Human lens coloration and aging. Evidence for crystallin modification by the major ultraviolet filter, 3-hydroxy-kynurenine O-beta-D-glucoside.

The human lens becomes increasingly yellow with age and thereby reduces our perception of blue light. This coloration is associated with lens proteins (crystallins), but its molecular basis was unknown. Here we show that the coloration occurs because of the interaction of crystallins with a UV filter compound, 3-hydroxykynurenine glucoside (3-OHKG). Crystallin modification results from deamination of the 3-OHKG amino acid side chain, yielding an unsaturated ketone that is susceptible to nucleophilic attack by cysteine, histidine, and lysine residues. This novel protein modification contributes to age-related lens coloration and may play a role in human nuclear cataractogenesis.     

Novel protein modification by kynurenine in human lenses.

It is known that human lenses increase in color and fluorescence with age, but the molecular basis for this is not well understood. We demonstrate here that proteins isolated from human lenses contain significant levels of the UV filter kynurenine covalently bound to histidine and lysine residues. Identification was confirmed by synthesis of the kynurenine amino acid adducts and comparison of the chromatographic retention times and mass spectra of these authentic standards with those of corresponding adducts isolated from human lenses following acid hydrolysis. Using calf lens proteins as a model, covalent binding of kynurenine to lens proteins has been shown to proceed via side chain deamination in a manner analogous to that observed for the related UV filter, 3-hydroxykynurenine O-beta-D-glucoside. Levels of histidylkynurenine and lysylkynurenine were low in human lenses in subjects younger than 30, but thereafter increased in concentration with the age of the individual. Post-translational modification of lens proteins by tryptophan metabolites therefore appears to be responsible, at least in part, for the age-dependent increase in coloration and fluorescence of the human lens, and this process may also be important in other tissues in which up-regulation of tryptophan catabolism occurs.     

Age‐related cleavages of crystallins in human lens cortical fiber cells generate a plethora of endogenous peptides and high molecular weight complexes

Low molecular weight peptides derived from the breakdown of crystallins have been reported in adult human lenses. The proliferation of these LMW peptides coincides with the earliest stages of cataract formation, suggesting that the protein cleavages involved may contribute to the aggregation and insolubilization of crystallins. This study reports the identification of 238 endogenous LMW crystallin peptides from the cortical extracts of four human lenses representing young, middle and old‐age human lenses. Analysis of the peptide terminal amino acids showed that Lys and Arg were situated at the C‐terminus with significantly higher frequency compared to other residues, suggesting that trypsin‐like proteolysis may be active in the lens cortical fiber cells. Selected reaction monitoring analysis of an endogenous αA‐crystallin peptide (αA_57‐65) showed that the concentration of this peptide in the human lens increased gradually to middle age, after which the rate of αA_57‐65 formation escalated significantly. Using 2D gel electrophoresis/nanoLC‐ESI‐MS/MS, 12 protein complexes of 40–150 kDa consisting of multiple crystallin components were characterized from the water soluble cortical extracts of an adult human lens. The detection of these protein complexes suggested the possibility of crystallin cross‐linking, with these complexes potentially acting to stabilize degraded crystallins by sequestration into water soluble complexes. Proteins 2015; 83:1878–1886. © 2015 Wiley Periodicals, Inc.     

Modifications of human betaA1/betaA3-crystallins include S-methylation, glutathiolation, and truncation

Disulfide bonding of lens crystallins contributes to the aggregation and insolubilization of these proteins that leads to cataract. A high concentration of reduced glutathione is believed to be key in preventing oxidation of crystallin sulfhydryls to form disulfide bonds. This protective role is decreased in aged lenses because of lower glutathione levels, especially in the nucleus. We recently found that human gamma-crystallins undergo S-methylation at exposed cysteine residues, a reaction that may prevent disulfide bonding. We report here that betaA1/A3-crystallins are also methylated at specific cysteine residues and are the most heavily methylated of the human lens crystallins. Among the methylated sites, Cys 64, Cys 99, and Cys 167 of betaA1-crystallin, methylation at Cys 99 is highest. Cys 64 and Cys 99 are also glutathiolated, even in a newborn lens. These post-translational modifications of the exposed cysteines may be important for maintaining the crystallin structure required for lens transparency. Previously unreported N-terminal truncations were also found.     

Formation mechanism of cross‐linking Maillard compounds in peptide–xylose systems

The formation mechanism of Maillard peptides was explored in Maillard reaction through diglycine/glutathione(GSH)/(Cys‐Glu‐Lys‐His‐Ile‐Met)–xlyose systems by heating at 120 °C for 30–120 min. Maximum fluorescence intensity of Maillard reaction products (MRPs) with an emission wavelength of 420~430 nm in all systems was observed, and the intensity values were proportional to the heating time. Taken diglycine/GSH–[¹³C₅]xylose systems as a control, it was proposed that the compounds with high m/z values of 379 and 616 have the high molecular weight (HMW) products formed by cross‐linking of peptides and sugar. In (Cys‐Glu‐Lys‐His‐Ile‐Met)–xylose system, the m/z value of HMW MRPs was not observed, which might be due to the weak signals of these products. According to the results of gel permeation chromatography, HMW MRPs were formed by Maillard reaction, especially in (Cys‐Glu‐Lys‐His‐Ile‐Met)–xylose system, the percentage of Maillard peptides reached 52.90%. It was concluded that Maillard peptides can be prepared through the cross‐linking of sugar and small peptides with a certain MW range. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Human protein aging: modification and crosslinking through dehydroalanine and dehydrobutyrine intermediates

Nonenzymatic post‐translational modification (PTM) of proteins is a fundamental molecular process of aging. The combination of various modifications and their accumulation with age not only affects function, but leads to crosslinking and protein aggregation. In this study, aged human lens proteins were examined using HPLC–tandem mass spectrometry and a blind PTM search strategy. Multiple thioether modifications of Ser and Thr residues by glutathione (GSH) and its metabolites were unambiguously identified. Thirty‐four of 36 sites identified on 15 proteins were found on known phosphorylation sites, supporting a mechanism involving dehydroalanine (DHA) and dehydrobutyrine (DHB) formation through β‐elimination of phosphoric acid from phosphoserine and phosphothreonine with subsequent nucleophilic attack by GSH. In vitro incubations of phosphopeptides demonstrated that this process can occur spontaneously under physiological conditions. Evidence that this mechanism can also lead to protein–protein crosslinks within cells is provided where five crosslinked peptides were detected in a human cataractous lens. Nondisulfide crosslinks were identified for the first time in lens tissue between βB2‐ & βB2‐, βA4‐ & βA3‐, γS‐ & βB1‐, and βA4‐ & βA4‐crystallins and provide detailed structural information on in vivo crystallin complexes. These data suggest that phosphoserine and phosphothreonine residues represent susceptible sites for spontaneous breakdown in long‐lived proteins and that DHA‐ and DHB‐mediated protein crosslinking may be the source of the long‐sought after nondisulfide protein aggregates believed to scatter light in cataractous lenses. Furthermore, this mechanism may be a common aging process that occurs in long‐lived proteins of other tissues leading to protein aggregation diseases.     

Tryptophan-derived ultraviolet filter compounds covalently bound to lens proteins are photosensitizers of oxidative damage

The human eye is chronically exposed to light of wavelengths >300 nm. In the young human lens, light of wavelength 300-400 nm is predominantly absorbed by the free Trp derivatives kynurenine (Kyn), 3-hydroxykynurenine (3OHKyn), and 3-hydroxykynurenine-O-beta-D-glucoside (3OHKynG). These ultraviolet (UV) filter compounds are poor photosensitizers. With age, the levels of the free UV filters in the lens decreases and those of protein-bound UV filters increases. The photochemical behavior of these protein-bound UV filters and their role in UV damage are poorly elucidated and are examined here. UVA illumination of protein-bound UV filters generated peroxides (principally H2O2) in a metabolite-, photolysis-time-, and wavelength-dependent manner. Unmodified proteins, free Trp metabolites, and Trp metabolites that do not bind to lens proteins gave low peroxide yields. Protein-bound 3OHKyn (principally at Cys residues) yielded more peroxide than comparable Kyn and 3OHKynG adducts. Studies using D2O and sodium azide implicated 1O2 as a key intermediate. Illumination of the protein-bound adducts also yielded protein-bound Tyr oxidation products (DOPA, di-tyrosine) and protein cross-links via alternative mechanisms. These data indicate that the covalent modification of lens proteins by Kyn derivatives yields photosensitizers that may enhance oxidation in older lenses and contribute to age-related nuclear cataract.     

Novel cyclization chemistry especially suited for biologically derived, unprotected peptides.

A novel method is described for the cyclization of peptides--or segments of polypeptides--which requires a free N-terminal alpha-amino group and a distal amino acid residue containing a nucleophilic side chain. The reaction is conducted in two steps, both in the aqueous phase. The first step involves acylation of the N-terminal alpha-amino group with iodoacetic anhydride at pH 6. This acylation reaction has greater than 90% specificity for peptide alpha-amino groups and gives no alkylation of Arg, His, Lys or Met by the iodoacetate side product (R. Wetzel et al., Bioconjugate Chem., 1, 114-122, 1990). In the second step, the acylation reaction mixture or the isolated iodoacetyl-peptide is incubated at room temperature to give the cyclic peptide formed by reaction of the nucleophilic side chain with the iodoacetyl moiety. The pH dependence of the cyclization reaction by Met, Lys, Arg or His is consistent with the pKa of the nucleophilic side chain. Thus, peptides containing Met plus other nucleophilic amino acids should preferentially cyclize via Met at low pH. In this paper, preparation of cyclic peptides containing 3-6 amino acids is described; the full range of ring sizes and sequences which can undergo this cyclization has not been further explored. Preliminary results suggest that this method is also fairly general with respect to the amino acid sequence being cyclized. The reaction appears to be particularly suited for cyclization via Lys and Met side chains. All of the cyclized products are sufficiently stable for many biological applications.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polypeptide modification and cross-linking by oxidized 3-hydroxykynurenine

3-Hydroxykynurenine (3OHKyn) is present in the mammalian lens as a UV filter and is formed from kynurenine in the tryptophan metabolic pathway. 3OHKyn is a readily autoxidized o-aminophenol which binds to proteins in vitro. The lens, particularly its central region, the nucleus, becomes increasingly oxidized with age. Under such conditions, the oxidation products of 3OHKyn may bind to lens proteins and contribute to nuclear cataract formation. The purpose of this study was to determine the structures of in vitro reaction products of 3OHKyn with model peptides as a general model for 3OHKyn modification of proteins. 3OHKyn was incubated with the dipeptide glycylglycine (GG) and the tetrapeptide tuftsin (sequence TKPR) under oxidizing conditions, and the reaction products were characterized by a variety of spectroscopic techniques. The major 3OHKyn-GG reaction product involves formation of a benzimidazole moiety between the GG N-terminus and the oxidized amino and/or phenol groups of 3OHKyn. In contrast, tuftsin, which has an N-terminal threonine, forms predominantly a cross-linked dimer with oxidized 3OHKyn. This product is analogous in structure to the dimeric reaction product, quinilinobenzoxamine, formed between oxidized 3OHKyn and glycyllysine [Aquilina, J. A., et al. (1999) Biochemistry 38, 11455-11464], which contains a benzoxazole moiety. The identification of a tuftsin dimer suggests that 3OHKyn can react with any peptide having a free alpha-amino group, via a general side chain elimination mechanism. The identification of both benzimidazole and benzoxazole adducts in peptides with a free N-terminus suggests that peptide amino groups can react initially at either the aromatic amino or hydroxyl group of oxidized 3OHKyn. The proportion of each adduct may change, however, depending on the amino acid sequence at the N-terminus.     

Protein oxidation and loss of protease activity may lead to cataract formation in the aged lens

Over 95% of the dry mass of the eye lens consists of specialized proteins called crystallins. Aged lenses are subject to cataract formation, in which damage, cross-linking, and precipitation of crystallins contribute to a loss of lens clarity. Cataract is one of the major causes of blindness, and it is estimated that over 50,000,000 people suffer from this disability. Damage to lens crystallins appears to be largely attributable to the effects of UV radiation and/or various active oxygen species (oxygen radicals, ¹O₂, H₂O₂, etc.). Photooxidative damage to lens crystallins is normally retarded by a series of antioxidant enzymes and compounds. Crystallins which experience mild oxidative damage are rapidly degraded by a system of lenticular proteases. However, extensive oxidation and cross-linking severely decrease proteolytic susceptibility of lens crystallins. Thus, in the young lens the combination of antioxidants and proteases serves to prevent crystallin damage and precipitation in cataract formation. The aged lens, however, exhibits diminished antioxidant capacity and decreased proteolytic capabilities. The loss of proteolytic activity may actually be partially attributable to oxidative damage which proteases (like any other protein)_can sustain. We propose that the rate of crystallin damage increases as antioxidant capacity declines with age. The lower protease activity of aged lens cells may be insufficient to cope with such rates of crystallin damage, and denatured crystallins may begin to accumulate. As the concentration of oxidatively denatured crystallins rises, cross-linking reactions may produce insoluble aggregates which are refractive to protease digestion. Such a scheme could explain many events which are known to contribute to cataract formation, as well as several which have appeared to be unrelated. This hypothesis is also open to experimental verification and intervention.     

Cysteine is the initial site of modification of alpha-crystallin by kynurenine

Tryptophan metabolites, such as kynurenine, are spontaneously unstable at neutral pH. They undergo side-chain deamination yielding reactive alpha, beta unsaturated ketones. In the lens, where these compounds act as UV filters, reaction of the breakdown products with lens proteins (crystallins) may be largely responsible for age-dependent colouration of this tissue. In previous research, where high pH (pH 9) was used to promote deamination and conjugation with lens protein, histidine, lysine, and cysteine residues were found to be modified. In this study we show that, at pH 7, site of reaction with the major lens chaperone alpha-crystallin, is the single cysteine residue of the alphaA subunit. This apparent selectivity has important ramifications because the cysteine-kynurenine adduct is itself unstable under physiological conditions.     

Aggregation of Trp > Glu point mutants of human gamma‐D crystallin provides a model for hereditary or UV‐induced cataract

Numerous mutations and covalent modifications of the highly abundant, long‐lived crystallins of the eye lens cause their aggregation leading to progressive opacification of the lens, cataract. The nature and biochemical mechanisms of the aggregation process are poorly understood, as neither amyloid nor native‐state polymers are commonly found in opaque lenses. The βγ‐crystallin fold contains four highly conserved buried tryptophans, which can be oxidized to more hydrophilic products, such as kynurenine, upon UV‐B irradiation. We mimicked this class of oxidative damage using Trp→Glu point mutants of human γD‐crystallin. Such substitutions may represent a model of UV‐induced photodamage—introduction of a charged group into the hydrophobic core generating “denaturation from within.” The effects of Trp→Glu substitutions were highly position dependent. While each was destabilizing, only the two located in the bottom of the double Greek key fold—W42E and W130E—yielded robust aggregation of partially unfolded intermediates at 37°C and pH 7. The αB‐crystallin chaperone suppressed aggregation of W130E, but not W42E, indicating distinct aggregation pathways from damage in the N‐terminal vs C‐terminal domain. The W130E aggregates had loosely fibrillar morphology, yet were nonamyloid, noncovalent, showed little surface hydrophobicity, and formed at least 20°C below the melting temperature of the native β‐sheets. These features are most consistent with domain‐swapped polymerization. Aggregation of partially destabilized crystallins under physiological conditions, as occurs in this class of point mutants, could provide a simple in vitro model system for drug discovery and optimization.     

Identifying sites of attachment of UV filters to proteins in older human lenses

Recent results indicate that covalent modification of proteins by tryptophan-derived UV filters may explain the age-dependent coloration of human lenses, and play a role in age-related cataract. The sites of attachment of the UV filters to the lens crystallins, however, have not been determined. This study utilized a database of predicted masses of UV filter-modified tryptic peptides to target sites of UV filter attachment. Proteins were isolated from old normal lenses and digested with trypsin at pH 6, in order to preserve the integrity of the sites of modification. Peptides were separated by high-performance liquid chromatography and characterized by mass spectrometry. Major colored and fluorescent peaks in the digest were found to correspond to cysteine-containing peptides in which the sulfur atom of the sidechain was linked to the major UV filter compound, 3-hydroxykynurenine glucoside. Three of the peptides originated from gammaS-crystallin and one from betaB1-crystallin. These results show that a predicted mass database can be used to facilitate the identification of sites of UV filter modification in human lens crystallins. Furthermore, this work represents the first evidence that UV filters bind to specific residues on lens proteins in vivo, and suggests that sulfhydryl groups may be important sites for the attachment of UV filters.     

Lactate Dehydrogenase Like Crystallin: A Potentially Protective Shield for Indian Spiny-Tailed Lizard (Uromastyx hardwickii) Lens Against Environmental Stress?

Taxon specific lens crystallins in vertebrates are either similar or identical with various metabolic enzymes. These bifunctional crystallins serve as structural protein in lens along with their catalytic role. In the present study, we have partially purified and characterized lens crystallin from Indian spiny-tailed lizard (Uromastyx hardwickii). We have found lactate dehydrogenase (LDH) activity in lens indicating presence of an enzyme crystallin with dual functions. Taxon specific lens crystallins are product of gene sharing or gene duplication phenomenon where a pre-existing enzyme is recruited as lens crystallin in addition to structural role. In lens, same gene adopts refractive role in lens without modification or loss of pre-existing function during gene sharing phenomenon. Apart from conventional role of structural protein, LDH activity containing crystallin in U. hardwickii lens is likely to have adaptive characteristics to offer protection against toxic effects of oxidative stress and ultraviolet light, hence justifying its recruitment. Taxon specific crystallins may serve as good models to understand structure–function relationship of these proteins.     

Structural characterization of a thiazoline-containing chromophore in an orange fluorescent protein, monomeric Kusabira Orange

Monomeric Kusabira Orange (mKO) is a green fluorescent protein (GFP)-like protein that emits orange light at a peak of 559 nm. We analyzed its X-ray structure at 1.65 A and found a novel three-ring chromophore that developed autocatalytically from a Cys65-Tyr66-Glu67 tripeptide in which the side chain of Cys65 formed the third 2-hydroxy-3-thiazoline ring. As a result, the chromophore contained the CNCOH group at the 2-position of the imidazolinone moiety such that the conjugated pi-electron system of the chromophore was more extended than that of GFP but less extended than that of the Discosoma sp. red fluorescent protein (DsRed). Since a sulfur atom has potent nucleophilic character, the third 3-thiazoline ring is rapidly and completely cyclized. Furthermore, our structure reveals the presence of a pi-pi stacking interaction between His197 and the chromophore as well as a pi-cation interaction between Arg69 and the chromophore. These structural findings are sufficient to account for the orange emission, pH tolerance, and photostability of mKO.     

A novel crystallin from octopus lens

Lens crystallins were isolated from cephalopods, octopus and squid. Two protein fractions were obtained from the octopus in contrast to only one crystallin from the squid. The native molecular mass for these purified fractions and their polypeptide compositions were determined by gel filtration, sedimentation analysis, and SDS-gel electrophoresis. Octopod and decapod lenses share one common major squid-type crystallin of 29 kDa, with one additional novel crystallin present only in the octopus lens. This newly-characterized crystallin (termed omega-crystallin) exists as a tetrameric protein of 230 kDa, consisting of 4 identical subunits of approx. 59 kDa. It is distinct from the previously known crystallins both in amino acid composition and subunit structure. N-terminal sequence analysis indicated that the omega-crystallin is N-terminally blocked, whereas the major octopus crystallin is identical to the reported squid crystallin with regard to the first 25 residues of protein sequence. Sequence similarity between this major cephalopod crystallin and glutathione S-transferase were found, which suggested some enzymatic role of crystallins inside the cephalopod lens.     

Protein-bound kynurenine is a photosensitizer of oxidative damage

Human lens proteins become progressively modified by tryptophan-derived UV filter compounds in an age-dependent manner. One of these compounds, kynurenine, undergoes deamination at physiological pH, and the product binds covalently to nucleophilic residues in proteins via a Michael addition. Here we demonstrate that after covalent attachment of kynurenine, lens proteins become susceptible to photo-oxidation by wavelengths of light that penetrate the cornea. H2O2 and protein-bound peroxides were found to accumulate in a time-dependent manner after exposure to UV light (lambda > 305-385 nm), with shorter-wavelength light giving more peroxides. Peroxide formation was accompanied by increases in the levels of the protein-bound tyrosine oxidation products dityrosine and 3,4-dihydroxyphenylalanine, species known to be elevated in human cataract lens proteins. Experiments using D2O, which enhances the lifetime of singlet oxygen, and azide, a potent scavenger of this species, are consistent with oxidation being mediated by singlet oxygen. These findings provide a mechanistic explanation for UV light-mediated protein oxidation in cataract lenses, and also rationalize the occurrence of age-related cataract in the nuclear region of the lens, as modification of lens proteins by UV filters occurs primarily in this region.     

Absence of small-angle maximums on the x-ray images of ocular lens tissue

X-ray small-angle scattering study of bovine lens tissue was carried out. It was shown that X-ray patterns of lens cortical and nuclear native tissues did not contain the small-angle maxima. The maximum in the range of 15-20 nm Bragg distance appeared as a result of the lens tissue partial dehydration. Earlier such maximum was considered by some authors as the evidence of crystallin proteins short-range order in the native lens. Now it is confirmed to be a preparative artefact of dehydration. It was shown also that similar maximum in 15-20 range existed in the case of concentrated crystallin solutions. This indicates that supramolecular organization of crystallins in the native lens is not similar to that in the concentrated solution.     

Physicochemical characterization of a crystallin from the squid lens and its comparison with vertebrate lens crystallins

A crystallin was isolated from the homogenate of the Squid (Loligo pealii) lens by gel filtration on a Sepharose CL-6B (2.5 X 170 cm) column. Biochemical characterization showed it is a dimeric protein with a molecular weight of (5.1 +/- 0.4) X 10(4) and a Stokes' radius of 26A. Electrophoresis on a cellulose acetate membrane indicated it is a basic protein with an isoelectric point higher than 8.6. High resolution two-dimensional gel in 8 M urea/2% NP-40 resolved this crystallin into 6 charge isomers, each with a major subunit of molecular weight 29,000 daltons and a minor subunit of 27,000 daltons in a molar ratio of 3:1. The extreme susceptibility of the protein to denaturation and precipitation even at low temperature hampered further characterization of this crystallin under nondenaturing conditions. Amino acid analysis indicated it contains an unusually high content of methionine (12.8 mol%) which may have some bearing on the instability of this crystallin in vitro. Biochemical comparison of the squid crystallin with mammalian lens crystallins shows that it is a crystallin distinguishable from all reported vertebrate lens crystallins. A detailed study of this protein may shed light on the evolution of lens crystallins in general.     

Production of crystallins and lens-like structures in differentiation-induced neoplastic pigment cells (goldfish erythrophoroma cells) in vitro

We examined the crystallins present in lens-like cell aggregates produced by goldfish erythrophoroma (tumors of integumental erythrophores) cells in vitro using a combination of Sephadex-G-200 gel filtration, one- and two-dimensional sodium-dodecyl-sulfate/polyacryl-amide gel electrophoresis, immunoblotting, and indirect immunofluorescence assays. The two studied neoplastic pigment cell lines, GEM 81 and GEM 218, formed small, spherical, transparent cell aggregates, resembling lentoid bodies, within the cell mounds of monolayer cultures after treatment with dimethylsulfoxide (DMSO) and autologous serum. Partial purification of a water-soluble extract of such lens-like cell aggregates and subsequent immunoblotting using antibodies (polyclonal) against newt whole lens proteins revealed the presence of about 20 unequivocally conjugated peptides with molecular masses of 19-27 kilodaltons. From their antigenicity and their behavior during gel filtration and electrophoresis, most of these peptides were identified as either alpha- or beta-form crystallins. Immunofluorescence microscopy using antibodies to newt whole lens proteins revealed intense fluorescence in the lens-like cell aggregates formed by these erythrophoroma cells, whereas the cell mounds in cultures of the same cell lines that had not been subjected to differentiation induction were almost unlabeled. Thus, goldfish erythrophoroma cells appear to be capable of crystallin production as well as the formation of lens-like cell aggregates upon the induction of differentiation. There is little available information indicating that normal pigment cells are capable of lens formation and crystallin synthesis during vertebrate ontogeny, and thus it is possible that neoplastic transformation of pigment cells is associated with the acquisition of the ability to produce crystallins.