAGE, fluorescence spectroscopy and mass spectrometry. Results showed that 3OHKyn, under the conditions used, does not crosslink lens protein. Proof of the hypothesis that 3OHKyn crosslinks proteins in the lens requires the isolation of characteristic chemical markers from cataract lens proteins that contain the modified 3OHKyn molecules. In Chapter 5, the aim was to isolate novel compounds from the hydrolysates of human cataract lens proteins and to determine their chemical properties. Overall this thesis provides evidence that 3OHKyn plays a role in the post-translational modification of normal human lens proteins, and it also provides preliminary data on the role of 3OHKyn in human cataract

Protein-bound and free UV filters in cataract lenses. The concentration of UV filters is much lower than in normal lenses

In human cataract lenses the UV filters, 3-hydroxykynurenine glucoside (3OHKG) and kynurenine (Kyn) were found to be covalently bound to proteins and the levels in the nucleus were much higher than in the cortex. The levels of the bound UV filters in cataract nuclei were much lower than those in age-matched normal lenses. 3-Hydroxykynurenine could not be detected in cataract lenses. As with normal lenses, protein-bound 3OHKG in cataract lenses was found at the highest levels followed by Kyn. Free UV filter concentrations were also markedly reduced in cataract lenses. This feature may well contribute to the lower protein-bound levels; however, there was no clear relationship between free and bound UV filter contents when individual lenses were examined. We propose that since cysteine is a major site for UV filter binding, the well-documented oxidation of protein sulfhydryl groups during the progression of nuclear cataract may account, in part, for the pronounced decrease in bound UV filters in cataract lenses

Connexin 30 expression and frequency of connexin heterogeneity in astrocyte gap junction plaques increase with age in the rat retina.

We investigated age-associated changes in retinal astrocyte connexins (Cx) by assaying Cx numbers, plaque sizes, protein expression levels and heterogeneity of gap junctions utilizing six-marker immunohistochemistry (IHC). We compared Wistar rat retinal wholemounts in animals aged 3 (young adult), 9 (middle-aged) and 22 months (aged). We determined that retinal astrocytes have gap junctions composed of Cx26, -30, -43 and -45. Cx30 was consistently elevated at 22 months compared to younger ages both when associated with parenchymal astrocytes and vascular-associated astrocytes. Not only was the absolute number of Cx30 plaques significantly higher (P<0.05) but the size of the plaques was significantly larger at 22 months compared to younger ages (p<0.05). With age, Cx26 increased significantly initially, but returned to basal levels; whereas Cx43 expression remained low and stable with age. Evidence that astrocytes alter connexin compositions of gap junctions was demonstrated by the significant increase in the number of Cx26/Cx45 gap junctions with age. We also found gap junctions comprised of 1, 2, 3 or 4 Cx proteins suggesting that retinal astrocytes use various connexin protein combinations in their gap junctions during development and aging. These data provides new insight into the dynamic and extensive Cx network utilized by retinal astrocytes for communication within both the parenchyma and vasculature for the maintenance of normal retinal physiology with age. This characterisation of the changes in astrocytic gap junctional communication with age in the CNS is crucial to the understanding of physiological aging and age-related neurodegenerative diseases

Protein-Bound UV Filters in Normal Human Lenses: The Concentration of Bound UV Filters Equals That of Free UV Filters in the Center of Older Lenses

PURPOSE. To survey the levels of protein-bound UV filters in the cortices and nuclei of normal human lenses as a function of age and to relate this to the concentration of free UV filters. METHODS. Levels of each of the three kynurenine (Kyn) UV filters, 3-hydroxykynurenine glucoside (3OHKG), Kyn, and 3-hydroxykynurenine (3OHKyn), covalently attached to proteins, were determined by using a newly developed method of reductive capture, after base treatment of the intact lens proteins. RESULTS. The data show that, in the normal lens, each of the three UV filters became bound to proteins to a significant extent only after age 50 and, further, that the levels in the nucleus were much higher than in the cortex. These findings are consistent with the lens barrier that forms in middle age. 3OHKG was present at the highest levels followed by Kyn, with 3OHKyn being attached in the lowest amount. The ratio was 145:4:1 (3OHKG-Kyn-3OHKyn), with a total proteinbound UV filter concentration in the lens nucleus after age 50 of approximately 1300 picomoles/mg protein. This ratio is in agreement with 3OHKG being the most abundant free UV filter in the human lens and 3OHKyn being present in the lowest concentration with free Kyn present in intermediate amounts. CONCLUSIONS. The three Kyn UV filters are bound to the nuclear proteins of all normal lenses over the age of 50. Indeed in the center of older normal lenses, the concentration of UV filters bound to proteins is approximately equal to that of the free filters. Since bound UV filters promote oxidation of proteins after exposure to wavelengths of light that penetrate the cornea, lenses in middle-aged and older individuals may be more prone to photooxidation than those of young people. (Invest Ophthalmol Vis Sci. 2007;48:1718 -1723 DOI: 10.1167DOI: 10. /iovs.06-1134 T he primate lens is unique among both other primate tissues and the lenses of other species, in that it synthesizes 3-hydroxykynurenine glucoside (3OHKG) as a UV filter from the amino acid tryptophan (Trp). 1 The immediate precursors kynurenine (Kyn) and 3-hydroxykynurenine (3OHKyn) are metabolites found in all organs of the body. All three of these Trp metabolites are unstable at physiological pH and undergo side chain deamination to yield ␣,␤-unsaturated ketones that are prone to nucleophilic attack. 2 In tissues that contain sufficient glutathione (GSH), it is likely that GSH will react with the deamination products before they bind to proteins 3 and that the adducts thus formed will diffuse out of the lens. In the interior of the lens, once the barrier to diffusion forms at middle age, 4 -6 the nucleus becomes a partially uncoupled region in which metabolites spend a longer time than in the young lens. This factor leads to an environment in the older lens nucleus that favors greater decomposition of intrinsically unstable molecules. Coupled with a diminished flux of GSH from the cortex, this results in increased covalent binding of UV filters to nuclear proteins after middle age. In this study, we set out to examine the levels of all three bound UV filters in normal lenses using a novel assay system in which lens proteins were incubated with excess GSH at pH 9.5. 9 Under these conditions, the UV filters that are attached to proteins are released, and the GSH adducts thus formed can be quantified by HPLC. UV filters were found to be covalently bound to proteins from all lenses older than 50 years. Such posttranslational modifications may have important consequences in terms of the susceptibility of the proteins to photooxidation and, in the case of 3OHKyn, the sensitivity to an oxidative environment such as that in the nuclei of lenses with age-related nuclear cataract. METHODS Purified water (purified to 18.2 M⍀/cm 2 ; Milli-Q; Millipore, Bedford, MA) was used in the preparation of all solutions. All organic solvents were HPLC grade (Ajax, Auburn, NSW, Australia). 3OHKyn, reduced GSH, trifluoroacetic acid (TFA), and guanidine HCl were obtained from Sigma-Aldrich (St. Louis, MO). Reversed-Phase HPLC Reversed-phase (RP)-HPLC was performed on a Shimadzu system (Kyoto, Japan). For analytical scale separations, a column (Jupiter, 5 m, C18, 300 Å, 250 ϫ 4.6 mm; Phenomenex, Torrance, CA) was used with the following mobile phase conditions: solvent A (aqueous 0.1% vol/vol TFA) for 5 minutes followed by a linear gradient of 0% to 50% solvent B (80% vol/vol acetonitrile/H 2 O, 0.1% vol/vol TFA) over 20 minutes, followed by a linear gradient of 50% to 100% B over 15 minutes and re-equilibration in the aqueous phase for 15 minutes. The flow rate was 0.5 mL/min and with detection at 360 nm. From th

Protein aging: Truncation of aquaporin 0 in human lens regions is a continuous age-dependent process

The human lens is ideal for the study of macromolecular aging because cells in the centre, along with their constituent proteins, are present for our entire lives. We examined the major membrane protein, aquaporin 0 (AQP0), in regions of the lens formed at different times during our lifespan, to determine if similar changes could be detected and if they were progressive. Membrane fractions from three concentric lens regions were examined by SDS-PAGE coupled with densitometry, and Western blotting, to assess the time course of truncation. The overall extent of modification was also examined by MALDI mass spectrometry of the undigested proteins. In all regions, AQP0 became progressively more truncated, specifically by the loss of a 2 kDa intracellular C-terminal peptide. The proteolysis increased steadily in all regions such that half of the AQP0 in the barrier region (that part of the lens formed immediately after birth) had been cleaved by age 40–50. MALDI mass spectrometry revealed that in all regions, AQP0 not only was shortened, it also became progressively more heterogeneous with age. Since the lens interior is devoid of active enzymes, it is very likely that the cleavage of AQP0 is chemically induced. We speculate that the loss of this C-terminal peptide ‘spacer’ may allow occlusion of AQP0 pores on the cytoplasmic face of the fibre cell membranes. Once a significant proportion of AQP0 has been cleaved, this occlusion may contribute to the formation of the lens permeability barrier that develops at middle age

Reversible binding of kynurenine to lens proteins : potential protection by gluthathione in young lenses

PURPOSE. Human ultraviolet light (UV) filters, such as kynurenine (Kyn), readily deaminate to reactive unsaturated ketones that covalently modify proteins in older human lenses. The aim of this study was to examine in vitro rates of formation and decomposition of the three major Kyn-amino acid adducts and possible consequences for the lens. METHODS. The t-Boc–protected Kyn-His, Kyn-Lys, and Kyn-Cys adducts and Kyn-Cys were synthesized from the corresponding amino acids and Kyn. Calf lens proteins were modified with Kyn by incubation at pH 7. Stability and competition studies of the adducts were conducted under physiological conditions. Kyn-amino acids and their decomposition products were quantified using HPLC. RESULTS. At physiological pH, Kyn-Cys adducts formed more rapidly than either Lys or His adducts, but they also decomposed readily. By contrast, His adducts were stable. Cysteine (Cys) residues in ß-crystallins were major sites of modification. The Kyn moiety, initially bound to Cys residues, was found to transfer to other amino acids. Glutathione promoted the breakdown of Kyn-Cys. CONCLUSIONS. These data may help explain why proteins in young lenses are not modified by UV filters in situ. The initial phase of the modification of proteins in the human lens by UV filters may be a dynamic process. In lenses, Cys residues of crystallins modify preferentially, but these adducts also decompose to release deaminated Kyn. This can then potentially react with other amino acids. Glutathione, which is present in high concentrations in the lenses of young people, may play a vital role in keeping proteins free from modification by intercepting reactive deaminated kynurenines formed by the spontaneous breakdown of free UV filters, promoting the decomposition of Kyn-Cys residues, and sequestering the unsaturated ketones once they are released from modified proteins.9 page(s

Tight binding of proteins to membranes from older human cells

The lens is an ideal model system for the study of macromolecular aging and its consequences for cellular function, since there is no turnover of lens fibre cells. To examine biochemical processes that take place in the lens and that may also occur in other long-lived cells, membranes were isolated from defined regions of human lenses that are synthesised at different times during life, and assayed for the presence of tightly bound cytosolic proteins using quantitative iTRAQ proteomics technology. A majority of lens beta crystallins and all gamma crystallins became increasingly membrane bound with age, however, the chaperone proteins alpha A and alpha B crystallin, as well as the thermally-stable protein, βB2 crystallin, did not. Other proteins such as brain-associated signal protein 1 and paralemmin 1 became less tightly bound in the older regions of the lens. It is evident that protein–membrane interactions change significantly with age. Selected proteins that were formerly cytosolic become increasingly tightly bound to cell membranes with age and are not removed even by treatment with 7 M urea. It is likely that such processes reflect polypeptide denaturation over time and the untoward binding of proteins to membranes may alter membrane properties and contribute to impairment of communication between older cells