Scallop lens Omega-crystallin (ALDH1A9): a novel tetrameric aldehyde dehydrogenase

Scallop eye lens Omega-crystallin is an inactive aldehyde dehydrogenase (ALDH1A9) related to cytoplasmic ALDH1A1 and mitochondrial ALDH2 that migrates by gel filtration chromatography as a homodimer. Because mammalian ALDH1A1 and ALDH2 are homotetramers, we investigated the native molecular mass of scallop Omega-crystallin by multi-angle laser light scattering. The results indicate that the scallop Omega-crystallin is a tetrameric, not a dimeric protein. Moreover, phylogenetic tree analysis shows that scallop Omega-crystallin clusters with the mitochondrial ALDH2 and ALDH1B1 rather than the cytoplasmic ALDH1A, yet it lacks the mitochondrial N-terminal leader sequence characteristic of the mitochondrial ALDHs. The mitochondrial grouping, enzymatic inactivity, and anomalous gel filtration behavior make scallop cytoplasmic Omega-crystallin an interesting protein for structural studies of evolutionary adaptations to become an enzyme-crystallin.     

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.     

Structure and expression of the scallop Omega-crystallin gene. Evidence for convergent evolution of promoter sequences.

Omega-crystallin of the scallop lens is an inactive aldehyde dehydrogenase (1A9). Here we have cloned the scallop Omega-crystallin gene. Except for an extra novel first exon, its 14-exon structure agrees well with that of mammalian aldehyde dehydrogenases 1, 2, and 6. The -2120/+63, -714/+63, and -156/+63 Omega-crystallin promoter fragments drive the luciferase reporter gene in transfected alphaTN4-1 lens cells and L929 fibroblasts but not in Cos7 cells. Putative binding sequences for cAMP-responsive element-binding protein (CREB)/Jun, alphaACRYBP1, AP-1, and PAX-6 in the Omega-crystallin promoter are surprisingly similar to the cis-elements used for lens promoter activity of the mouse and chicken alphaA-crystallin genes, which encode proteins homologous to small heat shock proteins. Site-specific mutations in the overlapping CREB/Jun and Pax-6 sites abolished activity of the Omega-crystallin promoter in transfected cells. Gel shift experiments utilizing extracts from the alphaTN4-1, L929, and Cos7 cells and the scallop stomach and oligonucleotides derived from the putative binding sites of the Omega-crystallin promoter showed complex formation. Gel shift experiments showed binding of recombinant Pax-6 and CREB to their respective sites. Our data suggest convergent evolutionary adaptations that underlie the preferential expression of crystallin genes in the lens of vertebrates and invertebrates.     

A novel crystallin from octopus lens

Lens crystallins were isolated from cephalopods, octopus and squid. Two protein fractions were obtained from the octopus in contrast to only one crystallin from the squid. The native molecular mass for these purified fractions and their polypeptide compositions were determined by gel filtration, sedimentation analysis, and SDS-gel electrophoresis. Octopod and decapod lenses share one common major squid-type crystallin of 29 kDa, with one additional novel crystallin present only in the octopus lens. This newly-characterized crystallin (termed omega-crystallin) exists as a tetrameric protein of 230 kDa, consisting of 4 identical subunits of approx. 59 kDa. It is distinct from the previously known crystallins both in amino acid composition and subunit structure. N-terminal sequence analysis indicated that the omega-crystallin is N-terminally blocked, whereas the major octopus crystallin is identical to the reported squid crystallin with regard to the first 25 residues of protein sequence. Sequence similarity between this major cephalopod crystallin and glutathione S-transferase were found, which suggested some enzymatic role of crystallins inside the cephalopod lens.     

Gene sharing, lens crystallins and speculations on an eye/ear evolutionary relationship

The crystallins comprise 80–90% of the water-soluble proteinsof the transparent, cellular, refractive eye lens and are responsiblefor its optical properties. Comparative studies have establishedthat the crystallins are surprisingly diverse and often differamong species in a taxon-specific fashion. In general, the crystallinsare derived from or identical to metabolic enzymes or stress(small heat shock) proteins that are expressed to a lesser extentin other tissues where they have non-refractive roles. We callthe phenomenon of having the small heat shock protein or enzymeand lens crystallin encoded in the identical gene "gene sharing";examples include small heat shock protein/     

Lens protein expression in mammals: Taxon-specificity and the recruitment of crystallins

Summary Vertebrate lenses show remarkably taxon-specific patterns of protein composition, most obviously in the recruitment of enzymes as major crystallins. Phylogenetic relationships are particularly apparent in mammals. Here we describe ν-crystallin, which is probably identical to cytosolic aldehyde dehydrogenase, lens-specifically expressed at high abundance in the elephant shrews, primitive eutherians of the family Macroscelidae, and μ-crystallin, a novel lens protein expressed in some marsupials. We have also observed that enzymes that have been recruited as crystallins in some species are also moderately abundant in the lenses of other species. This hints that the origins of enzyme-crystallins may lie in a pool of enzymes widely expressed in lenses at fairly high levels, perhaps because they have important developmental or functional roles in the tissue.     

The muscle-derived lens of a squid bioluminescent organ is biochemically convergent with the ocular lens. Evidence for recruitment of aldehyde dehydrogenase as a predominant structural protein.

Many of the structural proteins of ocular lenses, commonly referred to as crystallins, are identical to specific enzymes or the result of a recent gene duplication (Piatigorsky, J., and Wistow, G. (1991) Science 252, 1078-1079). One such enzyme, aldehyde dehydrogenase (ALDH), has been recruited as a lens crystallin in certain mammals (Wistow, G., and Kim, H. (1991) J. Mol. Evol. 32, 262-269) and cephalopods (Tomarev, S., Zinovieva, R., and Piatigorsky, J. (1991) J. Biol. Chem. 266, 24226-24231). We report here that a transparent tissue, derived from muscle but functioning as a lens in the light-emitting organ of a squid, Euprymna scolopes, shows striking biochemical convergence with the epidermally derived ocular lenses of some mammals and cephalopods. In the light organ lens of E. scolopes, an ALDH-like protein is the predominant molecular component. The typical muscle-specific proteins are replaced as the dominant species by a protein composed of 54-kDa subunits. This protein, which we designate as L-crystallin, constitutes approximately 70% of the total soluble protein of the light organ lens. The amino acid sequences of three peptides of L-crystallin (approximately 9% of the total protein) showed 54.5% sequence identity with human cytosolic ALDH. Using polyclonal antiserum made against L-crystallin, we found that it is present in low abundance in other tissues of the squid, including muscle and the ocular lens. This polyclonal antiserum also cross-reacted with the ALDH-like crystallins found in the ocular lenses of certain mammals and cephalopods. L-Crystallin showed no ALDH activity, which is similar to several other enzyme/crystallins, including ALDH/eta-crystallin (Wistow, G., and Kim, H. (1991) J. Mol. Evol. 32, 262-269). The characteristics of this muscle-derived lens are evidence that a common biochemical basis underlies transparency and that certain proteins may possess properties that promote their selection as lens structural proteins.     

Lactate Dehydrogenase Like Crystallin: A Potentially Protective Shield for Indian Spiny-Tailed Lizard (Uromastyx hardwickii) Lens Against Environmental Stress?

Taxon specific lens crystallins in vertebrates are either similar or identical with various metabolic enzymes. These bifunctional crystallins serve as structural protein in lens along with their catalytic role. In the present study, we have partially purified and characterized lens crystallin from Indian spiny-tailed lizard (Uromastyx hardwickii). We have found lactate dehydrogenase (LDH) activity in lens indicating presence of an enzyme crystallin with dual functions. Taxon specific lens crystallins are product of gene sharing or gene duplication phenomenon where a pre-existing enzyme is recruited as lens crystallin in addition to structural role. In lens, same gene adopts refractive role in lens without modification or loss of pre-existing function during gene sharing phenomenon. Apart from conventional role of structural protein, LDH activity containing crystallin in U. hardwickii lens is likely to have adaptive characteristics to offer protection against toxic effects of oxidative stress and ultraviolet light, hence justifying its recruitment. Taxon specific crystallins may serve as good models to understand structure–function relationship of these 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.     

Crystal structure of eta-crystallin: adaptation of a class 1 aldehyde dehydrogenase for a new role in the eye lens

Eta-crystallin is a retinal dehydrogenase that has acquired a role as a structural protein in the eye lens of elephant shrews, members of an ancient order of mammals. While it retains some activity toward retinal, which is oxidized to retinoic acid, the protein has acquired a number of specific sequence changes that have presumably been selected to enhance the lens role. The crystal structure of eta-crystallin, in common with class 1 and 2 ALDHs, is a dimer of dimers. It has a better-defined NAD binding site than those of related mammalian ALDH1 enzymes with the cofactor bound in the "hydride transfer" position in all four monomers with small differences about the dimer dyads. Although the active site is well conserved, the substrate-binding site is larger in eta-crystallin, and there are some mutations to the substrate access tunnel that might affect binding or release of substrate and product. It is possible that eta-crystallin has lost flexibility to improve its role in the lens. Enhanced binding of cofactor could enable it to act as a UV/blue light filter in the lens, improving visual acuity. The structure not only gives a view of a "natural mutant" of ALDH1 illustrating the adaptive conflict that can arise in multifunctional proteins, but also provides a well-ordered NAD binding site structure for this class of enzymes with important roles in development and health.     

Molecular cloning and baculovirus expression of the rabbit corneal aldehyde dehydrogenase (ALDH1A1) cDNA

Most mammalian species express high concentrations of ALDH3A1 in corneal epithelium with the exception of the rabbit, which expresses high amounts of ALDH1A1 rather than ALDH3A1. Several hypotheses that involve catalytic and/or structural functions have been postulated regarding the role of these corneal ALDHs. The aim of the present study was to characterize the biochemical properties of the rabbit ALDH1A1. We have cloned and sequenced the rabbit ALDH1A1 cDNA, which is 2,073 bp in length (excluding the poly(A+) tail), and has 5' and 3' nontranslated regions of 46 and 536 bp, respectively. This ALDH1A1 cDNA encodes a protein of 496 amino acids (Mr = 54,340) that is: 86-91% identical to mammalian ALDH1A1 proteins, 83-85% identical to phenobarbital-inducible mouse and rat ALDH1A7 proteins, 84% identical to elephant shrew ALDH1A8 proteins (eta-crystallins), 69-73% identical to vertebrate ALDH1A2 and ALDH1A3 proteins, 65% identical to scallop ALDH1A9 protein (omega-crystallin), and 55-57% to cephalopod ALDH1C1 and ALDH1C2 (omega-crystallins). Recombinant rabbit ALDH1A1 protein was expressed using the baculovirus system and purified to homogeneity with affinity chromatography. We found that rabbit ALDH1A1 is catalytically active and efficiently oxidizes hexanal (Km = 3.5 microM), 4-hydroxynonenal (Km = 2.1 microM) and malondialdehyde (Km = 14.0 microM), which are among the major products of lipid peroxidation. Similar kinetic constants were observed with the human recombinant ALDH1A1 protein, which was expressed and purified using similar experimental conditions. These data suggest that ALDH1A1 may contribute to corneal cellular defense against oxidative damage by metabolizing toxic aldehydes produced during UV-induced lipid peroxidation.     

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.     

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.     

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.     

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.     

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.