Ubiquitous lens alpha-, beta-, and gamma-crystallins accumulate in anuran cornea as corneal crystallins

Corneal epithelium is known to have high levels of some metabolic enzymes such as aldehyde dehydrogenase in mammals, gelsolin in zebrafish, and alpha-enolase in several species. Analogous to lens crystallins, these enzymes and proteins are referred to as corneal crystallins, although their precise function is not established in any species. Although it is known that after lentectomy, the outer cornea undergoes transdifferentiation to regenerate a lens only in anuran amphibians, major proteins expressed in an anuran cornea have not been identified. This study therefore aimed to identify the major corneal proteins in the Indian toad (Bufo melanostictus) and the Indian frog (Rana tigrina). Soluble proteins of toad and frog corneas were resolved on two-dimensional gels and identified by matrix-assisted laser desorption ionization time-of-flight/time-of-flight and electrospray ionization quadrupole time-of-flight. We report that anuran cornea is made up of the full complement of ubiquitous lens alpha-, beta-, and gamma-crystallins, mainly localized in the corneal epithelium. In addition, some taxon-specific lens crystallins and novel proteins, such as alpha- or beta-enolase/tau-crystallin, were also identified. Our data present a unique case of the anuran cornea where the same crystallins are used in the lens and in the cornea, thus supporting the earlier idea that crystallins are essential for the visual functions of the cornea as they perform for the lens. High levels of lens alpha-, beta-, and gamma-crystallins have not been reported in the cornea of any species studied so far and may offer a possible explanation for their inability to regenerate a lens after lentectomy. Our data that anuran cornea has an abundant quantity of almost all the lens crystallins are consistent with its ability to form a lens, and this connection is worthy of further studies.     

Vertebrate crystallins- from proteins to genes

A review of literature on tissue-specific proteins of the vertebrate eye lens and genes coding for these proteins is presented. Particular attention is paid to the most heterogeneous family of crystallins: beta- and gamma-crystallins, their nomenclature, and the structure of their genes. It is pointed out that mutations in gene coding for ubiquitous crystallins may be related to some forms of cataracts.     

Immunochemical markers of embryonic lens differentiation in Rana temporaria. II. Immunohistochemical analysis of the manifestation and localization of individual classes of lens proteins]

Individual lens proteins were studied during development of Rana temporaria. Antisera to alpha-, beta-crystallins of chicks and gamma-crystallins of Rana ridibunda were used as immunochemical markers. Besides the main crystallins, a new antigen was found in the R. temporaria lens tentatively called alphabeta-crystallin. It appears to be characteristic only for the amphibian lens. Using the indirect method of fluorescent antibodies, it was shown that all the antigens under study appeared in the lens of the R. temporaria tadpoles within 1--2 days (at 20 degrees). The crystallins are found initially only in the developing lens fibers and later in the lens epithelium. It was established that the lens epithelium contained gamma-crystallins which appeared somewhat earlier than alpha- and beta-crystallins, but simultaneously with alphabeta-crystallin.     

Immunochemical markers of embryonic lens differentiation in Rana temporaria. I. Composition and properties of water-soluble lens antigens]

Antisera were obtained to the total extract and individual electrophoretic fractions of lens proteins: alpha-, beta-, gamma1- and gamma2-crystallins. The crystallins under study are immunochemically heterogenous: each class of lens proteins contains 2--4 antigens. Using the indirect method of fluorescent antibodies, it was established that the appearance of crystallins during development coincided with the onset of formation of the presumptive lens fibers. No crystallins were found in the lens placode and early lens vesicle. gamma-Crystallins appear later than the other lens proteins and are characteristic, mainly, for the lens fibers; at the advanced stages of organogenesis gamma-crystallins are regularly found in the epithelial cells of the developing lens as well.     

Surface interactions of gamma-crystallins in the crystal medium in relation to their association in the eye lens

A comparative study of intermolecular interactions in crystals of two homologous low molecular weight proteins, gamma-II and gamma-IIIb crystallins, from calf eye lens was carried out. Crystal packings for these proteins are very different: intermolecular contact areas compose about 33% of the total accessible surface area of gamma-II as compared with 13% in gamma-III. Two key residues seem to be mainly responsible for the differences in protein association in the crystal medium. These are Ser 103 and Leu 155 in gamma-II, which are replaced by Met 103 and His 155 in gamma-IIb. A similar substitution of these residues is observed in different gene products of gamma-crystallins from a number of vertebrates. This is consistent with the existence of a genetically controlled mechanism for determining intermolecular association of gamma-crystallins in the native medium of the lens.     

Solution properties of γ‐crystallins: Compact structure and low frictional ratio are conserved properties of diverse γ‐crystallins

γ‐crystallins are highly specialized proteins of the vertebrate eye lens where they survive without turnover under high molecular crowding while maintaining transparency. They share a tightly folded structural template but there are striking differences among species. Their amino acid compositions are unusual. Even in mammals, γ‐crystallins have high contents of sulfur‐containing methionine and cysteine, but this reaches extremes in fish γM‐crystallins with up to 15% Met. In addition, fish γM‐crystallins do not conserve the paired tryptophan residues found in each domain in mammalian γ‐crystallins and in the related β‐crystallins. To gain insight into important, evolutionarily conserved properties and functionality of γ‐crystallins, zebrafish (Danio rerio) γM2b and γM7 were compared with mouse γS and human γD. For all four proteins, far UV CD spectra showed the expected β‐sheet secondary structure. Like the mammalian proteins, γM7 was highly soluble but γM2b was much less so. The heat and denaturant stability of both fish proteins was lower than either mammalian protein. The ability of full‐length and truncated versions of human αB‐crystallin to retard aggregation of the heat denatured proteins also showed differences. However, when solution behavior was investigated by sedimentation velocity experiments, the diverse γ‐crystallins showed remarkably similar hydrodynamic properties with low frictional ratios and partial specific volumes. The solution behavior of γ‐crystallins, with highly compact structures suited for the densely packed environment of the lens, seems to be highly conserved and appears largely independent of amino acid composition.     

gammaN-crystallin and the evolution of the betagamma-crystallin superfamily in vertebrates

The beta and gamma crystallins are evolutionarily related families of proteins that make up a large part of the refractive structure of the vertebrate eye lens. Each family has a distinctive gene structure that reflects a history of successive gene duplications. A survey of gamma-crystallins expressed in mammal, reptile, bird and fish species (particularly in the zebrafish, Danio rerio) has led to the discovery of gammaN-crystallin, an evolutionary bridge between the beta and gamma families. In all species examined, gammaN-crystallins have a hybrid gene structure, half beta and half gamma, and thus appear to be the 'missing link' between the beta and gamma crystallin lineages. Overall, there are four major classes of gamma-crystallin: the terrestrial group (including mammalian gammaA-F); the aquatic group (the fish gammaM-crystallins); the gammaS group; and the novel gammaN group. Like the evolutionarily ancient beta-crystallins (but unlike the terrestrial gammaA-F and aquatic gammaM groups), both the gammaS and gammaN crystallins form distinct clades with members in fish, reptiles, birds and mammals. In rodents, gammaN is expressed in nuclear fibers of the lens and, perhaps hinting at an ancestral role for the gamma-crystallins, also in the retina. Although well conserved throughout vertebrate evolution, gammaN in primates has apparently undergone major changes and possible loss of functional expression.     

Homology models of human gamma-crystallins: structural study of the extensive charge network in gamma-crystallins

The lens is composed of highly stable and long-lived proteins, the crystallins which are divided into alpha-, beta-, and gamma-crystallins. Human gamma-crystallins belong to the betagamma superfamily. A large number of gamma-crystallins have been sequenced and have been found to share remarkable sequence homology with each other. Some of the gamma-crystallins from various sources have also been elucidated structurally by X-ray crystallographic or NMR spectroscopic experiments. Their three-dimensional structures are also similar having consisted of two domains each possessing two Greek key motifs. In this study we have constructed the comparative or homology models of the four major human gamma-crystallins, gammaA-,gammaB-, gammaC-, and gammaD-crystallins and studied the charge network in these crystallins. Despite an overall structural similarity between these crystallins, differences in the ion pair formation do exist which is partly due to the differences in their primary sequence and partly due to the structural orientation of the neighboring amino acids. In this study, we present an elaborate analysis of these charged interactions and their formation or loss with respect to the structural changes.     

Thermodynamics of thermal and athermal denaturation of gamma-crystallins: changes in conformational stability upon glutathione reaction

The conformational stabilities of bovine lens gamma-crystallin fractions II, IIIA, IIIB, and IVA and those modified with glutathione were compared by studying the thermal and guanidine hydrochloride (Gdn-HCl) denaturation behavior. The conformational state was monitored by both far-UV CD and fluorescence measurements. All the gamma-crystallins studied showed a sigmoidal order-disorder transition with varied melting temperatures. The thermal denaturation of these proteins is reversible up to a temperature 3 or 4 degrees C above T 1/2; above this temperature, irreversible aggregation occurs. The validity of a two-state approximation of both thermal and Gdn-HCl denaturation was tested for all four crystallins, and the presence of one or more intermediates was evident in the unfolding of IVA. delta GDH2O values of these crystallins range from 4 to 9 kcal/mol. Upon glutathione treatment IVA showed the maximum decrease in T 1/2 by approximately 9 degrees C and in delta GDH2O value by 29%; the smallest decrease in T 1/2 was for IIIA by 2 degrees C and in delta GDH2O by 15%. We have demonstrated that the glutathione reaction can dramatically reduce the conformational stability of gamma-crystallins and, thus, that the thermodynamic quantities of the unreacted crystallins can be used to evaluate the stability of these proteins when modified during cataract formation.     

Immunochemical study of the water-soluble lens proteins in the embryo of Xenopus laevis with the mutation of periodic albinism]

The crystallins of ap mutants of Xenopus laevis have been studied in comparison with those of normal embryos and adults using the complex of immunochemical methods (immunoelectrophoresis, immunodiffusion, immunoadsorption, immunofluorescence, isoelectrofocusing with immunoidentification). The analysis was carried out with antisera to electrophoretic fractions of the mutant lens. 11 organ-specific antigens were found in the lens of both the normal and mutant animals. These proteins are heterogenous by electrophoretic mobility, isoelectrical point, antigenic and species specificity. Each class of crystallins contains antigens which are specific: a) for amphibians only, b) for lower vertebrates, c) for vertebrates in general. No qualitative differences were found between crystallins of the normal and mutant animals. Immunofluorescence analysis has shown that crystalins appear in the normal and mutant embryos practically at the same time. No significant differences in the appearance of specific immunofluorescence between the normal and mutant embryos were found (with various antisera). gamma-crystallins and, perhaps, a part of the primary lens fibers. Alpha-crystallins appear later. gamma-crystallins are first identified the synthesis of which manifests itself at the advanced developmental stages. The quantitative predominance of some beta--gamma-crystallins in the mutant lens detected by us (electrophoresis, isoelectrofocusing) is not related to their earlier synthesis in the embryogenesis.     

Lactate dehydrogenase A as a highly abundant eye lens protein in platypus (Ornithorhynchus anatinus): upsilon (upsilon)-crystallin

Vertebrate eye lenses mostly contain two abundant types of proteins, the alpha-crystallins and the beta/gamma-crystallins. In addition, certain housekeeping enzymes are highly expressed as crystallins in various taxa. We now observed an unusual approximately 41-kd protein that makes up 16% to 18% of the total protein in the platypus eye lens. Its cDNA sequence was determined, which identified the protein as muscle-type lactate dehydrogenase A (LDH-A). It is the first observation of LDH-A as a crystallin, and we designate it upsilon (upsilon)-crystallin. Interestingly, the related heart-type LDH-B occurs as an abundant lens protein, known as epsilon-crystallin, in many birds and crocodiles. Thus, two members of the ldh gene family have independently been recruited as crystallins in different higher vertebrate lineages, suggesting that they are particularly suited for this purpose in terms of gene regulatory or protein structural properties. To establish whether platypus LDH-A/upsilon-crystallin has been under different selective constraints as compared with other vertebrate LDH-A sequences, we reconstructed the vertebrate ldh-a gene phylogeny. No conspicuous rate deviations or amino acid replacements were observed.     

Lens crystallins and oxidation: the special case of gammaS

Among lens crystallins, gamma-crystallins are particularly sensitive to oxidation, because of their high amount of Cys and Met residues. They have the reputation to induce, upon ageing, lens structural modifications leading to opacities. A combination of small angle X-ray scattering and chromatography was used to study the oxidation of gamma-crystallins. At pH 7.0, all the gamma-crystallins under study were checked to have the same structure in solution. Under gentle oxidation conditions at pH 8.0, human gammaS (hgammaS) and bovine gammaS (bgammaS) formed disulfide-linked dimers, whereas the other bgamma-crystallins did not. Cys20 was shown to be responsible for dimer formation since the C20S mutant only formed monomers. The hgammaS dimers were stable for weeks and did not form higher oligomers. In contrast, monomeric gammaS-crystallins freshly prepared at pH 8.0, and submitted to more drastic oxidation by X-ray induced free radicals, were rapidly transformed into higher oligomers. So, only extensive oxidation causing partial unfolding could be detrimental to the lens and linked to cataract formation. The gammaS-crystallins lack the temperature-induced opacification observed with the other gamma-crystallins and known as cold cataract. The oxidation-induced associative behaviour and cold cataract are therefore demonstrated to be uncoupled.     

Acetylation of lens crystallins: A possible mechanism by which aspirin could prevent cataract formation

The calf eye lens homogenate incubated with [1-¹⁴C-acetyl] aspirin and separated into HMW, α, β_H, β_L and γ-crystallins by means of Sepharose 6B and Bio-Gel P2 columns showed radioactivity in all the crystallins. In contrast, no radioactivity was found in the crystallins when the lens homogenate was incubated with [¹⁴C-carboxyl] aspirin. These experiments clearly indicated that the eye lens crystallins are acetylated with aspirin. Furthermore, no decrease in the radioactivity in the crystallins after exhaustive dialysis against 0.15M NaCl suggests a covalent type of binding of acetyl moiety of aspirin to the lens crystallins. The significant decrease in the free ε-amino groups of aspirin-treated crystallins further suggests the probable sites of acetylation in the crystallins. It may be concluded that acetylation of free ε-amino groups of lens crystallins by aspirin may confer protection against crystallin aggregation in cataractogenesis.     

Synchronous scan fluorescence spectroscopy of proteins and human eye lenses

Scanning both the excitation and emission monochromators synchronously while recording the fluorescence spectrum results in a considerable decrease in the apparent band width and shift in the peak position. We demonstrate the potential of this approach in the studies on proteins and their interactions as well as fluorophores in condensed media. We have chosen crystallins, eye lens proteins and human lenses. Synchronous scan spectra of alpha-, beta- and gamma-crystallins are clearly distinguishable and appear to provide specific signatures. The spectrum of the mixed solution could be simulated by the linear combination of components indicating that these proteins might not have any specific interaction in the dilute solutions. Synchronous spectra of the human lenses, both normal and cataractous, show several distinguishable features.     

Evolution of crystallins: expression of lens-specific proteins in the blind mammals mole (Talpa europaea) and mole rat (Spalax ehrenbergi)

The mole (Talpa europaea; Insectivora) and the mole rat (Spalax ehrenbergi; Rodentia) both have degenerated eyes as a convergent adaptation to subterranean life. The rudimentary eye lenses of these blind mammals no longer function in a visual process. The crystallin genes, which display a lens-specific expression pattern, were studied in these blind mammals and in related species with normal eyes by hybridizing their genomic DNAs with probes obtained from cDNA clones for alpha A-, alpha B-, and beta Bp-crystallins from calf and gamma 3- crystallin from the rat. For all crystallin genes examined, the hybridization signals of mole and mole rat genomic DNA were comparable, respectively, with those of shrew and of rat and mouse, normal-vision representatives of the orders Insectivora and Rodentia. The expression of the crystallins at the protein level was tested by using antiserum specific for alpha-crystallin in immunofluorescence reactions on lens sections of mole and mole rat eyes and by using antisera against the beta- and gamma-crystallins on sections of the mole eye. All antisera gave positive fluorescence reactions exclusively with lens tissue of these blind mammals, indicating that the crystallins are still normally expressed despite the fact that these lenses have had no function in a visual process in these mammals for at least many million years. These findings apparently imply that some unknown selective advantage has conserved the crystallin genes and their expression after the loss of normal function of the lenses.      

Crystallins: recruiting proteins to perform new functions

The main properties of major water-soluble proteins of the lens, i.e., crystallins, include high ability to aggregation and stability. It is these properties of crystallins which are required for the formation of the transparent lens and maintenance of lens transparency over the whole lifespan of the animal.     

Comparative study of crystallins from the nucleus and cortex of the bovine ocular lens by the gel filtration and x-ray diffraction methods

Water--soluble proteins (alpha-, beta H-, beta L- and gamma-crystallins) from the bovine lens nucleus and cortex were fractionated and compared by gel filtration on Sephadex G-200. X-ray diffraction patterns from concentrated gels of these proteins were obtained. It allowed to compare qualitatively the structures of different crystallins and also to identify the maxima on X-ray diffraction patterns of the lens intact tissue.     

Glycation by ascorbic acid oxidation products leads to the aggregation of lens proteins

Previous studies from this laboratory have shown that there are striking similarities between the yellow chromophores, fluorophores and modified amino acids released by proteolytic digestion from calf lens proteins ascorbylated in vitro and their counterparts isolated from aged and cataractous lens proteins. The studies reported in this communication were conducted to further investigate whether ascorbic acid-mediated modification of lens proteins could lead to the formation of lens protein aggregates capable of scattering visible light, similar to the high molecular aggregates found in aged human lenses. Ascorbic acid, but not glucose, fructose, ribose or erythrulose, caused the aggregation of calf lens proteins to proteins ranging from 2.2 x 10(6) up to 3.0 x 10(8 )Da. This compared to proteins ranging from 1.8 x 10(6) up to 3.6 x 10(8 )Da for the water-soluble (WS) proteins isolated from aged human lenses. This aggregation was likely due to the glycation of lens crystallins because [U-(14)C] ascorbate was incorporated into the aggregate fraction and because NaCNBH(3), which reduces the initial Schiff base, prevented any protein aggregation. Reactions of ascorbate with purified crystallin fractions showed little or no aggregation of alpha-crystallin, significant aggregation of beta(H)-crystallin, but rapid precipitation of purified beta(L)- and gamma-crystallin. The aggregation of lens proteins can be prevented by the binding of damaged crystallins to alpha-crystallin due to its chaperone activity. Depending upon the ratios between the components of the incubation mixtures, alpha-crystallin prevented the precipitation of the purified beta(L)- and gamma-crystallin fractions during ascorbylation. The addition of at least 20% of alpha-crystallin by weight into glycation mixtures with beta(L)-, or gamma-crystallins completely inhibited protein precipitation, and increased the amount of the high molecular weight aggregates in solution. Static and dynamic light scattering measurements of the supernatants from the ascorbic acid-modified mixtures of alpha- and beta(L)-, or gamma-crystallins showed similar molar masses (up to 10(8 )Da) and hydrodynamic diameter (up to 80( )nm). These data support the hypothesis, that if the lens reducing environment is compromised, the ascorbylation of lens crystallins can significantly change the short range interactions between different classes of crystallins leading to protein aggregation, light scattering and eventually to senile cataract formation.     

Rat lens gamma-crystallins. Characterization of the six gene products and their spatial and temporal distribution resulting from differential synthesis

We have isolated, purified and characterized six individual gamma-crystallin polypeptides present in the rat lens. Comparison of their amino acid compositions with the known structure of the six gamma-crystallin genes permits a one-to-one correspondence to be made between each protein synthesized and the encoding gene. This demonstrates that each of the six genes is actually expressed in vivo. Two classes of three gamma-crystallins each, which we have designated classes gamma ABC and gamma DEF, are known to exist, on the basis of internal sequence homology. We have measured the temperature-dependent phase-separation characteristics of solutions of the six purified gamma-crystallins, and find that the three members of the gamma DEF class (gamma 2-2, gamma 3-1 and gamma 4-1) are all cryo-proteins with relatively high phase-separation temperatures, whereas the three gamma ABC crystallins (gamma 1-1, gamma 1-2 and gamma 2-1) do not show phase separation above -7 degrees C. We have measured the spatial distribution in rat lens of each of the alpha-, beta- and gamma-crystallins as a function of age from 1 to 420 days, using size-exclusion and ion-exchange high-pressure liquid chromatography (HPLC). Our findings in the cortical layer permit us to establish the differential synthesis of each of the crystallins during lens development. Particular attention has been devoted to the spatial and temporal distribution of the six individual gamma-crystallins. Up to birth, synthesis of the three components of the gamma DEF class predominates, and in particular that of gamma 2-2. In subsequent development the three components of the gamma ABC class assume a greater proportion of monomeric crystallins synthesized, while beta s-crystallin synthesis predominates in late development. Our analysis of different layers within single lenses provides novel information on spatial gradients of the water-soluble and water-insoluble protein fractions as a function of age. We consider the consequences of these findings for lens transparency and opacity in both rat and mouse lens. We show that the high concentrations of gamma DEF-crystallins appear to be responsible for the opacity known to occur in young rat lenses. We conclude from these observations that close control of the differential synthesis of gamma-crystallins plays an important role in maintaining lens transparency during development.     

Crystallin distribution patterns in Litoria infrafrenata and Phyllomedusa sauvagei lenses

The eye lens remains transparent because of soluble lens proteins known as crystallins. For years γ-crystallins have been known as the main lens proteins in lower vertebrates such as fish and amphibians. The unique growth features of the lens render it an ideal structure to study ageing; few studies have examined such changes in anuran lenses. This study aimed to investigate protein distribution patterns in Litoria infrafrenata and Phyllomedusa sauvagei species. Lenses were fractionated into concentric layers by controlled dissolution. Water-soluble proteins were separated into high (HMW), middle (MMW) and low molecular weight (LMW) fractions by size-exclusion HPLC and constituents of each protein class revealed by 1DE and 2DE. Spots were selected from 2DE gels on the basis of known ranges of subunit molecular weights and pH ranges and were identified by MALDI-TOF/TOF MS following trypsin digestion. Comparable lens distribution patterns were found for each species studied. Common crystallins were detected in both species; the most prominent of these was γ-crystallin. Towards the lens centre, there was a decrease in α- and β-crystallin proportions and an increase in γ-crystallins. Subunits representing taxon-specific crystallins demonstrating strong sequence homology with ζ-crystallin/quinone oxidoreductase were found in both L. infrafrenata and P. sauvagei lenses. Further work is needed to determine which amphibians have taxon-specific crystallins, their evolutionary origins, and their function.