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.      

Metabolism of crystallin fragments in cell-free extracts of bovine lens: effects of ageing and oxygen free-radicals.

1. The ability of cell-free preparations from bovine lens to degrade fragments of alpha-crystallin has been studied. Crystallin fragments, produced by either chemical cleavage with cyanogen bromide or prolonged treatment with H2O2 and Cu2+ to produce hydroxyl radicals, were labelled with 125I and incubated with preparations obtained from lenses from animals of different age. 2. Results showed that the ability of the preparations obtained from the lens cores (the innermost part of the lens composed of enucleated non-dividing cells incapable of protein synthesis) to degrade crystallin fragments decreased with animal age. No such age-related correlation was obtained with preparations obtained from the cortex (the outer region of the lens surrounding the core). 3. The effect of incubation of the various lenticular preparations with H2O2 and Cu2+ on subsequent ability to catabolise crystallin fragments was also examined. Preparations from the oldest lenses were found to be the least resistant to free-radical attack. 4. The relative susceptibility of the crystallins and non-lenticular proteins to H2O2/Cu(2+)-mediated free-radical attack was examined. Not only were the various crystallins (alpha, beta and gamma) far more resistant to cleavage under these conditions, they also protected the non-lenticular proteins from free-radical-mediated attack. The comparative resistance of the crystallins to attack and their ability to protect other proteins appeared to be dependent on their structural integrity as prior denaturation with acid and/or cleavage with cyanogen bromide eliminated these properties. 5. It is suggested that crystallins (which show sequence homology to some heat-shock proteins) possess homeostatic functions which could protect other proteins (e.g. proteases) from certain forms of free-radical-mediated damage; crystallins may therefore be important in ageing in general where aberrant polypeptides accumulate.     

Characterization of lens crystallins and their mRNA from the carp lenses

Crystallins from carp eye lenses have been isolated and characterized by gel permeation chromatography, SDS-gel electrophoresis, immunodiffusion and amino acid analysis. gamma-Crystallin is the most abundant class of crystallins and constitutes over 55% of the total lens cytoplasmic proteins. It is immunologically distinct from the alpha- and beta-crystallins isolated from the same lens and its antiserum shows a very weak cross-reaction to total pig lens antigens. Comparison of the amino acid compositions of carp gamma-crystallin with those of bovine gamma-II, haddock gamma- and squid crystallins indicates that gamma-crystallin from the carp is very closely related to that of the haddock, and probably also related to the invertebrate squid crystallin. In vitro translation of total mRNAs isolated from carp lenses confirms the predominant existence of gamma-crystallin. The genomic characterization of carp crystallin genes should provide some insight into the mechanism of crystallin evolution in general.     

Epidermal fucosylation of cell surface glycoprotein

The formation of covalently linked, high molecular weight protein aggregates has been thought to play an important role in opacification of the human lens. Antisera were used in Western blot analysis to demonstrate the involvement of all major classes of lens proteins (alpha, beta and gamma crystallin; the major intrinsic membrane polypeptide) in covalent aggregation. Of these classes, aggregation of gamma and beta crystallins via intermolecular disulfide bonding and aggregation of the major intrinsic membrane polypeptide via intermolecular, non-disulfide bonding were more pronounced in cataractous as compared with normal lenses.     

The structure and expression of a distantly related member of the beta-gamma crystallin super gene family from Xenopus.

In an attempt to understand amphibian crystallin gene regulation, we have isolated and partially characterized several genomic clones which hybridized to the gamma 1 cDNA probe from Rana temporaria. A complete sequence of one of these clones showed slight homology to the mammalian beta and gamma crystallins. The deduced amino acid sequence of the coding region and its alignment as a folding unit indicated that all the topologically equivalent residues involved in maintaining the protein folding pattern are highly conserved. Northern blot analysis of total RNAs derived from several adult tissues, including eye lens, suggested that an RNA of approximately 700 nucleotides long is present in lens, heart, spleen and embryos of later stages of development but not in retina, oocytes and embryos of early developmental stages.     

Comparative x-ray diffraction study of the crystalline lens in a number of vertebrates including man

X-ray diffraction method has been applied for comparative investigation of native structure of eye lens proteins (crystallins). X-ray diffraction patterns of the whole lenses and/or their nuclear parts were obtained for man and vertebrate animals. Crystalline lenses of the fishes Acerina cernua and Pelmatochromis kribensis, frog Rana temporaria, bull and man contain crystallins with a very similar secondary and tertiary structure, whereas lenses of chicks and the tortoise Testudo horsfieldi contain mainly crystallins with other structure. The results obtained reveal evolutionary conservatism of crystallin structure in fishes, amphibians and mammals. It was also concluded that there is no correlation between crystallin structure of the lens, elasticity of the latter and accommodation mechanism.     

Crystallins of the octopus lens. Recruitment from detoxification enzymes.

The eye lens crystallins of the octopus Octopus dofleini were identified by sequencing abundant proteins and cDNAs. As in squid, the octopus crystallins have subunit molecular masses of 25-30 kDa, are related to mammalian glutathione S-transferases (GST), and are encoded in at least six genes. The coding regions and deduced amino acid sequences of four octopus lens cDNAs are 75-80% identical, while their non-coding regions are entirely different. Deduced amino acid sequences show 52-57% similarity with squid GST-like crystallins, but only 20-25% similarity with mammalian GST. These data suggest that the octopus and squid lens GST-like crystallin gene families expanded after divergence of these species. Northern blot hybridization indicated that the four octopus GST-like crystallin genes examined are lens-specific. Lens extracts showed about 40 times less GST activity using 1-chloro-2,4-dinitrobenzene as substrate than liver extracts of the octopus, indicating that the major GST-like crystallins are specialized for a lens structural role. A prominent 59-kDa crystallin polypeptide, previously observed in octopus but not squid and called omega-crystallin (Chiou, S.-H. (1988) FEBS Lett. 241, 261-264), has been identified as an aldehyde dehydrogenase. Since cytoplasmic aldehyde dehydrogenase is a major protein in elephant shrew lenses (eta-crystallin; Wistow, G., and Kim, H. (1991) J. Mol. Evol. 32, 262-269) the octopus aldehyde dehydrogenase crystallin provides the first example of a similar enzyme-crystallin in vertebrates and invertebrates. The use of detoxification stress proteins (GST and aldehyde dehydrogenase) as cephalopod crystallins indicates a common strategy for recruitment of enzyme-crystallins during the convergent evolution of vertebrate and invertebrate lenses. For historical reasons we propose that the octopus GST-like crystallins, like those of the squid, are called S-crystallins.     

Comparative study of lens proteins of gray squirrel and human

1. The four crystallins of the gray squirrel lens have been characterized using gel filtration chromatography, polyacrylamide gel electrophoresis, and immunoblotting. Alpha, beta-heavy, beta-light, and gamma crystallins of squirrel lenses have been identified immunologically, and they cross-react strongly with rabbit polyclonal antibodies. The gamma-24 crystallin of the squirrel lens also reacts strongly with monoclonal anti-human lens gamma-24, as shown by its inhibition of the ELISA reaction by 85%. 2. The water-insoluble urea soluble proteins represent non-covalently associated species of soluble crystallins and the lens cytoskeletal proteins. The membrane intrinsic protein in the urea insoluble pellet has a mol. wt of 27,000 but other lower and higher mol. wt components are also present, which were removed by washing with 0.1 NaOH. The N-terminal 30 amino acid of squirrel lens gamma crystallin was found to be identical to that of the bovine (and human) lens. 3. Measurements of the distribution and state of SH and SS compounds in the squirrel lens have shown greater similarities to those of primates than those of rodents. The findings show that on the basis of both protein and sulfur chemistry the squirrel lens is a representative model for studies of oxidative lens changes in diurnal animals, including man.     

Lens crystallin changes associated with amphibian metamorphosis: involvement of a beta-crystallin polypeptide

Lens crystallins isolated from the tadpole and frog lenses were compared with regard to the developmental changes of crystallin compositions. The major changes during the process of metamorphosis were (1) the total contents of alpha- and gamma-crystallins decrease from more than 70% to less than 60% and (2) one of the major beta-crystallin polypeptides increases from less than 1% to about 6% and (3) an amphibian-specific rho-crystallin also increases from about 6% to more than 10% of total soluble proteins of the lens. We have characterized the metamorphosis-dependent beta-crystallin polypeptide by peptide mapping and sequence determination of the protease-digested fragments. This polypeptide showed very high sequence homology to that of the major beta Bp-crystallin chain reported for the mammalian lenses. The changes of the relative abundance of various crystallins and the gradually-elevated levels of the expression of this beta Bp-like crystallin in the developing lens during metamorphosis may also have some bearing on the maintenance of lens stability in the adult frog lenses.     

Crystallin distribution patterns in concentric layers from toad eye lenses

Protein distribution patterns across eye lenses from the Asiatic toad Bufo gargarizans were investigated and individual crystallin classes characterised. Special fractionation that follows the growth mode of the lens was used to yield nine fractions corresponding to layers laid down at different chronological (developmental) stages. Proportions of soluble and insoluble crystallins within each fraction were measured by Bradford assay. Water‐soluble proteins in all fractions were separated by size‐exclusion HPLC and constituents of each class further characterised by electrophoresis, RP‐HPLC and MS analysis. In outer lens layers, α‐crystallin is the most abundant soluble protein but is not found in soluble proteins in the lens centre. Water‐soluble β‐crystallins also decrease from their highest level in the outer lens to negligible mounts in the central lens. The proportion of soluble γ‐crystallin increases significantly towards the lens centre where this is the only soluble protein present. Insoluble protein levels increase significantly towards the lens centre. In B. gargarizans lenses, as with other anurans, the predominant water‐soluble protein class is γ‐crystallin. No taxon‐specific crystallins were found. The relationship between the protein distribution patterns and the functional properties of the lens this species is discussed.     

A RADIOIMMUNOASSAY FOR THE DETECTION OF DELTA CRYSTALLIN IN CHICK LENS DIFFERENTIATION

Delta crystallin was isolated from 10–13 day chick embryo lens fiber cells. The lens fiber cell extract was isoelectrically precipitated at pH 5.1 to remove alpha and beta crystallins. Further purification by filtration through Sephadex G-150 and then acrylamide gel electrophoresis yielded a single, homogeneous preparation of delta crystallin, as characterized by gel electrophoresis. This purified delta crystallin was injected into rabbits to produce a potent antiserum to chick lens delta crystallin. The purified delta crystallin was iodinated with ¹²⁵Iodine, using the chloramine-T procedure. A radioimmunoassay for delta crystallin was then developed, using the principles of competitive protein binding analysis. The radioimmunoassay developed here had a minimum sensitivity of 50 nanograms, and effectively ranged to 1000 nanograms.
Developing lens rudiments from early chick embryos, beginning from 24 hr incubation up to 72 hr were examined at 6 hr intervals. All determinations from 24 hr through the 48 hr sample showed less than 10 nanograms per 100 lens rudiments. This was below the effective minimum detection limits of the assay. The first accumulation of delta crystallin was detected in the 54 hr sample, and increased thereafter.     

Solution properties of γ‐crystallins: Hydration of fish and mammal γ‐crystallins

Lens γ crystallins are found at the highest protein concentration of any tissue, ranging from 300 mg/mL in some mammals to over 1000 mg/mL in fish. Such high concentrations are necessary for the refraction of light, but impose extreme requirements for protein stability and solubility. γ‐crystallins, small stable monomeric proteins, are particularly associated with the lowest hydration regions of the lens. Here, we examine the solvation of selected γ‐crystallins from mammals (human γD and mouse γS) and fish (zebrafish γM2b and γM7). The thermodynamic water binding coefficient B₁ could be probed by sucrose expulsion, and the hydrodynamic hydration shell of tightly bound water was probed by translational diffusion and structure‐based hydrodynamic boundary element modeling. While the amount of tightly bound water of human γD was consistent with that of average proteins, the water binding of mouse γS was found to be relatively low. γM2b and γM7 crystallins were found to exhibit extremely low degrees hydration, consistent with their role in the fish lens. γM crystallins have a very high methionine content, in some species up to 15%. Structure‐based modeling of hydration in γM7 crystallin suggests low hydration is associated with the large number of surface methionine residues, likely in adaptation to the extremely high concentration and low hydration environment in fish lenses. Overall, the degree of hydration appears to balance stability and tissue density requirements required to produce and maintain the optical properties of the lens in different vertebrate species.     

Lens aging: Effects of crystallins

The primary function of the eye lens is to focus light on the retina. The major proteins in the lens—α, β, and γ-crystallins—are constantly subjected to age-related changes such as oxidation, deamidation, truncation, glycation, and methylation. Such age-related modifications are cumulative and affect crystallin structure and function. With time, the modified crystallins aggregate, causing the lens to increasingly scatter light on the retina instead of focusing light on it and causing the lens to lose its transparency gradually and become opaque. Age-related lens opacity, or cataract, is the major cause of blindness worldwide. We review deamidation, and glycation that occur in the lenses during aging keeping in mind the structural and functional changes that these modifications bring about in the proteins. In addition, we review proteolysis and discuss recent observations on how crystallin fragments generated in vivo, through their anti-chaperone activity may cause crystallin aggregation in aging lenses. We also review hyperbaric oxygen treatment induced guinea pig and ‘humanized’ ascorbate transporting mouse models as suitable options for studies on age-related changes in lens proteins.     

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

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

Crystallins in Retinal Ganglion Cell Survival and Regeneration

Crystallins are heterogeneous proteins classified into alpha, beta, and gamma families. Although crystallins were first identified as the major structural components of the ocular lens with a principal function to maintain lens transparency, further studies have demonstrated the expression of these proteins in a wide variety of tissues and cell types. Alpha crystallins (alpha A and alpha B) share significant homology with small heat shock proteins and have chaperone-like properties, including the ability to bind and prevent the precipitation of denatured proteins and to increase cellular resistance to stress-induced apoptosis. Stress-induced upregulation of crystallin expression is a commonly observed phenomenon and viewed as a cellular response mechanism against environmental and metabolic insults. However, several studies reported downregulation of crystallin gene expression in various models of glaucomatous nerodegeneration suggesting that that the decreased levels of crystallins may affect the survival properties of retinal ganglion cells (RGCs) and thus, be associated with their degeneration. This hypothesis was corroborated by increased survival of axotomized RGCs in retinas overexpressing alpha A or alpha B crystallins. In addition to RGC protective functions of alpha crystallins, beta and gamma crystallins were implicated in RGC axonal regeneration. These findings demonstrate the importance of crystallin genes in RGC survival and regeneration and further in-depth studies are necessary to better understand the mechanisms underlying the functions of these proteins in healthy RGCs as well as during glaucomatous neurodegeneration, which in turn could help in designing new therapeutic strategies to preserve or regenerate these cells.     

Biochemical and immunological studies of lentoid formation in cultures of embryonic chick neural retina and day-old chick lens epithelium

During long-term cell culture of 8-day embryonic chick neural retina, lentoid bodies containing lens crystallins are developed. Although very low levels of crystallin can be detected in the embryonic neural retina, gross synthesis of each major crystallin class (α, anodal β, cathodal β, and δ) begins only after 12–16 days in culture. This occurs at least 10 days before lentoid bodies can be distinguished by eye. The concentration of each crystallin class was determined during lentoid development in cultures of both neural retina and lens epithelium. The proportions of crystallins in lentoid-containing cultures do not resemble those of embryonic lens fibres. Comparisons between two chick strains (N and Hy-1) differing in their growth rates revealed several differences in the crystallin compositions of lentoid bodies. These differences imply independent quantitative regulation for most or all of the crystallins.     

Isolation and cell-free translation of chick lens crystallin mRNA during normal development and transdifferentiation of neural retina

Messenger RNA has been isolated from day-old chick lens. Size characterization and heterologous cell-free translation demonstrate that the predominant species of mRNA present code for α-, β- and δ-crystallins. Total polysomal RNA and polysomal RNA which did not bind to oligo (dT)-cellulose translate in the cell-free system to give a crystallin profile qualitatively similar to that of poly(A)⁺ mRNA. RNA from postribosomal supernatant which binds to oligo(dT)-cellulose also translates to give crystallins, but the products are enriched for β-crystallins. Messenger RNAs isolated from 15-day embryo lens fiber and lens epithelium cells give products on translation which reflect the different protein compositions of these two cell types, as do mRNAs isolated from chick lenses at various developmental stages. Messenger RNAs were isolated from freshly excised 8-day embryo neural retina and from this tissue undergoing transdifferentiation into lens cells in cell culture. Cell-free translation demonstrates no detectable crystallin mRNAs in the freshly excised material, but by 42 days in cell culture, crystallin mRNAs are the most prominent species.