Developmental expression of crystallin genes: in situ hybridization reveals a differential localization of specific mRNAs

The time and place of the accumulation of alpha A-, beta B1- and gamma-crystallin RNA in the developing rat lens have been studied by in situ hybridization. alpha A- and gamma-crystallin RNA were first detected in the lens vesicle, while beta B1-crystallin RNA could be seen only after elongation of the primary fiber cells. Both beta B1- and gamma-crystallin RNA were confined to the fiber cells of fetal lenses, while alpha A-crystallin mRNA could also be detected in the epithelial cells. A quantification of the hybridization pattern obtained in the differentiation zone of the newborn rat lens showed that alpha A-crystallin RNA is concentrated in the cortical zone. alpha B-crystallin mRNA has the same distribution pattern. beta B1-crystallin RNA was relatively poorly detectable by in situ hybridization in both fetal and newborn rat lenses. The grain densities obtained with this probe increased from the periphery of the lens toward the interior, indicating that beta B1-crystallin RNA accumulated during differentiation of the secondary fiber cells. A similar accumulation pattern was obtained for gamma-crystallin mRNA, but, unexpectedly, this RNA could also be detected in the elongating epithelial cells. Our results show that gamma-crystallin RNA starts to accumulate as soon as visible elongation of epithelial cells occurs, during differentiation of the primary as well as the secondary fiber cells.     

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.     

Distribution of mRNA and binding sites of adrenoceptors and muscarinic receptors in the rat heart

Since there exist some obscurities in the expression of mRNAs and their receptors in the heart, we have investigated the gene expression (mRNA levels) of adrenoceptors (alpha1A-, alpha1B-, beta1-, beta2-, beta3-) and muscarinic receptors (M2) and the density of receptor binding sites (alpha1A-, alpha1B-, beta1-, beta2-adrenoceptors, muscarinic receptors). Moreover, the heart regions consist of tissue rich in ganglion cells (that are of importance in heart neural circuits) and those virtually free of them (myocytes). Therefore, we have examined the differences in the distribution of mRNAs/receptor binding sites in the atrial samples of the heart rich in ganglion cells vs. those are virtually free of them. Binding sites and mRNAs of muscarinic receptors and alpha1B-adrenoceptors differ in their distribution in different heart regions. The mRNAs for beta1- and beta2-adrenoceptors were almost equally distributed herein, while the amount of beta-adrenoceptors significantly differs in the heart regions. The alpha1A- and beta3-adrenoceptors mRNAs were also found in all investigated heart regions, but at significantly lower level and have not shown region differences. This is a new finding, especially to beta3-adrenoceptors, as they were not regularly found in each heart regions. alpha1B-adrenoceptors have similar distribution of their mRNAs and binding sites in some heart parts. Thus, we can conclude that there are noticeable differences in the presence of receptors in heart regions that contain ganglion cells in comparison to those are virtually free of them.     

Cloning and sequencing of a carp beta s-crystallin cDNA

The mRNAs were extracted from common carp (Cyprinus carpio) lenses, purified, reverse transcribed, dC tailed and cloned into Escherichia coli with pBR322 as vector. The cloning efficiency was around 1 X 10(7) colonies per micrograms of mRNA. A clone (pC20) was found by hybrid-arrested to contain the cDNA related to carp crystallins. However, comparison of the derived amino-acid sequence with bovine gamma-II and beta s-crystallins indicates that this carp crystallin sequence resembles closely the bovine beta s-crystallin and should be better classified as such except that this fish sequence does not contain the N-terminal 'arm' of four amino-acid residues present in bovine beta s-crystallin.     

Structural homology of lens crystallins. II. Homology expressed by correlation coefficients and hydropathy profiles

Bovine lens alpha A- and alpha B-crystallin polypeptides show extensive sequence homology with each other, but apparently none with beta Bp- and gamma 2-crystallin. Despite only 30% sequence homology, the latter two proteins are assumed to have a strong correspondence in tertiary structure, consisting of four structurally similar folding units of antiparallel beta-sheet. We have tested for internal structural repeats in all crystallins, and structural homology between crystallins, by comparing various physical properties of the amino acid residues, such as bulkiness and propensity to form beta-sheet and beta-turn structure. Two procedures used a combination of five physical parameters to calculate correlation coefficients. The 4-fold structural repeat in gamma 2-crystallin and the internal duplication in beta Bp-crystallin were readily detectable, as was also the strong structural homology between corresponding folding units in beta Bp- and gamma 2-crystallin. However, for alpha-crystallin polypeptides, no conclusive support was obtained for either a four-unit or a six-unit folding, the two models previously considered by us. The third procedure compared smoothened hydropathy plots, representing hydrophilic and hydrophobic regions along the polypeptide sequences. Hydropathy profiles were found to show strong correspondence, particularly between alpha B-crystallin and beta Bp-crystallin. These observations support a similar 4-fold folding pattern for all bovine crystallins. A possible role in subunit interactions of the N-terminal folding unit, which has hydrophobic surface characteristics in both alpha- and beta-crystallin polypeptides, is proposed.     

Cholesterol-derived bile acids enhance the chaperone activity of α-crystallins

Human lens membranes contain the highest cholesterol concentration of any known biological membranes, but it significantly decreases with age. Oxygenation of cholesterol generates numerous forms of oxysterols (bile acids). We previously showed that two forms of the bile acid components—ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA)—suppressed lens epithelial cell death and alleviated cataract formation in galactosemic rat lenses. We investigated whether these compounds also suppress the thermal aggregation of human lens crystallins. Total water-soluble (WS) proteins were prepared from human lenses, and recombinant human crystallins (αA-, αB-, βB2-, and γC-crystallin) were generated by a prokaryotic expression system and purified by liquid chromatography. The light scattering of proteins in the presence or absence of UDCA or TUDCA was measured using a spectrofluorometer set at Ex/Em = 400/400 nm. Protein blot analysis was conducted for detection of α-crystallins in the human lens WS proteins. High concentrations of UDCA and TUDCA significantly suppressed thermal aggregation of total lens WS proteins, which contained a low level of αA-/αB-crystallin. Spectroscopic analysis with each recombinant human lens crystallin indicated that the bile acids did not suppress the thermal aggregation of γC-, βB2-, αA-, or αB-crystallin. Combination of α-crystallin and bile acid (either UDCA or TUDCA) suppressed thermal aggregation of each individual crystallin as well as a non-crystallin protein, insulin. These results suggest that UDCA or TUDCA protects the chaperone activity of α-crystallin. It is believed that these two naturally occurring intermediate waste products in the lens enhance the chaperone activity of α-crystallin. This finding may lead to the development of UDCA and TUDCA as anticataract agents.     

Differential susceptibility of alpha A- and alpha B-crystallin to gamma-ray irradiation

Alpha-crystallin, a major protein of all vertebrate lenses, consists of two different subunits, alpha A and alpha B, which form polymeric aggregates with an average molecular mass of 300-800 kDa. Both the alpha A and alpha B subunit have a molecular mass of about 20 kDa. It is not known why alpha crystallin aggregates comprise two different subunits, given that the physicochemical properties of these proteins are very similar. The present study compares the susceptibility of the alpha A and alpha B subunits to gamma-rays. We prepared a recombinant form of human alpha A- and alpha B-crystallin and then irradiated the proteins with gamma-rays. Based on far-UV CD spectra, alpha A-crystallin retained beta-sheet conformation after gamma irradiation up to 3.0 kGy, whereas alpha B-crystallin lost beta-sheet conformation upon exposure to gamma irradiation at >1.0 kGy. Size exclusion chromatography showed that the aggregation and polydispersity of recombinant alpha A-crystallin increased slightly after >1.0 kGy irradiation. In contrast, irradiation of alpha B-crystallin at 1.0 kGy resulted in the formation of huge aggregates and a marked increase in heterogeneity. We have also compared the chaperone activities of gamma-irradiated alpha A- and alpha B-crystallin aggregates. The activity of irradiated alpha A-crystallin was retained while that of the irradiated alpha B-crystallin was became inactive after irradiation of >0.5 kGy. Our results indicate that alpha A-crystallin is more stable to gamma irradiation than alpha B-crystallin.     

Study of subunit interactions of alpha A- and alpha B-crystallins and the effects of gamma-irradiation on their interactions by surface plasmon resonance

Alpha-crystallin, a major protein of mammalian lens, consists of two subunits, alpha A-crystallin and alpha B-crystallin. They interact to form an aggregate and play a prominent role in the maintenance of lens transparency. We evaluated the interaction between these subunits via surface plasmon resonance (SPR) using four combinations of immobilized protein and analyte: 1) AA: alpha A-crystallin was ligand immobilized onto the sensor and alpha A-crystallin was passed over the ligand, 2) AB: ligand - alpha A-crystallin, analyte - alpha B-crystallin, 3) BB: ligand - alpha B-crystallin, analyte- alpha B-crystallin, 4) BA: ligand - alpha B-crystallin, analyte - alpha A-crystallin. The order of rate of dissociation was AA approximately BA>BB approximately AB. We also examined the dissociation of gamma irradiated alpha A- and alpha B-crystallins. As radiation dose increased, so did the dissociation rate of all of the crystallins. The order of rate of dissociation of irradiated crystallins was BB>AB approximately BA>AA. The results indicate that BB is the most susceptible to gamma-irradiation and that alpha B-crystallin forms a more stable aggregate than alpha A-crystallin under normal conditions. However, when alpha B is irradiated the aggregate becomes unstable.     

Functional Elements in Molecular Chaperone α-Crystallin: Identification of Binding Sites in αB-Crystallin

α-Crystallin, the predominant eye lens protein with sequence homology to small heat shock proteins, acts like a molecular chaperone by suppressing the aggregation of damaged crystallins and proteins. To gain an insight into the amino acid sequences in α-crystallin involved in chaperone-like function, we used a cleavable, fluorescent, photoactive, crosslinking agent, sulfosuccinimidyl-2(7-azido-4-methylcoumarin-3-acetamido)-ethyl-1,3′ dithiopropionate (SAED), to derivatize yeast alcohol dehydrogenase (ADH) and allowed it to complex with bovine α-crystallin at 48°C. The complex was photolyzed and reduced with DTT and the subunits of α-crystallin, αA- and αB-, were separated. Fluorescence analysis showed that both αA- and αB-crystallins interacted with ADH during chaperone-like function. Tryptic digestion, amino acid sequencing, and mass spectral analysis of αB-crystallin revealed that APSWIDTGLSEMR (57-69) and VLGDVIEVHGKHEER (93-107) sequences were involved in binding with ADH.     

Sense and antisense modification of glial alpha B-crystallin production results in alterations of stress fiber formation and thermoresistance

The phenotypic effects of selectively altering the levels of alpha B- crystallin in cultured glial cells were analyzed using sense and antisense approaches. Rat C6 glioma cells and human U-373MG glioma cells were transfected with a rat alpha B-crystallin sense cDNA or an antisense cDNA regulated by a Rous sarcoma virus promoter to alter cellular levels of alpha B-crystallin. The antisense strategy resulted in decreased alpha B-crystallin levels, as revealed by Western blot and immunocytochemical analyses. The reduced alpha B-crystallin expression was accompanied by alterations in cellular phenotype: (a) a reduction of cell size and/or a slender cell morphology; (b) a disorganized microfilament network; and (c) a reduction of cell adhesiveness. Like HSP27, the presence of additional alpha B-crystallin protein confers a thermoresistant phenotype to stable transfectants. Thus, alpha B- crystallin in glioma cells plays a role in their thermal resistance and may contribute to the stability of cytoskeletal organization.     

Isolation and characterization of beta- and gamma-crystallin genes from rat genomic cosmid libraries

Two libraries, together containing about 10(6) colonies, have been constructed by cloning different size fractions of a partial Sau3A digest of rat genomic DNA in the cosmid vector pTM. Upon screening with two cDNA clones, one containing alpha A2-crystallin and one containing beta B1-crystallin sequences, 14 cosmid clones were isolated which were beta B1-crystallin-specific; none was found which contained alpha A2-crystallin sequences. The inserts of the beta B1 clones, which range from 35 to 45 kb in length, contain overlapping DNA segments covering more than 60 kb of rat genomic DNA. The composite BamHI restriction map of this region shows a single beta B1-crystallin gene, which is interrupted by several intronic sequences. Five recombinants hybridizing with two different rat lens gamma-crystallin cDNA clones were also isolated from these libraries. Four of these contain 31- to 41-kb inserts, whereas the fifth recombinant contains a 12.2-kb insert. Hybridization analysis with 5' and 3'-specific cDNA fragments indicates that altogether these inserts contain six gamma-crystallin genes, three of which are located on one insert of only 31 kb.     

The carboxy-terminal lysine of alpha B-crystallin is an amine-donor substrate for tissue transglutaminase.

A hexapeptide, corresponding to the sequence around the glutamine in beta A3-crystallin that functions as amine-acceptor for transglutaminase, was synthesized. This peptide was biotinylated and used as a probe to identify amine-donor substrates for transglutaminase among lens proteins. It was found that Ca(2+)-activated transglutaminase linked this peptide not only to several beta-crystallins but, unexpectedly, also to alpha B-crystallin. The C-terminal lysine residue of alpha B-crystalline could be identified as the site of linkage. This strengthens the notion that, at least in crystallins, all transglutaminase substrate residues are located in terminal extensions of the polypeptides. It was shown that in lens homogenate, alpha B-crystallin can be covalently crosslinked to beta-crystallins by transglutaminase. The transglutaminase-mediated crosslinking of alpha B-crystallin may have implications for its involvement in normal and pathological processes in lens and other tissues.     

Regulation of mouse lens fiber cell development and differentiation by the Maf gene

Maf is a basic domain/leucine zipper domain protein originally identified as a proto-oncogene whose consensus target site in vitro, the T-MARE, is an extended version of an AP-1 site normally recognized by Fos and Jun. Maf and the closely related family members Neural retina leucine zipper (Nrl), L-Maf, and Krml1/MafB have been implicated in a wide variety of developmental and physiologic roles; however, mutations in vivo have been described only for Krml1/MafB, in which a loss-of-function causes abnormalities in hindbrain development due to failure to activate the Hoxa3 and Hoxb3 genes. We have used gene targeting to replace Maf coding sequences with those of lacZ, and have carried out a comprehensive analysis of embryonic expression and the homozygous mutant phenotype in the eye. Maf is expressed in the lens vesicle after invagination, and becomes highly upregulated in the equatorial zone, the site at which self-renewing anterior epithelial cells withdraw from the cell cycle and terminally differentiate into posterior fiber cells. Posterior lens cells in Maf(lacZ) mutant mice exhibit failure of elongation at embryonic day 11.5, do not express (&agr;)A- and all of the (beta)-crystallin genes, and display inappropriately high levels of DNA synthesis. This phenotype partially overlaps with those reported for gene targeting of Prox1 and Sox1; however, expression of these genes is grossly normal, as is expression of Eya1, Eya2, Pax6, and Sox2. Recombinant Maf protein binds to T-MARE sites in the (alpha)A-, (beta)B2-, and (beta)A4-crystallin promoters but fails to bind to a point mutation in the (alpha)A-crystallin promoter that has been shown previously to be required for promoter function. Our results indicate that Maf directly activates many if not all of the (beta)-crystallin genes, and suggest a model for coordinating cell cycle withdrawal with terminal differentiation.     

Temperature-dependent coaggregation of eye lens αB- and β-crystallins

Crystallin is essential not only for the maintenance of eye lens transparency, but also in the biology of other tissues. Eye lens α-crystallin exists as a heteropolymer composed of two homologous subunits, αA and αB. Despite the critical role of α-crystallin in many tissues, little is known regarding structural and functional significance of the two subunits. Herein, we describe a unique feature of αB-crystallin. At high temperatures (>70 °C) not only αB-crystallin aggregates but also enhances the aggregation of other lens proteins. Intriguingly, αB-crystallin-mediated coaggregation at and above 70 °C involves β- but not γ-crystallin. Further, αA-crystallin, but not a mutant (F71L) αA-crystallin, prevented aggregation of αB-crystallin and also reduced coaggregation of αB- and β-crystallin. These studies explain the rationale for the existence of α-crystallin heteropolymer with αA subunit as a major partner that is vital for lens transparency and provide insights into αB-crystallin-induced coaggregation which may have a bearing in some pathological conditions where αB-crystallin is overexpressed.