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.     

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.     

用蛋白质印迹转移技术分析正常及硒性白内障大鼠晶状体及房水中蛋白质的性质

本文用蛋白质印迹转移技术分析了正常及硒性白内障大鼠晶状体及房水中蛋白质的性质。结果表明,晶状体中的脲溶性蛋白质可被抗α及抗γ晶体蛋白血清识别,提示α及γ晶体蛋白均为脲溶性蛋白质的主要成份。患白内障时房水中的蛋白质含量明显增加,且主要被抗γ血清识别,而被抗α血清识别的成份很少,表明在大鼠硒性白内障形成过程中,有较多低分子量蛋白质漏出到房水中,且其主要成份为γ晶体蛋白。此外,我们还发现正常及硒性白内障大鼠晶状体膜蛋白质与抗α及抗γ血清起反应的程度及分布有所不同,提示晶状体细胞膜与晶体蛋白之间存在着相互作用。     

Covalent change in alpha crystallin during human senile cataractogenesis

The high molecular weight aggregates (HMWA) obtained from normal and cataractous human lens nuclei have been resolved by SDS-polyacrylamide gel electrophoresis, and the alpha crystallin band has been probed with antisera made against the whole alpha crystallin molecule and with antisera made against synthetic peptides of alpha crystallin (alpha A2 147-161 and alpha A2 163-173). Quantitation of these antisera binding demonstrated that the anti-alpha A2 163-173 serum and the anti-alpha whole sera bound equally well to the alpha crystallin band from the HMWA fraction from normal and cataractous lenses. In contrast, the anti-alpha A2 147-161 serum bound little, if at all, to alpha crystallin from normal lenses, while it bound well to alpha crystallin from cataractous lenses. These results demonstrate a covalent alteration in the alpha crystallin molecule, and suggest a possible location of a covalent change that may occur during the cataractogenic process in the aged human lens.     

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.     

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.     

The appearance of α, β and γ crystallins in an anophthalmic strain of mice

Abstract. A certain percentage of congenitally anophthalmic mouse embryos have the ability to generate small lens vesicles that have previously been shown to produce alpha crystallin at 13-day gestation. Further immunohistological analysis of 13- and 15-day-gestation anophthalmia embryos indicates that beta crystallin is present in those 13-day embryos which have lens vesicles with lens-fiber formation. Also, 15-day embryos with lenses demonstrating fiber elongation can produce both beta and gamma crystallins. The conclusion is drawn that the genetic potential to produce at least three characteristic biochemical markers of normal lens differentiation is present in the anophthalmia mutant. The spatial distribution patterns of the crystallins in normal and anophthalmia embryos were similar. However, there appeared to be a transposition in the temporal appearance of beta and gamma crystallins in the anophthalmia mutant. Optic cups and associated lenses in 15-day anophthalmia specimens were much smaller than those in controls. The optic and lens rudiments in these anophthalmia embryos were fairly proportional in size, which indicates that some degree of allometric growth compensation had occurred during the course of development. This ability for differential growth compensation in the mouse eye appears to be restricted to the predifferentiative stages of eye formation.     

Developmental constraints in regressive evolution: studies of the expression of the γs-crystallin gene in the developing lens of cave-dwelling Astyanax fasciatus (Cuvier, 1819) (Teleostei, Characidae) by in situ hybridization

The early morphogenesis of the lens and the expression of the γs-crystallin gene was studied in epigean Astyanax fasciatus and its cave-dwelling derivative. At early stages, the lens of the cave fish develops in a way that is similar to the epigean form. Later, the developmental timing is delayed and growth ceases in the cave-fish lens. With the beginning of cytodifferentiation, the development of the lens breaks down. Crystallin lens fibres are not produced at any time and the γs-crystallin gene, which is transcribed during a limited period in the lens of epigean fishes, is not active in cave specimens. This study confirms earlier immunofluorescence observations that demonstrated the lack of crystallin proteins in the cave-fish lens, but is in contrast to results on the blind mole rat, which showed a persistence of functioning crystallins in the degenerated lens of this species. The significance of developmental constraints in regressive evolution is discussed.     

Electrophoretic variation in low molecular weight lens crystallins from inbred strains of rats

Analysis of rat lens soluble proteins by analytical isoelectric focusing detected two inherited electrophoretic differences in low molecular weight (LM) crystallins from inbred strains of rats (Rattus norvegicus). The polymorphic lens crystallins were shown to be similar to a genetically variant LM crystallin, LEN-1, previously described in mice (Mus musculus) and encoded on chromosome 1, at a locus linked to Pep-3 (dipeptidase). Linkage analysis demonstrated that the rat crystallin locus was loosely linked to Pep-3 at a recombination distance of 38 +/- 4.5 U. These data suggest the conservation of a large chromosomal region during the evolution of Rodentia and support the hypothesis that the gamma-crystallins are evolving more rapidly than alpha- or beta-crystallins.     

Ageing in the chick lens: in vitro studies.

In principle, ageing may be due to the interaction of several factors, including the accumulation of random changes both genomic and non-genomic, secondary changes in a tissue contingent upon the changing function of other tissues, and programmed non-random changes in the tissue-specific expression of various genes. The use of a single tissue comprising one cell type only, in which the major gene products are well defined, in which there is a well attested series of developmental and age-related changes in cell properties and gene expression and which can be studied and compared in vivo and in vitro, offers advantages for investigation of these questions. The vertebrate eye lens possesses these advantages. The crystallins (proteins expressed at super-abundant levels in the lens) are well characterised. The lens epithelial cells (LEC) grow readily and can differentiate into the lens fibre cells in vitro, and, finally, such terminally differentiated cells may also be derived, by a process of transdifferentiation, from neural retina cells (NRC) in vitro. Thus the effect on ageing changes of the tissue of origin may also be studied. This article reviews our previous studies on long-term changes in growth potential, differentiation capacity and crystallin expression of chick lens cells in ageing cultures, their overall similarity to events in vivo and the effect on ageing changes of genotypes affecting the growth rate. It presents new information on these genetic aspects, and on crystallin expression in long-term ageing cultures of transdifferentiated neural retina, and compares the behaviour of ageing chick lens cells with that reported for mammals.     

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.     

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.     

Crystallin gene expression and lentoid body formation in quail embryo neuroretina cultures transformed by the oncogenic retrovirus Mill Hill 2 or Rous sarcoma virus.

The lens-specific proteins alpha and delta crystallins and lentoid bodies, structures that follow a differentiation pathway similar to that of the lens, regularly appear after 4 to 5 weeks in quail embryo neuroretina monolayer cultures. We have investigated the effects of the avian oncogenic retroviruses Mill Hill 2 and Rous sarcoma virus on this process. Quail embryo neuroretina cells transformed by Mill Hill 2 virus were established into permanent cultures that synthesized alpha and delta crystallins and contained stem cells for the production of lentoid bodies. In contrast, transformation with the Rous sarcoma virus mutant tsNY-68 blocked the appearance of mRNA crystallins, but cytoplasmic alpha and delta crystallin mRNA and alpha crystallin appeared 44 h after a shift to the nonpermissive temperature. However, delta crystallins and lentoid bodies were only present after 7 days. The crystallins of transformed quail neuroretina cultures were immunologically indistinguishable from those of quail lenses and of normal quail embryo neuroretina cultures.     

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.     

Age‐related cleavages of crystallins in human lens cortical fiber cells generate a plethora of endogenous peptides and high molecular weight complexes

Low molecular weight peptides derived from the breakdown of crystallins have been reported in adult human lenses. The proliferation of these LMW peptides coincides with the earliest stages of cataract formation, suggesting that the protein cleavages involved may contribute to the aggregation and insolubilization of crystallins. This study reports the identification of 238 endogenous LMW crystallin peptides from the cortical extracts of four human lenses representing young, middle and old‐age human lenses. Analysis of the peptide terminal amino acids showed that Lys and Arg were situated at the C‐terminus with significantly higher frequency compared to other residues, suggesting that trypsin‐like proteolysis may be active in the lens cortical fiber cells. Selected reaction monitoring analysis of an endogenous αA‐crystallin peptide (αA_57‐65) showed that the concentration of this peptide in the human lens increased gradually to middle age, after which the rate of αA_57‐65 formation escalated significantly. Using 2D gel electrophoresis/nanoLC‐ESI‐MS/MS, 12 protein complexes of 40–150 kDa consisting of multiple crystallin components were characterized from the water soluble cortical extracts of an adult human lens. The detection of these protein complexes suggested the possibility of crystallin cross‐linking, with these complexes potentially acting to stabilize degraded crystallins by sequestration into water soluble complexes. Proteins 2015; 83:1878–1886. © 2015 Wiley Periodicals, Inc.