Effect of chronic hyperglycemia on crystallin levels in rat lens

Crystallins are the major structural proteins in the vertebrate eye lens that contribute to lens transparency. Although cataract, including diabetic cataract, is thought to be a result of the accumulation of crystallins with various modifications, the effect of hyperglycemia on status of crystallin levels has not been investigated. This study evaluated the effect of chronic hyperglycemia on crystallin levels in diabetic cataractous rat lens. Diabetes was induced in rats by injecting streptozotocin and maintained on hyperglycemia for a period of 10 weeks. At the end, levels of α-, β-, γ-crystallins and phosphoforms of αB-crystallins (αBC) were analyzed by immunoblotting. Further, solubility of crystallins and phosphoforms of αBC was analyzed by detergent soluble assay. Chronic diabetes significantly decreased the protein levels of α-, β- and αA-crystallins (αAC) in both soluble and insoluble fraction of lens. Whereas γ-crystallin levels were decreased and αBC levels were increased in lens soluble fraction with no change in insoluble fraction in diabetic rat lens. Although, diabetes activated the p38MAPK signaling cascade by increasing the p-p38MAPK in lens, the phosphoforms of αBC were decreased in soluble fraction with a concomitant increase in insoluble fraction of diabetic lens when compared to the controls. Moreover, diabetes strongly enhances the degradation of crystallins and phosphoforms of αBC in lens. Taken together, the decreased levels of crystallins and insolubilization of phosphoforms of αBC under chronic hyperglycemia could be one of the underlying factors in the development of diabetic cataract.     

Altered chaperone-like activity of alpha-crystallins promotes cataractogenesis

Despite the enormous number of studies demonstrating changes in the chaperone-like activity of α-crystallins in vitro, little is known about how these changes influence life-long lens transparency in vivo. Using the γB-crystallin I4F mutant protein as a target for αA-crystallins, we examined how cataract phenotypes are modulated by interactions between α-crystallins with altered chaperone-like activities and γB-I4F proteins in vivo. Double heterozygous α-crystallin knock-out αA(+/-) αB(+/-) mice with a decreased amount of α-crystallins were used to simulate reduced total α-crystallin chaperone-like activity in vivo. We found that triple heterozygous αA(+/-) αB(+/-) γB(I4F/+) mice developed more severe whole cataracts than heterozygous γB(I4F/+) mice. Thus, total chaperone-like activity of α-crystallins is important for maintaining lens transparency. We further tested whether mutant αA-crystallin Y118D proteins with increased chaperone-like activity influenced the whole cataract caused by the γB-I4F mutation. Unexpectedly, compound αA(Y118D/+) γB(I4F/+) mutant lenses displayed severe nuclear cataracts, whereas the lens cortex remained unaffected. Thus, the synergistic effect of αA-Y118D and γB-I4F mutant proteins is detrimental to the transparency only in the lens core. α-Crystallins with different chaperone-like activities are likely required in the lens cortex and nucleus for maintaining transparency.     

Truncation, cross-linking and interaction of crystallins and intermediate filament proteins in the aging human lens

The optical properties of the lens are dependent upon the integrity of proteins within the fiber cells. During aging, crystallins, the major intra-cellular structural proteins of the lens, aggregate and become water-insoluble. Modifications to crystallins and the lens intermediate filaments have been implicated in this phenomenon. In this study, we examined changes to, and interactions between, human lens crystallins and intermediate filament proteins in lenses from a variety of age groups (0-86years). Among the lens-specific intermediate filament proteins, filensin was extensively cleaved in all postnatal lenses, with truncated products of various sizes being found in both the lens cortical and nuclear extracts. Phakinin was also truncated and was not detected in the lens nucleus. The third major intermediate filament protein, vimentin, remained intact in lens cortical fiber cells across the age range except for an 86year lens, where a single ~49kDa breakdown product was observed. An αB-crystallin fusion protein (maltose-binding protein-αB-crystallin) was found to readily exchange subunits with endogenous α-crystallin, and following mild heat stress, to bind to filensin, phakinin and vimentin and to several of their truncated products. Tryptic digestion of a truncated form of filensin suggested that the binding site for α-crystallin may be in the N-terminal region. The presence of significant amounts of small peptides derived from γS- and βB1-crystallins in the water-insoluble fraction of the lens indicates that these interact tightly with cytoskeletal or membrane components. Interestingly, water-soluble complexes (~40kDa) contained predominantly γS- and βB1-crystallins, suggesting that cross-linking is an alternative pathway for modified β- and γ-crystallins in the lens.     

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.     

Ontogeny and localization of the α, β, and γ crystallins in newt eye lens development

The α-, β-, and γ-crystallins, proteins characteristic for the vertebrate eye lens, have been localized in the developing lens of Notophthalmus viridescens, the eastern spotted newt. Using the immunofluorescence technique, antibodies to the α-, β-, and γ-crystallin classes were applied to tissue sections through the eye region of developing N. viridescens embryos, Harrison (external) Stages 30 to 46+. β-Crystallins were the first of the crystallins to appear in a few cells of the lens vesicle even before the lengthening of the prospective primary fiber cells. γ-Crystallins were first detectable at a slightly more advanced stage in the prospective primary fibers, and α-crystallins in a few cells of the beginning primary fiber area. The external layer/epithelium was negative for β-crystallins until late in lens morphogenesis, and α- and γ-crystallins could not be detected in these cells at any time. This, the first use in amphibia of homologous antibodies specific for the crystallin classes, makes clear that phylogenetic differences exist as to the primacy and relevance of specific crystallins to events during morphogenesis of the eye lens.     

Rat lens β-crystallins are internally duplicated and homologous to γ-crystallins

The nucleotide sequence of two cloned rat lens β-crystallin cDNAs pRLβB3-2 and pRLβB1-3 has been determined. pRLβB3-2 contains the complete coding information for a β-crystallin, designated βB3, of 210 amino acid residues. pRLβB1-3 is incomplete at its 5′ end; the 5′ codogenic information which is not present in this cDNA clone was deduced from the cloned gene. pRLβB1-3 codes for a β-crystallin polypeptide, designated βB1, whose full length is 247 amino acid residues. Considerable sequence homology is noted between the amino- and carboxy-terminal halves of each protein. The two rat β-crystallins show a substantial sequence homology with each other (60%) as well as with the published sequences of rat γ-crystallin (37%) and bovine and murine β-crystallins (55 and 45%). All these proteins have a two-domain structure which, like the bovine γII-crystallin, might be folded into four remarkably similar protein motifs. Our data further indicate that the β-crystallins can be subdivided into two groups which are evolutionarily related. Both groups are, although more distantly, also related to the γ-crystallins.     

Partially Folded Aggregation Intermediates of Human γD-, γC-, and γS-Crystallin Are Recognized and Bound by Human αB-Crystallin Chaperone

Human γ-crystallins are long-lived, unusually stable proteins of the eye lens exhibiting duplicated, double Greek key domains. The lens also contains high concentrations of the small heat shock chaperone α-crystallin, which suppresses aggregation of model substrates in vitro. Mature-onset cataract is believed to represent an aggregated state of partially unfolded and covalently damaged crystallins. Nonetheless, the lack of cell or tissue culture for anucleate lens fibers and the insoluble state of cataract proteins have made it difficult to identify the conformation of the human γ-crystallin substrate species recognized by human α-crystallin. The three major human lens monomeric γ-crystallins, γD, γC, and γS, all refold in vitro in the absence of chaperones, on dilution from denaturant into buffer. However, off-pathway aggregation of the partially folded intermediates competes with productive refolding. Incubation with human αB-crystallin chaperone during refolding suppressed the aggregation pathways of the three human γ-crystallin proteins. The chaperone did not dissociate or refold the aggregated chains under these conditions. The αB-crystallin oligomers formed long-lived stable complexes with their γD-crystallin substrates. Using α-crystallin chaperone variants lacking tryptophans, we obtained fluorescence spectra of the chaperone-substrate complex. Binding of substrate γ-crystallins with two or three of the four buried tryptophans replaced by phenylalanines showed that the bound substrate remained in a partially folded state with neither domain native-like. These in vitro results provide support for protein unfolding/protein aggregation models for cataract, with α-crystallin suppressing aggregation of damaged or unfolded proteins through early adulthood but becoming saturated with advancing age.     

Simultaneous stereoinversion and isomerization at the Asp-4 residue in βB2-crystallin from the aged human eye lenses

The lens proteins are composed of α-, β-, and γ-crystallins that interact with each other to maintain the transparency and refractive power of the lens. Because the lens crystallins are long-lived proteins, they undergo various post-translational modifications including racemization, isomerization, deamidation, oxidation, glycation, and truncation. In βB2-crystallin, which is the most abundant β-crystallin, the deamidation of asparagine and glutamine residues has been reported. Here, we found that the aspartyl (Asp) residue at position 4 of βB2-crystallin in the lenses of elderly human individuals undergoes a significant degree of inversion and isomerization to the biologically uncommon residue D-β-Asp. Surprisingly, the D/L ratio of β-Asp at position 4 in βB2-crystallin from elderly donors (67-77 year old) was 0.88-3.21. A D/L ratio of amino acids greater than 1.0 is defined as an inversion of configuration from the L- to D-form, rather than a racemization. These extremely high D/L ratios are equivalent to those of Asp-58 and Asp-151 (D/L ratio: 3.1 for Asp-58 and 5.7 for Asp-151) in αA-crystallin from elderly donors (~80 year old) as reported previously. Initially, we identified specific Asp residues in the β-crystallin family of proteins that undergo a high degree of inversion. These results show that the isomerization and inversion of Asp residues occurs both in the α- and β-crystallins of the lens. Inversion of these Asp residues directly affects the higher order structure of the protein. Hence, this modification may change crystallin-crystallin interactions and disrupt the function of crystallins in the lens.     

A comparison of structural relationships among α-crystallin, human Hsp27, γ-crystallins and βB2-crystallin

The 3D structures of α-crystallin, a major eye lens protein, and related small heat shock proteins are unresolved. It has been assumed that α-crystallin is primarily a β-sheet globular protein similar to γ-crystallin (Siezen and Argos, Biochim. Biophys. Acta, 1983, 748, 56–67) containing sequence repeats in its two domains (Wistow, FEBS Lett. 1985, 181, 1–6). Positional flexibility of amino acid residues and far UV-circular dichroism spectroscopy were used to investigate structural relationships among these proteins. The utility of flexibility plots for predicting protein structure is demonstrated by the excellent correlation of these plots with the known 3D X-ray structures of β/γ-crystallins. Similar analyses of α-crystallin subunits, αA and αB, and human heat shock protein 27 show that the C-terminal domains and connecting segments of these proteins are very similar while the N-terminal domains have significant structural differences. Unlike β/γ-crystallins, both Hsp27 and α-crystallin subunits are asymmetrical with highly flexible C-terminal domains. Flexibility is considered essential for protein functional activity. Therefore, the C-terminal region may play an active role in α-crystallin and small heat shock protein function. Differences in flexibility profiles and estimated secondary structure distribution in α-crystallin by three recent/updated algorithms from far UV-CD spectra support our predicted 3D structure and the concept that α-crystallin and members of β/γ-superfamily are structurally dissimilar.     

α-Crystallin Acting as a Molecular Chaperonin Against Photodamage by UV Irradiation

α-Crystallin, a major protein of the eye lens, is known to have chaperone activity in preventing heat-induced aggregation of enzymes and other crystallins. In this study, we investigate the ability of α-crystallin to inhibit UV-light-induced aggregation of other lens proteins and the effect of exposure of α-crystallin to UV irradiation on its chaperone activity. The chaperone activities of α-crystallin preincubated at different temperatures were found to be different and could be correlated with its change in quaternary structure as determined by the fluorescence probe ANS (8-anilo-1-naphthalene sulfonate). α-Crystallin can inhibit the aggregation of γ-crystallin from UV irradiation at room temperature, and the preheated α-crystallins provide more protection than the native one. Upon irradiation by UV light, α-crystallin gradually lost its ability to protect β-crystallin against thermal aggregation. The loss of the chaperone efficacy of α-crystallin to protect other lens proteins may shed light on human cataract formation induced by long-term exposure to UV irradiation.     

sHSP in the eye lens: crystallin mutations, cataract and proteostasis

α-Crystallin, a major component of the eye lens cytoplasm, is a large multimer formed from two members of the small heat shock protein (sHsp) family. Inherited crystallin mutations are a common cause of childhood cataract, whereas miscellaneous changes to the long-lived crystallins cause age-related cataract, the most common cause of blindness worldwide. Newly formed eye lens cells use proteostasis to deal with the consequences of mutations, whereas mature lens cells, devoid of the ATP-driven folding and degradation machines, are hypothesized to have the α-crystallin "holdase" chaperone function to prevent protein aggregation. We discuss the impact of truncating and missense mutations on α-crystallin, based on recent progress towards determining sHsp 3D structure. Dominant missense mutations to the "α-crystallin domain" of αA- (HSPB4) or αB-crystallin (HSPB5) occur on residues predicted to facilitate domain dynamics. αB-Crystallin is also expressed in striated muscle and mutations cause myopathy. The impact on these cellular cytoplasms is compared where sHsp multimer partners and metabolic constraints are different. Selected inherited mutations of the lens β- and γ-crystallins are considered in the context of their possible dependence on the "holdase" chaperone function of α-crystallin. Looking at discrete changes to specific crystallin polypeptide chains that can function as chaperone or substrate provide insights into the workings of a cytoplasmic proteostatic system. These observations provide a framework for validating the function of α-crystallin as a chaperone, or as a lens space filler adapted from a chaperone function. Understanding the mechanistic role of α-crystallins will aid progress in research into age-related cataract and adult-onset myopathy. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology.     

Aggregation of crystallins induced by pulsed laser UV light (308 nm)

Here we compile and analyze the data on photoaggregation of a model protein carboanhydrase and the main eye lens proteins α-, β-, γ-crystallins under the action of pulsed UV irradiation from a Xe-Cl laser (308 nm) with broad variation of pulse energy density and repetition rate. The aggregation efficacy proves to be a nonlinear function of these parameters and protein concentration. A theoretical model is proposed that qualitatively explains the experimental data. It is shown that N-arm-truncated βA3-crystallin is more prone to UV-induced aggregation than the full-sized protein; such defects caused by mutation or aging may aggravate the development of lenticular opacity. Analyzed is the effect of some low-molecular compounds on the aggregation of β-crystallin and its mixture with α-crystallin. A combination of short peptides prepared on this basis markedly impedes crystallin aggregation and retards the development of UV-induced cataract in rats.     

Sequence Characterization of γ-Crystallins from Lip Shark (Chiloscyllium colax): Existence of Two cDNAs Encoding γ-Crystallins of Mammalian and Teleostean Classes

γ-Crystallin is a common lens protein of most vertebrate eye lenses and the major protein component in lenses of fishes and in many mammalian species during embryonic and neonatal stages. To facilitate the structural characterization of γ-crystallin possessing extensive charge heterogeneity, a cDNA mixture was constructed from the poly(A)⁺ mRNA isolated from shark eye lenses, and amplification by polymerase chain reaction (PCR) was carried out to obtain cDNAs encoding multiple shark γ-crystallins. Sequencing analysis of multiple positive clones containing PCR-amplified inserts revealed the presence of a multiplicity of isoforms in the γ-crystallin class of this cartilaginous fish. It was of interest to find that two shark cDNA sequences coexist, one encoding γ-crystallin (γM₁) of high methionine content (15.5%) and the other encoding one (γM₂) of low methionine content (5.1%), each corresponding to the major teleostean and mammalian γ-crystallins, respectively. Comparison of protein sequences encoded by these two shark cDNAs with published sequences of γ-crystallins from mouse, bovine, human, frog, and carp lenses indicated that there is about 61–80% sequence homology between different species of the piscine class, whereas only 47–66% is found between mammals and shark. A phylogenetic tree constructed on the basis of sequence divergence among various γ-crystallin cDNAs revealed the close relatedness between shark γM₂-crystallin and mammalian γ-crystallins and that between shark γM₁ and teleostean γ-crystallins. The results pointed to the fact that ancestral precursors of γ-crystallins were present in the sharp lens long before the appearance of modern-day mammalian and teleostean γ-crystallins.     

Resistance of α-crystallin quaternary structure to UV irradiation

The damaging effect of UV radiation (λ > 260 nm) on bovine α-crystallin in solution was studied by small-angle X-ray scattering, gel permeation chromatography, electrophoresis, absorption and fluorescence spectroscopy, and differential scanning calorimetry. The results obtained show that damage to even a large number of subunits within an α-crystallin oligomer does not cause significant rearrangement of its quaternary structure, aggregation of oligomers, or the loss of their solubility. Due to the high resistance of its quaternary structure, α-crystallin is able to prevent aggregation of destabilized proteins (especially of γ- and β-crystallins) and so to maintain lens transparency throughout the life of an animal (the chaperone-like function of α-crystallin).     

Effect of Trifluoroethanol on the Structural and Functional Properties of α-Crystallin

Alpha crystallin is an eye lens protein with a molecular weight of approximately 800 kDa. It belongs to the class of small heat shock proteins. Besides its structural role, it is known to prevent the aggregation of β- and γ-crystallins and several other proteins under denaturing conditions and is thus believed to play an important role in maintaining lens transparency. In this communication, we have investigated the effect of 2,2,2-trifluoroethanol (TFE) on the structural and functional features of the native α-crystallin and its two constituent subunits. A conformational change occurs from the characteristic β-sheet to the α-helix structure in both native α-crystallin and its subunits with the increase in TFE levels. Among the two subunits, αA-crystallin is relatively stable and upon preincubation prevents the characteristic aggregation of αB-crystallin at 20% and 30% (v/v) TFE. The hydrophobicity and chaperone-like activity of the crystallin subunits decrease on TFE treatment. The ability of αA-crystallin to bind and prevent the aggregation of αB-crystallin, despite a conformational change, could be important in protecting the lens from external stress. The loss in chaperone activity of αA-crystallin exposed to TFE and the inability of peptide chaperone—the functional site of αA-crystallin—to stabilize αB-crystallin at 20–30% TFE suggest that the site(s) involved in subunit interaction and chaperone-like function are quite distinct.     

Identification of Isomeric Aspartate residues in βB2-crystallin from Aged Human Lens

Many post-translational modifications such as oxidation, deamidation and isomerization of amino acid residues occur in lens proteins with aging. One such modification, isomerization of aspartate in lens α-crystallin, has been well studied by amino acid enantiomer analysis and LC-MS/MS. LC-MS/MS can quickly and easily identify D- and L-amino acid-containing peptides without purification of lens protein mixtures. However, this method has a weak point in that isomeric peptides of major components are detected predominantly, while those from minor proteins such as β- and γ-crystallins have not been fully determined. Therefore, the isomerization of amino acid residues in β- and γ-crystallin families has been little studied. To solve those problems and detect the isomerization of Asp residues in lens βB2-crystallin, the main component of the β-crystallin family, here we have developed steps for sample fractionation before d/l analysis based on either LC-MS/MS or amino acid derivatization to diastereoisomers followed by RP-HPLC. To capture a small amount of peptide, a multiple reaction monitoring (MRM) method based on quadrupole MS/MS (Q-MS) was applied to the water-soluble fraction of whole lens. The d/l analysis based on both LC-MS/MS and diastereoisomer formation showed the presence of multiple isomerization sites, including Asp4, Asp83, Asp92 and Asp192, in βB2-crystallin in aged lens. These isomerization sites were confirmed to exist in an age-dependent manner by Q-MS. Synthetic peptides of βB2-crystallin containing different isomers of Asp showed differential elution profiles during RP-HPLC, indicating differences in the local structure or hydrophobicity of Asp-isomer-containing peptides. These results suggest that the isomerization sites are distributed on exposed regions of βB2-crystallin and thus likely to have an impact on crystallin subunit–subunit interactions, induce abnormal crystallin aggregation, and contribute to senile cataract formation in aged lens.     

正常与患白内障大鼠晶状体中脲溶性蛋白质性质的研究

本文研究了正常及三种类型白内障大鼠晶状体中脲溶性蛋白质的含量及性质的变化,发现在每种类型白内障晶状体中,水溶性蛋白质均减少,水不溶性蛋白质则都相对增加。经SephadexG-200柱层析及SDS聚丙烯酰胺凝胶电泳发现,晶状体中脲溶性蛋白质主要是由二硫键交联而成的高分子聚合物。经巯基乙醇还原后,绝大部分高分子聚合物可分解成低分子量蛋白质,其分子量与水溶性的γ晶体蛋白相同。这提示晶状体中脲溶性蛋白质的主要成分很可能是以二硫键交联而成的γ晶体蛋白聚合物。此结果与本实验室所得白内障晶状体水溶性蛋白质的变化相吻合。     

Bovine β-crystallin complementary DNA clones: Alternating proline/alanine sequence of βB1 subunit originates from a repetitive DNA sequence

A library of recombinant plasmids carrying complementary DNA sequences synthesized from bovine lens messenger RNAs was constructed. Clones coding for five different β-crystallin subunits: βB1, βB3, βBp, βs, βA3 (and βA1), were identified by means of hybridization selection, followed by one- and two-dimensional gel electrophoresis of the translational products. Under rather stringent conditions each of these clones hybridizes with its corresponding mRNA and does not show significant cross-hybridization with mRNAs coding for other β-crystallins, except in the case of the homologous βA3 and βA1-crystalline. The βA3 and βA1 subunits seem to be encoded by one mRNA using two different AUG codons as start position for translation. We have also determined the nucleotide sequence of a βB1-crystallin cDNA (pBLβB1) which enabled us to deduce the complete amino acid sequence of the protein. The βB1-crystallin, a characteristic component of the high molecular weight crystallin aggregate (β_H), is internally homologous both at DNA and protein level as has been reported for γ-and other β-crystallins. This is in agreement with the idea that these proteins had a common ancestral precursor gene that internally duplicated. The G + C content of the coding sequence of βB1 is very high: 67% overall and even 84.2% for the first 170 nucleotides, due to a remarkable non-random codon usage. A proline/ alanine repetition in the N-terminal domain of the protein is encoded by a repetitive “simple” DNA sequence.     

α-Crystallin: molecular chaperone and protein surfactant

Bovine lens α-crystallin has recently been shown to function as a molecular chaperone by stabilizing proteins against heat denaturation (Horwitz, J. (1992) Proc. Natl. Acad. Sci. USA, 89, 10449–10453). An investigation, using a variety of physico-chemical methods, is presented into the mechanism of stabilization. α-Crystallin exhibits properties of a surfactant. Firstly, a plot of conductivity of α-crystallin versus concentration shows a distinct inflection in its profile, i.e., a critical micelle concentration (cmc), over a concentration range from 0.15 to 0.17 mM. Gel chromatographic and ¹H-NMR spectroscopic studies spanning the cmc indicate no change in the aggregated state of α-crystallin implying that a change in conformation of the aggregate occurs at the cmc. Secondly, spectrophotometric studies of the rate of heat-induced aggregation and precipitation of alcohol dehydrogenase (ADH), β_L- and γ-crystallin in the presence of α-crystallin and a variety of synthetic surfactants show that stabilization against precipitation results from hydrophobic interactions with α-crystallin and monomeric anionic surfactants. Per mole of subunit or monomer, α-crystallin is the most efficient at stabilization. α-Crystallin, however, does not preserve the activity of ADH after heating. After heat inactivation, gel permeation HPLC indicates that ADH and α-crystallin form a high molecular weight aggregate. Similar results are obtained following incubation of β_L- and γ-crystallin with α-crystallin. ¹H-NMR spectroscopy of mixtures of α- and β_L-crystallin, in their native states, reveals that the C-terminus of βB2-crystallin is involved in interaction with α-crystallin. In the case of γ- and α-crystallin mixtures, a specific interaction occurs between α-crystallin and the C-terminal region of γ_B-crystallin, an area which is known from the crystal structure to be relatively hydrophobic and to be involved in intermolecular interactions. The short, flexible C-terminal extensions of α-crystallin are not involved in specific interactions with these proteins. It is concluded that α-crystallin interacts with native proteins in a weak manner. Once a protein has become denatured, however, the soluble complex with α-crystallin cannot be readily dissociated. In the aging lens this finding may have relevance to the formation of high molecular weight crystallin aggregates.     

Crystallin synthesis in lens differentiation in cultures of neural retinal cells of chick embryos.

Dissociated cells of neural retinas of 3.5-day-old chick embryos differentiated into “lentoid bodies” within about 10–12 days when cultured in vitro. Protein synthesis of these cultured cells was studied with the use of SDS-polyacrylamide gel electrophoresis, affinity chromatography, and autoradiography combined with immunological techniques. Incorporation of [¹⁴C]leucine into total proteins, α-crystallin, and δ-crystallin was estimated after increasing times of culture up to about 30 days. Isotope incorporation into δ-crystallin was detected at 9 days, and it increased sevenfold after another 17 days. α-Crystallin was also first detected at 9 days, but its relative content reached a maximum at 12 days and then decreased gradually. The ratio of δ-crystallin synthesis to total protein synthesis increased up to 40% at 26 days, while that of α-crystallin synthesis remained 3% throughout the culture period. These results show that lens differentiation from neural retinal cells is associated with the preferential synthesis of lens crystallins, particularly of δ-crystallin.