Human Eye Lens Research Articles

Dynamic disulfide exchange in a crystallin protein in the human eye lens promotes cataract-associated aggregation

Increased light scattering in the eye lens due to aggregation of the long-lived lens proteins, crystallins, is the cause of cataract disease. Several mutations in the gene encoding human γD-crystallin (HγD) cause misfolding and aggregation. Cataract-associated substitutions at Trp42 cause the protein to aggregate in vitro from a partially unfolded intermediate locked by an internal disulfide bridge, and proteomic evidence suggests a similar aggregation precursor is involved in age-onset cataract. Surprisingly, WT HγD can promote aggregation of the W42Q variant while itself remaining soluble. Here, a search for a biochemical mechanism for this interaction has revealed a previously unknown oxidoreductase activity in HγD. Using in vitro oxidation, mutational analysis, cysteine labeling, and MS, we have assigned this activity to a redox-active internal disulfide bond that is dynamically exchanged among HγD molecules. The W42Q variant acts as a disulfide sink, reducing oxidized WT and forming a distinct internal disulfide that kinetically traps the aggregation-prone intermediate. Our findings suggest a redox "hot potato" competition among WT and mutant or modified polypeptides wherein variants with the lowest kinetic stability are trapped in aggregation-prone intermediate states upon accepting disulfides from more stable variants. Such reactions may occur in other long-lived proteins that function in oxidizing environments. In these cases, aggregation may be forestalled by inhibiting disulfide flow toward mutant or damaged polypeptides.

The effect of age on white light perception.

The way that persons from different age groups experience “white light” is investigated. Human eye lens transmission changes spectrally with age and this may influence the way that humans from different ages experiences light. Such a difference may be important in industrial and medical environments. Two different age groups, one group younger than 40 years of age and another group older than 50 years of age were subjected to the same “white” definition task. A conventional single-booth setup was used where observers were able to adjust the intensity of four coloured LED’s. Results of the psychophysical test procedure were used to generate specifications of two light sources, as selected by the two age groups. The two age groups selected two very different light sources when tasked to achieve a “perception” of white. Results show that the older group prefers a source with a colour rendering index number of 89 and the younger group prefers a source with a colour rendering index number of 74. The sources selected by the two age groups specifies correlated colour temperature values of 5150 K for the older age group and 6592 K for the younger group.

Differences In α-Crystallin Isomerization In The Human Eye Lens Expose New Details About Aging

Differences In α-Crystallin Isomerization In The Human Eye Lens Expose New Details About Aging

Sequence specific 1H, 13C and 15N resonance assignments of the C-terminal domain of human γS-crystallin.

The high solubility and stability of crystallins present in the human eye lens maintains its transparency and refractive index with negligible protein turnover. Monomeric γ-crystallins and oligomeric β-crystallins are made up of highly homologous double Greek key domains. These domains are symmetric and possess higher stability as a result of the complex topology of individual Greek key motifs. γS-crystallin is one of the most abundant structural βγ-crystallins present in the human eye lens. In order to understand the structural stability of individual domains of human γS-crystallin in isolation vis-à-vis full length protein, we set out to structurally characterize its C-terminal domain (abbreviated hereafter as γS-CTD) by solution NMR. In this direction, we have cloned, over-expressed, isolated and purified the γS-CTD. The 2D [15N-1H] HSQC recorded with uniformly 13C/15N labeled γS-CTD showed a highly dispersed spectrum indicating the protein to adopt an ordered conformation. In this paper, we report almost complete sequence-specific 1H, 13C and 15N resonance assignments of γS-CTD using a suite of heteronuclear 3D NMR experiments.

Anatomical-spatial dissection of single-cell retinal transcriptomics yields new insights in complement regulation

Human γd-crystallin (Hγd-crystallin), a major protein component of the human eye lens, is associated with the development of juvenile- and mature-onset cataracts. Evidence suggests that nonenzymatic protein glycation plays an important role in the aetiology of cataract and diabetic sequelae. This research compared the effects of various glycation modifiers on Hγd-crystallin aggregation, by treating samples of Hγd-crystallin with ribose, galactose, or methylglyoxal using several biophysical techniques. To measure advanced glycation end products, an Nε-(carboxyethyl)lysine enzyme-linked immunosorbent assay was performed on the glycating agent-treated Hγd-crystallin samples. Fructosamine production detection was performed for both ribose-treated and galactose-treated samples. Methylglyoxal-treated samples had the highest level of aggregation and the greatest extent of unfolding, and upon incubation for a minimum of 12 days, exhibited a marked enhancement in the amount of Nε-(carboxyethyl)lysine. The molecular profiles and morphological features of the glycated samples were highly correlated to the type of glycation agent used. These findings highlight a close connection between the type of glycation modifier and the various aggregation species that form. Thus, these results may facilitate deciphering of the molecular mechanism of diabetic cataractogenesis.

Effects of glycation on human γd-crystallin proteins by different glycation-inducing agents

Human γd-crystallin (Hγd-crystallin), a major protein component of the human eye lens, is associated with the development of juvenile- and mature-onset cataracts. Evidence suggests that nonenzymatic protein glycation plays an important role in the aetiology of cataract and diabetic sequelae. This research compared the effects of various glycation modifiers on Hγd-crystallin aggregation, by treating samples of Hγd-crystallin with ribose, galactose, or methylglyoxal using several biophysical techniques. To measure advanced glycation end products, an Nε-(carboxyethyl)lysine enzyme-linked immunosorbent assay was performed on the glycating agent-treated Hγd-crystallin samples. Fructosamine production detection was performed for both ribose-treated and galactose-treated samples. Methylglyoxal-treated samples had the highest level of aggregation and the greatest extent of unfolding, and upon incubation for a minimum of 12 days, exhibited a marked enhancement in the amount of Nε-(carboxyethyl)lysine. The molecular profiles and morphological features of the glycated samples were highly correlated to the type of glycation agent used. These findings highlight a close connection between the type of glycation modifier and the various aggregation species that form. Thus, these results may facilitate deciphering of the molecular mechanism of diabetic cataractogenesis.

Differences in α-Crystallin isomerization reveal the activity of protein isoaspartyl methyltransferase (PIMT) in the nucleus and cortex of human lenses

Although it is well-known that protein turnover essentially stops in mature lens fiber cells, mapping out the ensuing protein degradation and its effects on lens function over time remains challenging. In particular, isomerization is a common, spontaneous post-translational modification that occurs over long timescales and generates products invisible to most analytical methods. Nevertheless, isomerization can significantly impact protein structure, function, and solubility, which are all necessary to maintain clarity and proper refractive index within the lens. Herein, we examine the degree of isomerization occurring in crystallin proteins in the human eye lens as a function of both age and location within the lens. A novel mass spectrometric technique leveraging radical chemistry enables detailed characterization of proteins extracted from the cortex and nucleus of the lens. It is observed that the degree of isomerization increases significantly between the cortex and nucleus and between water-soluble and water-insoluble fractions. Interestingly, the abundance of L-isoAsp is low in the water-soluble cortex despite being the dominant product generated by isomerization of Asp in vitro, suggesting that Protein L-isoaspartyl methyltransferase (PIMT) is active in the cortex and suppresses the accumulation of L-isoAsp. The abundance of L-isoAsp increases dramatically in the nucleus, revealing that PIMT activity decreases over time in the center of the lens. In addition, the growth of L-isoAsp in the nuclear fraction suggests protein isomerization continues within the nucleus, despite the fact that most of the protein within the nucleus has become insoluble. Additionally, it is demonstrated that sequential Asp residues lead to isomerization hotspots in human crystallin proteins and that the isomerization profiles for αA and αB crystallin are notably different. Although αA is more prone to isomerization, αB loses solubility more rapidly upon modification. These differences are likely related to the distribution of Asp residues within αA and αB, which are in turn connected to refractive index. The high Asp content of αA is a hazard in terms of isomerization and aging, but it serves to enhance the refractive index of αA relative to αB, and may explain why αA is only found in the eye.

Bioinspired adaptive gradient refractive index distribution lens

Inspired by the soft, deformable human eye lens, a synthetic polymer gradient refractive index distribution (GRIN) lens with an adaptive geometry and focal power has been demonstrated via multilayer coextrusion and thermoforming of nanolayered elastomeric polymer films. A set of 30 polymer nanolayered films comprised of two thermoplastic polyurethanes having a refractive index difference of 0.05 were coextruded via forced-assembly technique. The set of 30 nanolayered polymer films exhibited transmission near 90% with each film varying in refractive index by 0.0017. An adaptive GRIN lens was fabricated from a laminated stack of the variable refractive index films with a 0.05 spherical GRIN. This lens was subsequently deformed by mechanical ring compression of the lens. Variation in the optical properties of the deformable GRIN lens was determined, including 20% variation in focal length and reduced spherical aberration. These properties were measured and compared to simulated results by placido-cone topography and ANSYS methods. The demonstration of a solid-state, dynamic focal length, GRIN lens with improved aberration correction was discussed relative to the potential future use in implantable devices.

Ultrafast excited state decay of natural UV filters: from intermolecular hydrogen bonds to a conical intersection.

Kynurenines (KNs) are natural UV filters of the human eye lens, protecting the eye tissues from solar UV radiation. Key points of their effective protection are the intramolecular charge transfer (ICT) in the excited state and the fast dissipation of absorbed light energy into heat via the intermolecular H-bonds. Herein we report that the introduction of an unsaturated double bond in the amino acid side chain, operating as an ICT-enhancing electron donor group, drastically accelerates the internal conversion (IC) due to a conical intersection (CI) between the potential energy surfaces of the excited and ground states. Our photophysical study of a deaminated KN (carboxyketoalkene, CKA), an intrinsic product of KN thermal decomposition, demonstrates an unusually fast excited state decay in a broad range of solvents of different polarity and proticity. The detailed analysis of interactions in the excited state by different computational techniques revealed that the changes in molecular structure - the twist of the carbonyl group from the plane of the aromatic ring followed by the formation of two mutually orthogonal conjugated substructures - are responsible for the CI of the excited and ground state potential energy surfaces. Intermolecular H-bonds facilitate the transition to the CI, but do not play a crucial role in the fast decay of the excited state. An extremely fast and efficient IC in CKA opens the way for the design of new types of organic UV filters and their applications in material science, cosmetics and medicine.

Sequence specific 1H, 13C and 15N resonance assignments of a cataract-related variant G57W of human γS-crystallin.

γS-crystallin is a major structural component of the human eye lens, which maintains its stability over the lifetime of an organism with negligible turnover. The G57W mutant of human γS-crystallin (abbreviated hereafter as γS-G57W) is associated with dominant congenital cataracts. In order to provide a structural basis for the ability of γS-G57W causing cataract, we have cloned, overexpressed, isolated and purified the protein. The 2D [15N-1H]-HSQC spectrum recorded with uniformly 13C/15N-labelled γS-G57W was highly dispersed indicating the protein to adopt an ordered conformation. In this paper, we report almost complete sequence-specific 1H, 13C and 15N resonance assignments of γS-G57W using a suite of heteronuclear 3D NMR experiments.

ESTABLISHMENT OF DETAILED EYE MODEL OF ADULT CHINESE MALE AND DOSE CONVERSION COEFFICIENTS CALCULATION UNDER NEUTRON EXPOSURE.

The human eye lens is sensitive to radiation. ICRP-118 publication recommended a reduction of the occupational annual equivalent dose limit from 150 to 20 mSv, averaged over defined periods of 5 y. Therefore, it is very important to build a detailed eye model for the accurate dose assessment and radiation risk evaluation of eye lens. In this work, a detailed eye model was build based on the characteristic anatomic parameters of the Chinese adult male. This eye model includes seven main structures, which are scleral, choroid, lens, iris, cornea, vitreous body and aqueous humor. The lens was divided into sensitive volume and insensitive volume based on different cell populations. The detailed eye model was incorporated into the converted polygon-mesh version of the Chinese reference adult male whole-body surface model. After the incorporation, dose conversion coefficients for the eye lens were calculated for neutron exposure atAP, PA and LAT geometries with Geant4, the neutron energies were from 0.001 eV to 10 MeV. The calculated lens dose coefficients were compared with those of ICRP-116 publication. Significant differences up to 97.47% were found at PA geometry. This could mainly be attributed to the different geometry characteristic of eye model and parameters of head in different phantom between the present work and ICRP-116 publication.

The (lack of) relation between straylight and visual acuity. Two domains of the point-spread-function.

The effect of cataract and other media opacities on functional vision is typically assessed clinically using visual acuity. In both clinical and basic research, straylight (the functional result of light scattering in the eye) is commonly measured. The purpose of the present study was to determine the link between these two measures: is visual acuity in cataract and other media opacities related to straylight? Interdependence between acuity and straylight is addressed from three different points of view: (1) Methodological: can acuity differences affect the measurement value of straylight, and vice versa? (2) Basic optics: does the optical process of light scattering in the human eye affect both straylight and visual acuity? (3) Statistical: how strongly are acuity and straylight correlated in the practice of important clinical conditions? Experimental and theoretical aspects will be considered, with a focus on normal ageing and cataract formation. (1) Methodological: testing potential effects of acuity, artificially manipulated with positive trial lenses, showed no effect on measured straylight values. Since light scattering in the eye involves a low percentage of the light and has large angular spreading, contrast reduction due to straylight is limited, resulting in virtually absent acuity effects. (2) Basic optics: light scattering from the human donor eye lens is found to have virtually no effect in the centre of the point-spread-function, also for cataractous lenses, resulting in virtually absent acuity effects. (3) Statistical: literature data on straylight and visual acuity show a weak correlation for the important groups of normal ageing and cataract populations. The point-spread-function of the normal ageing and cataractous human eye is built upon two rather independent basic parts. Aberrations control the central peak. Light scattering controls the periphery from about 1° onwards. The way acuity and straylight are measured ensures no confounding between them. Statistically within the normal ageing and cataract populations, visual acuity and straylight vary quite independently from each other. Visual acuity losses with cataract and other media opacities are not due to straylight, but caused by aberrations and micro-aberrations. Straylight defines disability glare, and causes symptoms of glare, haloes, hazy vision etc. Overall, visual acuity and straylight are rather independent aspects of quality of vision.

Albumin in the Vitreous Body, Retina and Lens of Human Fetal Eye.

The content of serum albumin was evaluated in the vitreous body, retina, and lens of human fetuses of 14, 16, 17, 18 and 24 weeks of gestation. Albumin was detected in these tissues. PCR analysis revealed no albumin mRNA in the retina or in the lens, while in fetal liver (control) mRNA for this protein was expressed. These findings suggest that serum albumin is not synthesized by cells of retina and lens, but is captured by them. The presence of serum albumin in human eye tissues suggests its involvement in the metabolism, maintenance of tissue volume, and antioxidant reactions.

Lipid-Protein Interactions in Fiber Cell Plasma Membrane Isolated From Human and Porcine Eye Lenses

The protein content in human lens fiber cell plasma membranes is extremely high, increases with age, and is higher in the nucleus than in cortex. This strongly affects the organization and physical properties of the lipid bilayer portion of intact fiber cell plasma membranes including the alkyl chain order, fluidity, hydrophobicity, and oxygen transport parameter. Investigated membranes were isolated from the eye lenses of pigs and human donors. They were studied using electron paramagnetic resonance spin-labeling method that provides an opportunity to discriminate coexisting lipid domains and to evaluate the relative amounts of phospholipids and cholesterol in them. Fiber-cell membranes were found to contain three distinct lipid environments: bulk, boundary and trapped domains that appear due to the presence of membrane proteins. In nuclear membranes the amount of boundary and trapped phospholipids as well as the amount of cholesterol in trapped lipid domains was greater than that in cortical membranes. Cholesterol was to a large degree excluded from trapped lipid domains in cortical membranes. The difference between the amounts of lipids in domains uniquely formed due to the presence of membrane proteins increased with the donors‘ age. The rigidity of nuclear membranes was greater than that of cortical membranes for all age groups. The nuclear fiber cell plasma membranes were less permeable to oxygen than cortical membranes and both of them become less permeable to oxygen with age. In nuclear porcine membranes the amounts of lipids in domains created due to the presence of membrane proteins were greater than those in cortical membranes and the differences were larger than the differences observed for human membranes. Lipids in porcine nuclear fiber cell membranes were more rigid and less permeable to oxygen than in human nuclear membranes.

Tryptophan and Non-Tryptophan Fluorescence of the Eye Lens Proteins Provides Diagnostics of Cataract at the Molecular Level

The chemical nature of the non-tryptophan (non-Trp) fluorescence of porcine and human eye lens proteins was identified by Mass Spectrometry (MS) and Fluorescence Steady-State and Lifetime spectroscopy as post-translational modifications (PTM) of Trp and Arg amino acid residues. Fluorescence intensity profiles measured along the optical axis of human eye lenses with age-related nuclear cataract showed increasing concentration of fluorescent PTM towards the lens centre in accord with the increased optical density in the lens nucleolus. Significant differences between fluorescence lifetimes of “free” Trp derivatives hydroxytryptophan (OH-Trp), N-formylkynurenine (NFK), kynurenine (Kyn), hydroxykynurenine (OH-Kyn) and their residues were observed. Notably, the lifetime constants of these residues in a model peptide were considerably greater than those of their “free” counterparts. Fluorescence of Trp, its derivatives and argpyrimidine (ArgP) can be excited at the red edge of the Trp absorption band which allows normalisation of the emission spectra of these PTMs to the fluorescence intensity of Trp, to determine semi-quantitatively their concentration. We show that the cumulative fraction of OH-Trp, NFK and ArgP emission dominates the total fluorescence spectrum in both emulsified post-surgical human cataract protein samples, as well as in whole lenses and that this correlates strongly with cataract grade and age.

Inhibition of copper-mediated aggregation of human γD-crystallin by Schiff bases.

Protein aggregation, due to the imbalance in the concentration of Cu2+ and Zn2+ ions is found to be allied with various physiological disorders. Copper is known to promote the oxidative damage of β/γ-crystallins in aged eye lens and causes their aggregation leading to cataract. Therefore, synthesis of a small-molecule 'chelator' for Cu2+ with complementary antioxidant effect will find potential applications against aggregation of β/γ-crystallins. In this paper, we have reported the synthesis of different Schiff bases and studied their Cu2+ complexation ability (using UV-Vis, FT-IR and ESI-MS) and antioxidant activity. Further based on their copper complexation efficiency, Schiff bases were used to inhibit Cu2+-mediated aggregation of recombinant human γD-crystallin (HGD) and β/γ-crystallins (isolated from cataractous human eye lens). Among these synthesized molecules, compound 8 at a concentration of 100 μM had shown ~95% inhibition of copper (100μM)-induced aggregation. Compound 8 also showed a positive cooperative effect at a concentration of 5-15 μM on the inhibitory activity of human αA-crystallin (HAA) during Cu2+-induced aggregation of HGD. It eventually inhibited the aggregation process by additional ~20%. However, ~50% inhibition of copper-mediated aggregation of β/γ-crystallins (isolated from cataractous human eye lens) was recorded by compound 8 (100 μM). Although the reductive aminated products of the imines showed better antioxidant activity due to their lower copper complexing ability, they were found to be non-effective against Cu2+-mediated aggregation of HGD.

Validation of the Nuclear Cataract Grading System BCN 10

Purpose: To evaluate a new nuclear cataract grading system which is intended as a surgical guidance system to predict lens hardness before cataract surgery. Methods: The new BCN 10 grading system consists of frontal and cross-sectional slit-lamp images of human eye lenses, ranging from a completely transparent lens nucleus to a totally black nuclear cataract. Validation was done with 9 observers for 110 cases. Two modalities were applied, and observers were asked to use only whole digits and then half digits for grading. Results: Repeatability with regard to test-retest differences showed a mean limit of agreement of 1.70 for whole digits and 1.32 for half digits. The absolute test-retest difference was close to zero for low as well high degrees of cataracts. Reliability for the entire group of 9 observers yielded an intraclass correlation coefficient which was within the same confidence interval, i.e., 0.991-0.995, for whole digits and half digits. Conclusions: BCN 10 grading repeatability was not affected by the severity of the cataract. It showed very good repeatability. Repeatability was significantly higher when the observers used half digits compared to whole digits. Reliability was found to be very good as well, independently of the use of whole or half digits.

Factors Determining the Oxygen Permeability of Biological Membranes: Oxygen Transport Across Eye Lens Fiber-Cell Plasma Membranes.

Electron paramagnetic resonance (EPR) spin-label oximetry allows the oxygen permeability coefficient to be evaluated across homogeneous lipid bilayer membranes and, in some cases, across coexisting membrane domains without their physical separation. The most pronounced effect on oxygen permeability is observed for cholesterol, which additionally induces the formation of membrane domains. In intact biological membranes, integral proteins induce the formation of boundary and trapped lipid domains with a low oxygen permeability. The effective oxygen permeability coefficient across the intact biological membrane is affected not only by the oxygen permeability coefficients evaluated for each lipid domain but also by the surface area occupied by these domains in the membrane. All these factors observed in fiber cell plasma membranes of clear human eye lenses are reviewed here.

Composition of phacoemulsificated human lenses analyzed by infrared spectroscopy

PurposePhacoemulsification is one of the most popular techniques of treating cataract. During the surgery the vibrating phaco‐probe emulsifies the lens into pieces that are vacuumed into a cassette. Afterwards the shredded lens material is usually disposed. Currently spectroscopic methods are ordinarily applied for studying biological materials due to their high sensitivity, reliability and non‐destructive character. The study attempts to determine whether the infrared spectroscopy technique is appropriate to reveal the differences in the structure of the lenses, especially focusing on the content of substances indicating changes in the secondary protein structure or mineralization process.MethodsIn the study 99 randomly selected dispersed human lenses were analyzed. The procedures of phacoemulsification were performed at the Military Hospital in Cracow and Military Institute in Warsaw. Obtained shredded lenses were forwarded to the Institute of Nuclear Physics at Polish Academy of Sciences in Cracow for further investigation. After appropriate preparation, the material was analyzed by FTIR spectroscopy using Nicolet spectrometer equipped with DTGS detector and ATR attachment. For the accurate interpretation of the spectra of lenses, exemplary samples of albumin, DNA, glucose and hydroxyapatite were also analyzed.ResultsThe FTIR spectra of lenses are characteristic for natural tissues and are dominated by intense bandwidth derived from amides and lipids. The FTIR spectra of the lenses are most similar to the spectrum of albumin.ConclusionsThe material obtained during phacoemulsification is suitable for infrared spectroscopic investigation. The FTIR method allows for the determination of human eye lenses structure and enables to indicate the differences in their composition.

Slices method to describe ray propagation in inhomogeneous media

We describe an alternative method that numerically calculates the trajectory followed by a light ray in rotationally symmetric inhomogeneous media in the paraxial approximation. The medium is divided into thin parallel slices and a radial quadratic refractive index is considered for each slice. The ABCD matrix is calculated in each slice and the trajectory of the ray was obtained. The method is demonstrated considering media with a refractive index distribution used to describe the human eye lens. The results are compared with the exact numerical solution for each particular distribution. In all cases, a good agreement is obtained for the proposed method and the exact numerical solution.