[Experimental study of influence of different damaging factors on lens. Report 3. Changes of lens protein composition].

Using differential electrophoresis protein composition of lens major proteins in hybrid mice F1 (C57B1XCBA) with cataracts of different etiology (senile, ultraviolet, radioactive and combined ultraviolet-radioactive exposure) was studied Changes that may be specific for cataract caused by aging, ultraviolet and/or gamma-irradiation were not revealed in water-soluble and water-insoluble protein fractions.

Simultaneous chemical and photochemical protein crosslinking induced by irradiation of eye lens proteins in the presence of ascorbate: the photosensitizing role of an UVA-visible-absorbing decomposition product of vitamin C.

Exposure to light has been implicated as a risk factor during aging of the eye lens and in cataract generation. In order to visualize the actual effect of UVA-visible light on this tissue, we incubated water-soluble eye lens proteins with ascorbate in the presence and absence of UVA-visible light for 3, 6 and 9 days at low oxygen concentration. The samples incubated in the presence of light were characterized by an initially small but continuous increase over time of the protein crosslinking. This was not the result of more extensive glycation because the decrease in amino group content of the proteins and the decomposition of ascorbate was the same in both irradiated and unirradiated samples. The augmented crosslinking capacity observed in the presence of UVA-visible light is due to the generation of a chromophore from the decomposition of ascorbate. This chromophore, obtained after 3, 6 and 9 days of incubation of solutions containing only ascorbate, induces both protein-crosslinking and oxidation after exposure to UVA-visible light in the presence of lens proteins. The extent of the crosslinking was proportional to the amount of the chromophore present in the solution. The presence of this chromophore was also determined when ascorbate was incubated with four-fold higher concentrations of N-α-acetyl lysine and N-α-acetyl arginine. When these samples were used as photosensitizers, the crosslinking degree was conditioned by the presence of this chromophore; nonetheless, the ascorbate-mediated advanced glycation end product (AGE) generation also made a contribution. The results of this work indicate that ascorbate oxidation, which generates the AGEs responsible for the chemical crosslinking of the lens proteins, also simultaneously produces a chromophore that can act as a photosensitizer, further increasing the protein crosslinking.

Mechanism of the very efficient quenching of tryptophan fluorescence in human gamma D- and gamma S-crystallins: the gamma-crystallin fold may have evolved to protect tryptophan residues from ultraviolet photodamage.

Proteins exposed to UV radiation are subject to irreversible photodamage through covalent modification of tryptophans (Trps) and other UV-absorbing amino acids. Crystallins, the major protein components of the vertebrate eye lens that maintain lens transparency, are exposed to ambient UV radiation throughout life. The duplicated beta-sheet Greek key domains of beta- and gamma-crystallins in humans and all other vertebrates each have two conserved buried Trps. Experiments and computation showed that the fluorescence of these Trps in human gammaD-crystallin is very efficiently quenched in the native state by electrostatically enabled electron transfer to a backbone amide [Chen et al. (2006) Biochemistry 45, 11552-11563]. This dispersal of the excited state energy would be expected to minimize protein damage from covalent scission of the excited Trp ring. We report here both experiments and computation showing that the same fast electron transfer mechanism is operating in a different crystallin, human gammaS-crystallin. Examination of solved structures of other crystallins reveals that the Trp conformation, as well as favorably oriented bound waters, and the proximity of the backbone carbonyl oxygen of the n - 3 residues before the quenched Trps (residue n), are conserved in most crystallins. These results indicate that fast charge transfer quenching is an evolved property of this protein fold, probably protecting it from UV-induced photodamage. This UV resistance may have contributed to the selection of the Greek key fold as the major lens protein in all vertebrates.

Cross-linking of lens crystallins in a photodynamic system: a process mediated by singlet oxygen.

In a dye-sensitized photooxidation system, lens crystallin polypeptides become cross-linked, and a blue fluorescence that is associated with the proteins is produced. These changes are similar to those seen in vivo in the aging human lens. Evidence implicating singlet oxygen as the causative agent of the effects in vitro is presented, and the possibility that this species may play a role in aging and cataractogenesis in vivo is discussed.

Eye lens color: formation and function.

Aromatic amino acids are photooxidized by near-ultraviolet light to colored products that are bound very tightly to protein amino groups. The resulting colored proteins absorb near-ultraviolet light more strongly and are rendered more hydrophobic than the untreated compounds, and they fluoresce at 440 nanometers when excited at 360 nanometers. Coloration in the lenses of diurnally active animals (including man) may be caused by this reaction, and senile cataracts may result. Such changes in many other proteins (as in the skin and retina) could lead to more serious consequences.

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.

[Chaperon-like anticataract agents, the antiaggregants of lens crystallin. Communication 4. Study of the effect of a mixture of di- and tetrapeptides on a prolonged rat model of UV-induced cataract].

There is a potential of therapeutic action on certain stages of caractogenesis, in particular on the aggregation of water-soluble proteins of cytoplasmic lens fiber cells, giving rise to insoluble protein complexes. The effect of a combined preparation (N-acetyl carnosine and D-patethine), acting by the chaperon-like mechanism, was studied in vivo on a prolonged rat model of UV-induced cataract. The use of the combined preparation consisting of a mixture of peptides of N-acetyl carnosine and D-patethine in a ratio of 1:1 as ocular instillations and intraperitoneal injections could slow down the development of UV-induced cataract in vivo. Pathomorphological studies suggest that the combined preparation has a protective effect on lens tissue when the rat model of UV-induced cataract is employed.

[Search for chaperon-like anticataract agents, the antiaggregants of lens crystallin. Communication 3. Possibilities of a follow-up of caractogenesis processes on a prolonged rat model of UV-induced cataract].

To study the mechanisms of action of new-generation anticataract drugs, it is necessary to have an accessible and adequate model of age-related cataract. A model of UV-induced cataract is pathogenetically closest to that of age-related cataract. A prolonged rat model of UV-induced cataract developing within 10 months is proposed; the clinical features of UV-induced cataract have been established at different stages of its development. A moderate homogeneous cloud-like lenticular opacity was observed at the end of the experiment; a less pronounced homogeneous opacity was seen in the anterior and posterior cortical layers. Cataract development was assessed by the appraisal method using the developed rat lenticular transparency scale, as well as by microdensitometry of biomicroscopic lenticular optical sections. Within the proposed model, the pathomorphological lenticular changes are largely similar to the histological pattern of age-related cataract.

Visible light neutralizes the effect produced by ultraviolet radiation in proteins.

The damage produced by UV-C radiation (100-280nm) in organisms and cells is a well known fact. The main reactions of proteins to UV-C radiation consist in the alteration of their secondary structures, exposure of hydrophobic residues, unfolding and aggregation. Furthermore, it has been found that electromagnetic radiation of lower energy (visible light, where wavelengths are between 400 and 750nm) also induces different disturbances in biomolecules. For instance, it has been observed that blue visible light from emitting diodes (LEDs) produces severe damage in murine cone photoreceptor-derived cells, and it can be even more harmful for some organisms than UV radiation. Recently, it has been found that the exposure of proteins to green and red light produces conformational changes, considerably increasing their cohesion enthalpies. This is presumably due to the strengthening of the hydrogen bonds and the formation of new ones. Therefore, it seems that visible light acts contrary to what it is observed for UV-C: instead of unfolding the proteins it folds them further, halting the damage produced by UV-C. This can be understood if we consider the modification of the folding energy-landscape; visible light induces the descent of the proteins into deeper states impeding the unfolding produced by UV-C.

Preventive role of lens antioxidant defense mechanism against riboflavin-mediated sunlight damaging of lens crystallins.

The main components of sunlight reaching the eye lens are UVA and visible light exerting their photo-damaging effects indirectly by the aid of endogenous photosensitizer molecules such as riboflavin (RF). In this study, lens proteins solutions were incubated with RF and exposed to the sunlight. Then, gel mobility shift analysis and different spectroscopic assessments were applied to examine the structural damaging effects of solar radiation on these proteins. Exposure of lens proteins to direct sunlight, in the presence of RF, leads to marked structural crosslinking, oligomerization and proteolytic instability. These structural damages were also accompanied with reduction in the emission fluorescence of Trp and Tyr and appearance of a new absorption peak between 300 and 400nm which can be related to formation of new chromophores. Also, photo-oxidation of lens crystallins increases their oligomeric size distribution as examined by dynamic light scattering analysis. The above mentioned structural insults, as potential sources of sunlight-induced senile cataract and blindness, were significantly attenuated in the presence of ascorbic acid and glutathione which are two important components of lens antioxidant defense system. Therefore, the powerful antioxidant defense mechanism of eye lens is an important barrier against molecular photo-damaging effects of solar radiations during the life span.

Increased sensitivity of amino-arm truncated betaA3-crystallin to UV-light-induced photoaggregation.

PURPOSE: Exposure to UV-B light (wavelength, 290-320 nm) is a well-documented risk factor for age-related cataracts. As the lens ages, beta-crystallins tend to undergo proteolytic cleavage of their terminal extensions. To delineate the effects of loss of terminal arms on beta-crystallin function, the sensitivity of purified recombinant wild-type (rbetaA3) to UV-irradiation induced aggregation was compared with that of betaA3-crystallin missing the N-terminal extension (rbetaA3tr). METHODS: Proteins were expressed in baculovirus-infected Sf9 cells and purified by chromatography. Purified protein solutions (pH 7.4) were reduced by using Tris (2-carboxyethyl) phosphine HCl and irradiated with a 308-nm excimer laser at physiologically relevant UV doses and wavelengths (308 nm), and light-scattering (633 nm) was measured. Irradiated crystallins were analyzed by matrix-assisted desorption ionization (MALDI) and tandem liquid chromatography/mass spectrometry (LC-MS/MS). RESULTS: UV-irradiation of both rbetaA3 and rbetaA3tr resulted in major loss of soluble protein, as shown by absorption at 280 nm, size-exclusion chromatography (SEC) and SDS-PAGE, with concomitant formation of insoluble aggregates producing light-scattering. Compared with wild-type rbetaA3, rbetaA3tr showed a significant tendency to begin scattering light at lower UV dose and had a higher aggregation rate with increasing UV exposure. Changes in irradiated crystallins include aggregation and cross-linking, photolysis, and oxidation of methionine and tryptophan residues. CONCLUSIONS: Loss of beta-crystallin terminal arms appears to increase their tendency to aggregate in response to UV irradiation, suggesting that this loss in the maturing lens may increase susceptibility to age-related cataract.

Red edge excitation shifts of crystallins and intact lenses. A study of segmental mobility and inter-protein interactions.

The shift that occurs in the fluorescence emission wavelength upon changing the excitation wavelength towards the red edge of the absorption band is termed red edge excitation shift (REES). We have monitored the REES of intrinsic protein fluorescence of freshly isolated intact lenses, of individual crystallins in their native, denatured and photodamaged states and also of crystallin mixtures. The observed REES values for the lenses from different species are different suggesting that the mobilities and packing of the crystallins may vary with the species. Lens photodamage in all the cases resulted in an increase of REES. Denaturation of crystallins in solution reduces REES and renaturation restores it. Mixtures of alpha- and beta-crystallins prepared either by directly mixing equimolar solutions or mixing them in 4 M urea followed by dialysis (reconstituting) gave similar REES values indicating the absence of any specific interactions in dilute solutions. Possible existence of induced alterations facilitating inter-crystallin interactions at high protein concentration is suggested.

Dose- and wavelength-dependent oxidation of crystallins by UV light--selective recognition and degradation by the 20S proteasome.

The lens of the human eye is a suitable model for age-related alterations at the molecular level. Age-related cataract formation is closely related to the accumulation of oxidatively altered proteins. In this study the influence of UV-A, UV-B, and UV-C irradiation on the proteolytic susceptibility of alpha-, betaL-, and betaH-crystallins by the isolated 20S proteasome was investigated. The proteins were irradiated with 280, 300, and 350 nm monochromatic light. Changes of the physical properties of the crystallins were characterized by absorbance measurements at 280 nm, fluorescence spectra, and SDS-PAGE-electrophoresis. The proteolytic susceptibility of crystallins was maximal after irradiation at 280 nm and three- to fivefold lower at 300 nm. Irradiation at 350 nm was not able to initiate proteolysis, probably due to protein-aggregate formation of higher molecular weight, as shown by SDS-PAGE. The damage of crystallins by UV-C light might be a signal for its proteolytic degradation by the 20S proteasome, whereas UV-B and UV-A do not increase the proteolytic susceptibility to the same extent but promote the formation of crosslinked proteins. Therefore, irradiation with UV, which is not followed by an increase in the proteolytic susceptibility, is accompanied by the formation of crosslinked proteins. It was concluded, that also long UV-B and UV-A may be involved in age-related alterations of the human lens and cataract formation.

Measures of leucine aminopeptidase can be used to anticipate UV-induced age-related damage to lens proteins: ascorbate can delay this damage.

This paper focuses on damage to soluble lens proteins during ultraviolet (UV) light exposure and its prevention by ascorbate (Vitamin C). Using 2.3 X 10(-3) W/cm2 UV A and 0.4 X 10(-4) W/cm2 UV B, aminopeptidase inactivation in lens supernatants is significant after 60 min. Protein aggregation and decreases in tryptophan levels, phenomena associated with UV-induced and cataract-related damage, are observed only after longer (6 h) UV exposure. Thus, it would appear that measurements of aminopeptidase activity can be used to anticipate damage to lens structural proteins. Ascorbate (15 mM) added to soluble lens proteins prior to photoirradiation can prevent some of these changes. The data presented suggest plausible relationships between impaired proteolysis and cataract formation.

The effect of UVA light on the anaerobic oxidation of ascorbic acid and the glycation of lens proteins.

PURPOSE: To determine whether UVA-excited human lens chromophores can cause the oxidation of ascorbic acid in the absence of oxygen, and whether these oxidation products are capable of glycating lens proteins. METHODS: The oxidation of ascorbic acid, mediated by UVA irradiation in the presence of aged human lens proteins, was measured in the absence of oxygen by the decrease in absorbance at 265 nm in vitro. An action spectrum from 320 to 400 nm was determined for both ascorbate oxidation and the photobleaching of the lens yellow pigments at lambda = 350 nm. The UVA-mediated oxidation products of [U-(14)C]ascorbate were quantified by HPLC. Glycation was assayed by the UVA-dependent incorporation of [U-(14)C]ascorbate into lens proteins with a water-insoluble (WI) fraction in vitro, with incubated whole human lenses, and with a WI fraction after a 5- to 7-day exposure to ambient sunlight. An enzymatic digest of [U-(14)C]ascorbate-labeled proteins was fractionated over HPLC columns and compared with the 330-nm absorbance profile of a proteolytic digest of aged human lens proteins. RESULTS: Aged human lens WI proteins absorbed UVA light (86 J/h per square centimeter) and oxidized 33 to 45 nanomoles of ascorbate over 1 hour in the absence of oxygen. No ascorbate oxidation was detected, however, in the dark control. An action spectrum showed that ascorbate oxidation occurred throughout the UVA region, with lambda(max) at 350 nm, which was similar to the action spectrum obtained for the photobleaching of the lens chromophores. Anaerobic UVA irradiation of aged human lens proteins for 2 hours with [U-(14)C]ascorbate resulted in a 40% loss of ascorbate with the accumulation of dehydroascorbic acid, diketogulonic acid, and oxalate. After subsequent incubation for 24 hours, the ascorbate oxidation products disappeared, with a corresponding incorporation of radioactivity into lens proteins. Chromatography of enzymatic digests of the labeled proteins produced peaks that coeluted with several of the 330-nm absorbing peaks in an aged human lens protein digest. Irradiation of whole human lenses for 2 hours caused a 33% loss of total lens ascorbate. UVA irradiation of aged human lenses for 2 hours resulted in the incorporation of ascorbate into lens proteins during the ensuing 24 hours in the dark. Exposure of aged human lens WI proteins to reflected ambient sunlight (1.1 J/h per square centimeter) for 5 to 7 days in the absence of oxygen also produced an increased incorporation of [(14)C]ascorbate into protein when compared with dark control samples. CONCLUSIONS: These data argue that UVA light can cause an oxidation of ascorbic acid in the absence of oxygen, due to the activation of the sensitizers present in aged human lens WI proteins. The oxidation products formed were the same as those seen in the presence of oxygen, and were rapidly incorporated into protein, apparently by Maillard-type chemistry. These data argue that ascorbate glycation can occur under the low oxygen levels thought to exist in the human lens nucleus in vivo.

Effect of chronic near-ultraviolet radiation on the gray squirrel lens in vivo.

The effects of ambient exposure to near-ultraviolet (near-UV) radiation (300-400 nm) on the ocular lens of the diurnal squirrel (Sciurus carolinensis) are reported. Gray squirrels lived in cages illuminated for 12 hr a day with near-UV light (6 mW/cm2, 365 nm) for 1 yr. The non-UV-exposed controls were housed separately. In the lenses of UV-exposed animals, anterior pole changes occurred. Central epithelial cells swelled, disappeared, or underwent proliferation. A band of disoriented degenerating fiber cells was seen in the midcortex, with a degree of liquefaction. When lens protein compartments were separated by centrifugation, water-insoluble but urea-soluble fractions were enhanced in the outer and inner cortex and the nucleus. Both high-performance liquid chromatography and polyacrylamide gel electrophoresis revealed that proteins mainly in the midcortex and nucleus were altered considerably. Evidence of a loss of sulfhydryl compounds (by chemical and Raman spectroscopic analyses) and an increase of protein-thiol mixed disulfides (chemically) was also observed. These data prove that repetitive ambient exposure of diurnal animals to near-UV radiation at subsolar levels damages the lens by interfering with the maintenance of epithelial cells and altering the structural proteins; some of this may be due to the conversion of sulfhydryls to mixed disulfides.

Crosslinking and photoreaction of ozone-oxidized calf-lens alpha-crystallin.

Direct-photo-oxidation, singlet oxygen-oxidation, or photosensitized oxidation can modify lens crystallins, causing an increase in blue fluorescence and covalent crosslinking. A relationship between these changes has not been elucidated. We now report results from experiments with ozone oxidation. When calf-lens alpha-crystallin is treated with zone oxidation. When calf-lens alpha-crystallin is treated with ozone, new absorption, fluorescence, and phosphorescence, which are characteristic of the oxidized product of tryptophan (N-formylkynurenine), appear at 320, 435, and 445 nm, respectively. In addition, in this ozonization of alpha-crystallin, its polypeptides are crosslinked by nondisulfide bonds. Irradiation of ozone-treated alpha-crystallin with near-ultraviolet (365 nm) light increases crosslinking and reduces the 320 nm absorbance with a concomitant appearance of a new absorption at about 420 nm. This photoproduct exhibits an intense fluorescence around 450 nm and a weak phosphorescence at 510 nm, with excitation peaks at 400, 415, and 422 nm. These findings are essentially the same as those observed in photo-oxidized alpha-crystallin, suggesting the involvement of the same tryptophan oxidized product in the modification of the lens protein.

Changes in the distribution of lens calcium during development of x-ray cataract.

The present study was designed to examine the possible role of calcium in the opacification of x-ray-induced cataract in rabbit. The results demonstrate that the concentration of calcium in x-rayed lenses, just prior to lens hydration (7.5 weeks postirradiation), was twice that present in contralateral control lenses. At this stage of immature cataract, the lens nucleus remained transparent and maintained a normal level of calcium, but the lens cortex, containing regions of subcapsular opacification, accumulated a level of calcium that was twice that of the control. In the completely opaque mature cataract, (8-9 weeks postx-ray), both the cortex and nucleus had gained significant amounts of calcium. As the concentration of total calcium increased in the immature x-ray cataract, the amount of the cation bound to membranes and insoluble proteins of the cytosol also increased comparably. However, the relative proportion of calcium in the various fractions remained unaltered in the immature cataract; in both control lenses and immature cataracts, 20% of the total calcium remained in the membrane pellet and 70% was located in the soluble protein fraction. Only in the mature stage of cataract was a shift in the distribution of calcium apparent, as the proportion of calcium in the soluble protein fraction increased to 90%. Although only 7% of the total calcium in a mature cataract was bound to membrane, the amount represented a fivefold increase over the control. The results of this study demonstrate that an elevation in lens calcium accompanies the opacification process in x-ray cataract. The work also suggests that changes in calcium levels are not likely to result from inactivation of Ca-ATPase.

In vitro cross-linking of bovine lens proteins photosensitized by promazines.

Promazine derivatives induce cross-linking of bovine lens crystallins in vitro by irradiation with near-ultraviolet (UV) light in the presence of O2, as revealed by electrophoresis after denaturation. With the five derivatives tested (promazine [PZ], chlorpromazine [CPZ], triflupromazine [ TFPZ ], methoxypromazine [ MTPZ ], and acepromazine [ ACPZ ] ), single-hit kinetics are observed. Evidence implicating the cation radicals of the PZ derivatives as the causative agent of this in vitro effect is presented. Hydroxyl radicals do not appear to be involved in the photo-cross-linking reaction. Sodium ascorbate protects against damage induced either by PZ derivatives plus light or by PZ cation radicals in the dark. These findings are discussed with respect to development of cataracts induced by these drugs in vivo.

Ascorbic acid and glucose oxidation by ultraviolet A-generated oxygen free radicals.

PURPOSE: To determine the relative rate of oxidation of ascorbic acid (ASA) and glucose under conditions used for glycation reactions in vitro and by ultraviolet A (UVA)-generated oxygen free radicals using human lens sensitizers. METHODS: ASA and [14C]glucose were incubated in 0.1 M phosphate buffer, and the rate of oxidation was determined by absorbance at 265 nm and by thin-layer chromatography, respectively. Oxidation also was measured during the UVA irradiation of 2 mg/ml solutions of human lens water-insoluble proteins. The role of individual reactive oxygen species was determined by the protective effects of superoxide dismutase, catalase, and sodium azide. RESULTS: ASA was oxidized rapidly in 0.1 M phosphate buffer. This loss was prevented by the addition of a metal chelator, by previous chelex resin treatment of the buffer, or by the addition of lens proteins. Glucose was not oxidized under any of the above conditions. UVA irradiation with 2 mg/ml human lens protein as sensitizer oxidized 1 mM ASA after several hours but oxidized, at most, only 2 microM glucose even after 8 hours of irradiation. Superoxide anion was responsible for 24%, and singlet oxygen for 40%, of the ASA oxidized. UVA-generated H2O2 caused little or no oxidation of ASA. H2O2 did accelerate the oxidation of ASA in phosphate buffer, but this was almost completely prevented by the addition of either a chelating agent or lens proteins. CONCLUSIONS: The conditions used for glycation reactions in vitro rapidly oxidized ASA, but not glucose. The UVA-dependent generation of oxygen free radicals also oxidized ASA at a 10(3) faster rate than glucose. Superoxide anion and singlet oxygen were identified as the principal oxidants of ASA in this process. These data argue that ASA may be the primary glycating agent in aging normal lenses.