Eye Lens Proteins Research Articles

A novel method for the amino acid sequence/configuration determination of peptides containing D/L-amino acids utilizing a fluorogenic Edman reagent, 7-N,N-dimethylaminosulphonyl-4-(2,1,3-benzoxadiazolyl)isothiocyanate (DBD-NCS).

A novel method for the amino acid sequence and configuration determination of peptides containing D- or L-amino acids is presented. An enkephalin analogue, [D-Ala2, D-Leu5]enkephalin (Tyr-Ala-Gly-Phe-Leu) was derivatized with a fluorogenic Edman reagent, 7-N,N-dimethylaminosulphonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS), cleaved and cyclized with trifluoroacetic acid at 50 degrees C for 1 min and the resultant thiazolinone derivative (DBD-thiazolinyl-L-Tyr) was separated from the racemized DBD-thiazolinyl-D-Tyr (about 20% as compared to the L-isomer) on a phenylcarbamylated cyclodextrin column: The separation factor (alpha) for D- and L-isomers was 1.09. The column eluate was monitored fluorometrically at 524 nm with excitation at 387 nm. The detection limit was about pmol range. The same treatment adopted for the residual peptide, Ala-Gly-Phe-Leu, gave DBD-thiazolinyl-D-Ala (alpha = 1.09 for D,L-Phe) with the lesser amount of L-analogue (about 20%). In the same manner, Gly and L-Phe (alpha = 1.09) were detected. The method might be useful for the study of aging of proteins such as eye lens and amyloid proteins derived from Alzheimer's disease.

Role of advanced glycation end-products (AGE) in late diabetic complications

To evaluate accumulation of advanced glycation end-products (AGE) in diabetes and its possible correlation with late diabetic complications, AGE levels were measured by spectrofluorimetry in eye lens and sciatic nerve proteins and isolated tail tendon collagen of rats with experimental diabetes of 3- and 6-month duration. The values obtained were compared to those from age-matched control rats and correlated with cataract presence and somatosensory evoked potential (SEP) alterations. Diabetic animals had increased AGE levels in all tissues at both times; cataract developed in 29% of diabetic rats at 3 months and in 57% at 6 months; SEP conduction velocity was reduced in diabetic animals both at 3 (54.5 ± 1.8 S.E.M. m/s vs. 73.9 ± 1.0, P < 0.0001) and 6 months (59.5 ± 1.4 vs. 71.5 ± 1.6, P < 0.0001) from diabetes induction. No eye lens AGE level differences were observed when cataract presence was considered. Interestingly, in diabetic rats, increased sciatic nerve AGE levels were associated with reduced SEP. These data show that: (1) AGE levels are increased as early as 3 months from development of hyperglycemia; (2) other factors, in addition to an enhanced rate of fluorescent AGE formation, might play important roles in the pathogenesis of diabetic cataract; (3) increased peripheral nerve AGE levels are associated with SEP alterations.

The avian eye lens protein delta-crystallin shows a novel packing arrangement of tetramers in a supramolecular helix.

Little is known of the intermolecular organization of crystallins in the protein-packed eye lens. The tetrameric structure of the 200,000 Da avian delta-crystallin, which is closely related to the enzyme argininosuccinate lyase and is characteristic of the accommodating, soft lens of birds, has recently been solved at atomic resolution at acidic pH. To help understand how delta-crystallin remains soluble at the very high concentrations found in the avian lens we have now crystallized turkey delta-crystallin at around neutral pH and examined its intermolecular interactions. Turkey delta-crystallin has been crystallized around neutral pH. The X-ray structure has been solved at 4.5 A resolution in space group C2 with three and a half tetramers in the asymmetric unit. The symmetrical 222 tetramers have a novel packing arrangement consisting of continuous helices, with 7(3)2 non-crystallographic symmetry, in an approximately hexagonal close-packed array. The internal 222 symmetry of the tetramers allows different polymeric chains to be constructed, based on the tetramer-tetramer association observed in the crystalline helix. It is possible to build a model of a tubule of diameter 212 A that is very similar to observed tubules of bovine argininosuccinate lyase. Elements of helical organization may occur in the concentrated solution of the avian eye lens where delta-crystallin is the prominent protein. The symmetry of the tetramer provides a choice in the direction of growth of a helix at each link so that highly hydrated irregular polymers may be formed rather than large compact regular structures that would not be compatible with a transparent lens.

New trends in photobiology (invited review) photodegradation of keratin and other structural proteins

Keratin and many other proteins are damaged when they are exposed to ultraviolet and visible radiation. In wool keratin, this is apparent as a colour change and a weakening or embrittlement of the fibre. Photodegradation of other structural proteins such as collagen, elastin and eye lens proteins (crystallins) have been linked with problems such as premature skin ageing and cataract formation; a possible involvement in skin cancer has even discussed. Despite several decades of research, the photochemistries of these processes are still imperfectly understood. This review deals with the photophysical and early photochemical events occurring in keratin upon exposure to ultraviolet and longer wavelength radiation. General aspects of the photochemistry of other proteins and model amino acid systems are also reviewed.

Age-at-Maturity Estimates for Atlantic Coast Female Striped Bass

Abstract This study was undertaken to estimate the percentage of mature female striped bass Morone saxatilis present in each age-class during annual coastal feeding migration. Migratory striped bass (N = 302) were sampled in coastal Rhode Island waters during spring (May–June) and fall (September–November) from 1985 to 1987. Stocks were identified by analysis of morphometric characters and isoelectric focusing of eye-lens proteins. Histological sections of ovarian tissue were used to categorize maturity state. Fish were considered mature if a class of oocytes measuring at least 150 μm and containing cytoplasmic inclusions was found in the ovarian sections. All females whose age at next potential spawning was 7 and older were mature. Our empirical observations indicated that 12% of fish in age-class 4, 34% of fish in age-class 5, and 77% of fish in age-class 6 were mature. The estimate of the proportion of mature fish in age-class 5 differs significantly from that of Merriman (1941), who also examined coas...

Oxidation of gamma II-crystallin solutions yields dimers with a high phase separation temperature.

Aqueous solutions of the bovine eye lens protein gamma II (or gamma B)-crystallin at neutral pH show a gradual increase in phase separation temperature, Tph, when allowed to stand for several weeks at room temperature without reducing agents. In a typical experiment, the Tph of the protein solution (218 mg/ml) increases from 2.5 +/- 1 degree C to 32.5 +/- 1 degree C after 21 days, and a new protein species, gamma IIH, is formed. The Tph of pure gamma IIH is at least 40 degrees C higher than that of pure gamma II. The average apparent hydrodynamic radius is 36 A for gamma IIH compared to 26 A for gamma II. The molecular mass of gamma IIH is approximately 41.5 kDa compared to 20 kDa for native gamma II. Therefore, gamma IIH is probably a dimer of gamma II crystallin. gamma IIH has a lower thiol content than gamma II and is not formed in the presence of dithiothreitol. We conclude that gamma IIH is a thiol oxidation product of gamma II-crystallin and is a dimer containing an intermolecular disulfide crosslink. Thus, some oxidative modifications of protein thiol groups lead to an increase in net attractive interactions between proteins. As a result, Tph increases and protein aggregates are formed. These two microscopic changes produce the increased light scattering associated with lens opacification.

Heterologous expression in Escherichia coli of native and mutant forms of the major intrinsic protein of rat eye lens (MIP26).

The complete cDNA of rat eye lens major intrinsic protein (MIP26) was sequenced using the dideoxy chain termination method. The sequence displayed 89% nucleotide identity and 95% identity at the amino acid level with bovine MIP26 [Gorin, Yancey, Cline, Revel and Horwitz (1984) Cell, 39, 49-54]. Both native and mutant cDNAs coding for rat MIP26 were amplified by PCR and subcloned into the pPOW expression vector for expression of Escherichia coli. A membrane signal peptide (PelB) was used for secretion of MIP26 into the cytoplasmic membrane. A hydrophilic octapeptide tail (FLAG) was fused to either the N- or C-terminus of MIP26 to aid monoclonal antibody-mediated identification and purification. Heterologously expressed MIP26 was identified by using a monoclonal antibody corresponding to the FLAG peptide located at the termini of MIP26. Immunofluorescently labelled monoclonal antibody was used to determine the localization of MIP26 in the cytoplasmic membrane. The majority of the protein was integrated into cell plasma membrane. MIP26 was extracted with n-octyl beta-D-glucopyranoside and then purified on an affinity gel column. Rat MIP26 cDNA contains an -Asn-Gly- sequence at the C-terminus, which has been shown in other proteins to be particularly susceptible to spontaneous deamidation [Takemoto and Emmons (1991) Curr. Eye Res. 10, 863-869]. We therefore modified the MIP26 molecule using a site-directed mutagenesis method to generate a mutant MIP26 at the appropriate asparagine residue (Asn244-->Asp) near the C-terminus. The mutation was confirmed by DNA sequencing. The mutant MIP26 protein was also expressed in E. coli and incorporated predominantly into the cytoplasmic membrane.

Dynamic critical phenomena in aqueous protein solutions.

An aqueous protein solution is a binary mixture of large particles (protein molecules) and small particles (water molecules). In contrast, a binary liquid mixture is a mixture of two different kinds of particles that are approximately the same size. Thus, a comparison of the critical behavior in these two systems allows one to determine the effect on critical behavior of a relative difference of size in the two kinds of particles in a binary mixture. The correlation length j, osmotic compressibility kT , and coexistence curve of aqueous protein solutions in the vicinity of the critical point for liquid-liquid phase separation have been reported recently [1‐3]. These experimental results established that the static critical properties of these solutions are consistent with the behavior of members of the static universality class of three-dimensional systems with short range interactions and scalar order parameters. This universality class includes binary liquid mixtures. In contrast to the equilibrium properties, the dynamic properties of aqueous protein solutions in the vicinity of the critical point have not been investigated as thoroughly. In this Letter, we report our use of quasielastic light scattering (QLS) to investigate the time decay of spontaneous concentration fluctuations, as a function of both wave number q of the fluctuations and temperature T, along the critical isochore of aqueous solutions of the bovine eye lens protein gII-crystallin. The only previous study of critical dynamics in a protein solution of which we are aware is the work of Ishimoto and Tanaka who made QLS measurements at a single fixed wave number of the fluctuations using a 19 channel digital correlator on aqueous solutions of lysozyme [4] along the critical isochore for sT 2 TcdyTc $ 5 3 10 23 (q j, 0.1), where Tc is the critical temperature. The present study is a significant experimental advance over the work of Ishimoto and Tanaka in that (i) we obtain data for fluctuations at up to 12 different wave numbers, (ii) we obtain data using a 144 channel digital correlator, and (iii) we obtain data much closer to the critical point [ sT 2 TcdyTc $ 1.56 3 10 24 and q j# 3.18]. The more thorough investigation of critical dynamics presented here has revealed new and unexpected behavior. Specifically, we find that the wave number and temperature dependence of the average rate of decay of the concentration fluctuations are consistent with the theory for critical dynamics in binary liquid mixtures [5] only if we allow both the background viscosity and the background contribution to the decay rate to be unusually large. Furthermore, in contrast to the behavior seen in binary liquid mixtures we find that the concentration fluctuations exhibit very significant deviation from exponential decay with time. Of course, these protein solutions may be viewed as

αA-crystallin confers cellular thermoresistance

The bovine eye lens protein αA-crystallin has been overexpressed both by stable transfection of HeLa cells and by transient transfection of NIH 3T3 cells. In both experimental systems αA-crystallin overexpression results in an increased cellular thermoresistance as judged by different clonal survival assays. In contrast, similar overexpression of another stable lens protein, βB2-crystallin, does not confer thermoresistance. These results indicate that the structural relationship of αA-crystallin to the small heat shock proteins HSP25/27 and to αB-crystallin is sufficient for the shared thermoprotective function of all of these molecules and strongly suggests that the chaperone-like properties that they have in common are responsible for the conferred cellular thermoresistance.

Conformational studies on δ-crystallin, the core protein of the bird eye lens

Proteins that perform other functions elsewhere appear to be recruited for structural purposes in the eye lens. The lens being a tissue with very little metabolic activity and little or no turnover, the lens proteins, crystallins, are long lived. In an effort to understand whether their recruitment might be related to their conformation and structural stability, we have examined these features of the avian lens protein δ-crystallin. The native molecule is a tetramer (molecular mass 200 kDa) that is highly α-helical in conformation, and with an unusually blue tryptophan fluorescence (315,325 nm), which is only partially quenched by conventional quenchers. We show that the fluorescence doublet arises due to Trp residues that are effectively buried inside the rigid hydrophobic core of the tetrameric aggregate. The protein is heat stable up to 91°C. Guanidinium chloride (GuHCl) effects the complete denaturation of δ-crystallin, whereas heat or urea treatment results in only partial unfolding or dissociation. The initial transition is the disruption of the quaternary structure by perturbing the intersubunit interactions, leading to exposure of hydrophobic contact surfaces (as monitored by extrinsic probe fluorescence). This initial transition is seen upon heating to 60°C as well as in 1 M GuHCl and 4 M urea. We show that in 2.2 M GuHCl the molecule is swollen but is still largely helical with the Trp residues being present in a somewhat more polar environment than in the native molecule. Beyond 4 M GuHCl there is a gradual unfolding of the molecule, which is complete in 6 M GuHCl. This structural robustness of δ-crystallin might be important in its recruitment as the core protein of the avian lens.

Eye-Lens Proteins?: Structure, Superstructure, Stability, Genetics

Eye-Lens Proteins?: Structure, Superstructure, Stability, Genetics

Eye-Lens proteins structure, superstructure, stability, genetics

The eye lens in vertebrates and invertebrates is an avascular tissue which allows one to focus objects on the retina. The lens grows throughout life, maintaining transparency without significant turnover of its densely packed proteins. Apart from cytoskeletal and taxon-specific components, these proteins belong mainly to the alpha- and beta gamma-crystallin families. The detailed structural analysis of beta gamma-crystallins can explain the anomalous stability by the specific supersecondary structure ("Greek key" topology) of the domains and by strong domain and subunit interactions. The spatial correlation of the molecules at the given high concentrations in the fiber cells gives rise to "short-range order" with minimum light scattering, thus providing optimum transparency of the eye lens.

Alpha-crystallin/small heat shock protein has autokinase activity.

The alpha-crystallins (alpha A and alpha B) are major water-soluble proteins of the transparent eye lens that are expressed in a variety of tissues and can function as molecular chaperones. alpha B-crystallin is also a small heat shock protein associated with numerous degenerative diseases and abnormal growth patterns. Previous experiments have shown that alpha A-and alpha B-crystallin are phosphorylated on specific serine residues by a cAMP-dependent pathway. Here we provide evidence that either total bovine alpha-crystallin or its isolated polypeptides can autophosphorylate serine by a cAMP-independent mechanism in the presence of Mg2+ and [gamma-32P]ATP; the autophosphorylated products isoelectrically focus with the authentic phosphorylated forms of the alpha-crystallin polypeptides. Thus, the alpha A- and alpha B-crystallin/small heat shock protein polypeptides are enzyme-crystallins which may be involved in metabolic pathways important for the development, maintenance, or pathology of the lens and other tissues.

Close packing of an oligomeric eye lens β-crystallin induces loss of symmetry and ordering of sequence extensions

β-Crystallins are oligomeric eye lens proteins that are related to monomeric γ-crystallins. The main sequence difference between the two families is the presence of sequence extensions in the β-crystallins. A major question concerns the role that these extensions play in mediating interactions at the high protein concentrations found in the lens. The predominant β-crystallin polypeptide, βB2, can be crystallized in two different space groups, I222 and C222. The I222 crystal structure revealed that the protein packed as a tetramer with perfect 222 symmetry but that the extensions were disordered. The X-ray structure of the C222 lattice of βB2 has now been refined at 3·3 Å, the structure analysed and compared with the I222 lattice. The protein is also a tetramer with 222 symmetry in the C222 lattice but differs in that parts of the N-terminal extensions have been visualized. In the asymmetric unit of the C222 lattice there are four subunits, each comprising a single polypeptide chain, in which certain flexible loops in the N-terminal domains and the N-terminal extensions have various conformations. The tetramers in the C222 lattice are more tightly packed than in the I222 form. Analysis of the tetramer contacts shows that the sites of interaction break the 222 symmetry of the tetramers. The N-terminal extensions play a major role in directing interactions between tetramers. One of the N-terminal extensions interacts with a hydrophobic patch on the N-terminal domain of another tetramer. These crystallographic observations obtained over a physiological concentration range indicate how, in β-crystallin oligomers, the N-terminal extensions of βB2 can switch from interacting with water to interacting with protein depending on their relative concentrations. This could be useful in maintaining a gradient of refractive index.

Ultrasonic spectroscopy of the porcine eye lens

The purpose of the work is to measure and study the acoustic characteristics of the porcine eye lens and find correlations with chemical and optical parameters, obtained from literature. Ultrasonic spectroscopy was performed by using a scanning acoustic macroscope (frequency 20 MHz, resolution 150 μm). The transducer performed a two-dimensional scan over a central slice (1 mm thickness) of porcine lens (number of lenses = 10). A double transmission pulse-echo method was used to acquire the ultrasonic data from the lens. Two-dimensional images were reconstructed of the local ultrasound velocity and the frequency-dependent ultrasound attenuation. Axial and equatorial profiles of these parameters were calculated from the images. The acoustic parameters are not constant, but show a systematic dependence on the location within the lens. The profiles of the acoustic parameters are similar in shape to profiles of the protein and water contents of eye lens and to the profiles of the optical refractive index. A thorough quantitative correlation study is indicated, which should be based on detailed protein content data in porcine lenses.

Elastase inhibition by the C-terminal domains of α-crystallin and small heat-shock protein

α-Crystallin, an abundant eye-lens protein and a stress protein in other tissues, shows structural and functional similarities with the small heat-shock proteins. One of the properties in common is the inhibition of elastase. We now report that the separated subunits of α-crystallin, αA and αB,also exhibit elastase inhibition, whereas phosphorylation of these subunits apparently has no influence on the inhibitory capacity. Furthermore, for both αA-crystallin and mouse HSP25 the putative C-terminal structural domain, comprising the major region of homology between these proteins, is sufficient to give elastase inhibition. With database search no homology could be found between the three proteins under investigation and any of the known consensus sequences of proteinase inhibitor families.

Rotational Inhibition and Magnetization Transfer in α-Crystallin Solutions

The magnetic field dependence (dispersion profile) of 1/ T 1 of solvent water protons of solutions of the mammalian eye lens protein α-crystallin has a reversible nonlinear dependence on concentration which, as protein concentration increases above ∼20 wt.%, changes rapidly from a profile characteristic of mobile protein solute to a profile characteristic of rotationally immobilized protein (e.g., chemically cross-linked). From quantitative comparisons of new measurements of K at 200.1 MHz, the rate of water-to-protein interfacial magnetization transfer, with earlier data for α-crystallin and bovine serum albumin (BSA), we conclude, for α-crystallin, that: (i) Brownian rotation is slowed by intermolecular interactions at unexpectedly low concentrations; (ii) K is mediated by the newly reported, long-lived, protein hydration sites with the same surface density as BSA; and (iii) 14N peaks seen in solvent 1/ T 1 arise from interfacial magnetization transfer plus diffusion to protein NH magnetization sinks.

Functional elements DE2A, DE2B, and DE1A and the TATA box are required for activity of the chicken alpha A-crystallin gene in transfected lens epithelial cells.

alpha A-crystallin is an abundant soluble protein of the vertebrate eye lens. In addition to the TATA box, four positive cis-regulatory elements of the chicken alpha A-crystallin gene have been identified by linker scanning mutagenesis, DNase I footprinting, and gel mobility shift experiments. The regulatory elements described here have been named DE2A (at positions -144 to -134), DE2B (at positions -128 to -118), and DE1A (at positions -114 to -103). DE2A and DE2B form a dyad of symmetry between positions -141 and -118 (5'-AGACTGTCAT....AGGTCAGTCT-3'), consistent with the close similarity in the mobility of complexes formed with lens nuclear proteins by these two elements. Mutations in DE2A, DE2B, and DE1A leading to loss of promoter activity using the bacterial chloramphenicol acetyltransferase reporter gene transfected into primary embryonic chicken lens epithelial cells resulted in a corresponding loss in the ability to compete for complex formation with lens nuclear proteins in gel mobility shift assays. Mutation of the alpha A-CRYBP1-like site (-67/-57), necessary for function of the mouse alpha A-crystallin promoter, did not affect the activity of the chicken promoter. The DNase I footprinting and gel mobility shift experiments confirmed the previously noted binding of nuclear proteins to a dyad of symmetry at positions -153 to -140. In contrast to DE2A, DE2B, and DE1A, mutagenesis and gel mobility shift experiments failed to correlate function and protein binding for the -153/-140 dyad. DE2A, DE2B, and DE1A agree well with the regulatory elements alpha CE1 (-162/-134), alpha CE3 (-135/-121), and alpha CE2 (-119/-99) (Matsuo, I., and Yasuda, K. (1992) Nucleic Acids Res. 20, 3701-3712) for this gene. The present results suggest, however, that the lens enhancer activity of alpha CE1 is due to the sequence -141/-134, which forms the upper half of the DE2A/DE2B dyad of symmetry, rather than the -153/-140 dyad as previously suspected.

α-Crystallin, a Molecular Chaperone, Forms a Stable Complex with Carbonic Anhydrase upon Heat Denaturation

Alpha-crystallin, a major eye lens protein of vertebrates has been characterized as a molecular chaperone based on its ability to inhibit the aggregation of proteins undergoing thermal denaturation (Horwitz, J., Proc. Natl. Acad. Sci. USA 1992, 89, 10449-10453). To understand the mechanisms underlying this chaperone-like activity, the present study addressed molecular interactions between α-crystallin and its target proteins. Using carbonic anhydrase as a model target protein, we demonstrate complex formation between the 2 proteins upon heating, as assessed by the criteria of agarose gel electrophoresis, immunoprecipitation, ultrafiltration and gel filtration chromatography. The complex of α-crystallin and carbonic anhydrase is stable, at room temperature and at 4°C, for over 18 hours, and is non-covalent in nature. The results also indicate that α-crystallin binds the early non-native form of the target protein.

Age-Related Changes of Water-Soluble Proteins of Human Eye Lens during the Prenatal Period

Saline extracts from human lenses were studied during prenatal eye development. Using immunodiffusion and immunoelectrophoresis with a specific antiserum against total extract of human adult lenses and monospecific antisera against the individual crystallin classes, it was established that alpha and beta crystallins were present in extracts from eye lenses from 5-week-old embryos while gamma crystallins could be detected in extracts from embryonic lenses at the beginning of the 6th gestational week. When analyzed by isoelectric focussing all alpha and beta crystallin components were found in extracts from 8-week-old embryos, while those of gamma crystallins were shown to appear successively within the period between the 8th and 14th gestational week. Some changes in the relative proportion of the different crystallin classes and subclasses were observed in the course of prenatal eye lens development between the 8th and 26th gestational week, as the percentage of alpha, high molecular weight and gamma crystallins was increased while the proportion of beta crystallins was gradually lowered.