Identification and characterization of six ß-crystallin gene mutations associated with congenital cataract in Chinese families.

BACKGROUND: This study aims to identify the underlying genetic defects of ß-crystallin (CRYB) genes responsible for congenital cataracts in a group of Chinese families. METHODS: Detailed family history and clinical data of six Chinese families with autosomal dominant congenital cataracts were recorded. Targeted exome sequencing was applied to detect the underlying genetic defects for the families. Generated variants were confirmed by PCR and sanger sequencing. Afterward, bioinformatic analysis through several computational predictive programs was performed to assess impacts of mutations on protein structure and function. RESULTS: A total of 53 participants (23 affected and 30 unaffected) from six unrelated Chinese families were recruited. Cataract phenotypes covered nuclear, total, posterior polar, pulverulent, snowflake-like, and zonular. Through targeted exome sequencing, six mutations in four ß-crystallin genes were revealed which included five missense mutations CRYBB1 p.Q70P, CRYBB2 p.E23Q, CRYBB2 p.A49V, CRYBB2 R188C, CRYBA4 p.M14K and one splice mutation CRYBB3 c.75+1 G>A. In silico results predicted pathogenic for all four missense variants except variant CRYBB2-p.A49V yielded results as tolerant. The CRYBB3 c.75+1 G>A splice site mutation was predicted to be deleterious by leading to a broken splice site, a premature stop codon, and subsequently resulting in a short peptide of 113 amino acids, which may affect protein features. CONCLUSION: The obtained results expanded mutational and phenotype spectrum of ß-crystallin genes and offer clues for pathogenesis of congenital cataracts. The data also demonstrated that targeted exome sequencing is valuable for providing molecular diagnostic information for congenital cataract patients.

ßγ-Crystallination Endows a Novel Bacterial Glycoside Hydrolase 64 with Ca2+-Dependent Activity Modulation.

The prokaryotic ßγ-crystallins are a large group of uncharacterized domains with Ca2+-binding motifs. We have observed that a vast number of these domains are found appended to other domains, in particular, the carbohydrate-active enzyme (CAZy) domains. To elucidate the functional significance of these prospective Ca2+ sensors in bacteria and this widespread domain association, we have studied one typical example from Clostridium beijerinckii, a bacterium known for its ability to produce acetone, butanol, and ethanol through fermentation of several carbohydrates. This novel glycoside hydrolase of family 64 (GH64), which we named glucanallin, is composed of a ßγ-crystallin domain, a GH64 domain, and a carbohydrate-binding module 56 (CBM56). The substrates of GH64, ß-1,3-glucans, are the targets for industrial biofuel production due to their plenitude. We have examined the Ca2+-binding properties of this protein, assayed its enzymatic activity, and analyzed the structural features of the ß-1,3-glucanase domain through its high-resolution crystal structure. The reaction products resulting from the enzyme reaction of glucanallin reinforce the mixed nature of GH64 enzymes, in contrast to the prevailing notion of them being an exotype. Upon disabling Ca2+ binding and comparing different domain combinations, we demonstrate that the ßγ-crystallin domain in glucanallin acts as a Ca2+ sensor and enhances the glycolytic activity of glucanallin through Ca2+ binding. We also compare the structural peculiarities of this new member of the GH64 family to two previously studied members.IMPORTANCE We have biochemically and structurally characterized a novel glucanase from the less studied GH64 family in a bacterium significant for fermentation of carbohydrates into biofuels. This enzyme displays a peculiar property of being distally modulated by Ca2+ via assistance from a neighboring ßγ-crystallin domain, likely through changes in the domain interface. In addition, this enzyme is found to be optimized for functioning in an acidic environment, which is in line with the possibility of its involvement in biofuel production. Multiple occurrences of a similar domain architecture suggest that such a "ßγ-crystallination"-mediated Ca2+ sensitivity may be widespread among bacterial proteins.

Dissimilarity in the Contributions of the N-Terminal Domain Hydrophobic Core to the Structural Stability of Lens ß/γ-Crystallins.

Vertebrate lens ß/γ-crystallins share a conserved tertiary structure consisting of four Greek-key motifs divided into two globular domains. Numerous inherited mutations in ß/γ-crystallins have been linked to cataractogenesis. In this research, the folding mechanism underlying cataracts caused by the I21N mutation in ßB2 was investigated by comparing the effect of mutagenesis on the structural features and stability of four ß/γ-crystallins, ßB1, ßB2, γC, and γD. Our results showed that the four ß/γ-crystallins differ greatly in solubility and stability against various stresses. The I21N mutation greatly impaired ßB2 solubility and native structure as well as its stability against denaturation induced by guanidine hydrochloride, heat treatment, and ultraviolet irradiation. However, the deleterious effects were much weaker for mutations at the corresponding sites in ßB1, γC, and γD. Molecular dynamics simulations indicated that the introduction of a nonnative hydrogen bond contributed to twisting Greek-key motif I outward, which might direct the misfolding of the I21N mutant of ßB2. Meanwhile, partial hydration of the hydrophobic interior of the domain induced by the mutation destabilized ßB1, γC, and γD. Our findings highlight the importance of nonnative hydrogen bond formation and hydrophobic core hydration in crystallin misfolding caused by inherited mutations.

Polyphenol-enriched fraction of Vaccinium uliginosum L. protects selenite-induced cataract formation in the lens of Sprague-Dawley rat pups.

Purpose: As the aging population is increasing, the incidence of age-related cataract is expected to increase globally. The surgical intervention, a treatment for cataract, still has complications and is limited to developed countries. In this study, we investigated whether the polyphenol-enriched fraction of Vaccinium uliginosum L. (FH) prevents cataract formation in Sprague-Dawley (SD) rat pups. Methods: Sixty rat pups were randomly divided into six groups: CTL, Se, FH40, FH80, FH120, and Cur80. The cataract was induced with subcutaneous injection of sodium selenite (18 µmol/kg bodyweight) on postnatal (P) day 10. All groups, except CTL, were injected with sodium selenite, and the FH40, FH80, and FH120 groups were given gastric intubation with FH40 mg/kg, 80 mg/kg, and 120 mg/kg on P9, P10, and P11. The Cur80 group was also given gastric intubation with curcumin 80 mg/kg on P9, P10, and P11. All rat pups were euthanized on P30. Results: Lens morphological analysis showed that FH dose-dependently inhibited cataract formation. In the Se group, soluble proteins were insolubilized, and the gene expression of the α-, ß-, and γ-crystallins was downregulated. However, FH treatment statistically significantly inhibited insolubilization of soluble proteins and downregulation of the gene expression of the α-, ß-, and γ-crystallins. In the Se group, the gene and protein levels of m-calpain were downregulated, which were attenuated with FH treatment. In addition, sodium selenite injection caused reduced antioxidant enzymes (superoxide dismutase (SOD) and glutathione peroxidase (GPx)), glutathione (GSH) depletion, and malondialdehyde (MDA) production in the lens. The administration of FH inhibited sodium selenite-induced oxidative stress in a dose-dependent manner. The mechanism of protection against oxidative stress by FH involves NF-E2-related factor (Nrf-2) and hemoxygenase-1 (HO-1). FH treatment inhibited decrease of Nrf-2 in the nucleus fraction and HO-1 in the cytosol fraction. Finally, the FH treatment protected poly (ADP)-ribose polymerase (PARP) from cleavage, determined with western blotting. Conclusions: FH showed a preventive effect against cataract formation by inhibiting m-calpain-mediated proteolysis and oxidative stress in the lens. These results suggest that FH could be a potential anticataract agent in age-related cataract.

Crystal Structure of Chicken γS-Crystallin Reveals Lattice Contacts with Implications for Function in the Lens and the Evolution of the ßγ-Crystallins.

Previous attempts to crystallize mammalian γS-crystallin were unsuccessful. Native L16 chicken γS crystallized avidly while the Q16 mutant did not. The X-ray structure for chicken γS at 2.3 Å resolution shows the canonical structure of the superfamily plus a well-ordered N arm aligned with a ß sheet of a neighboring N domain. L16 is also in a lattice contact, partially shielded from solvent. Unexpectedly, the major lattice contact matches a conserved interface (QR) in the multimeric ß-crystallins. QR shows little conservation of residue contacts, except for one between symmetry-related tyrosines, but molecular dipoles for the proteins with QR show striking similarities while other γ-crystallins differ. In γS, QR has few hydrophobic contacts and features a thin layer of tightly bound water. The free energy of QR is slightly repulsive and analytical ultracentrifugation confirms no dimerization in solution. The lattice contacts suggest how γ-crystallins allow close packing without aggregation in the crowded environment of the lens.

Transcriptome Analysis and Differential Gene Expression on the Testis of Orange Mud Crab, Scylla olivacea, during Sexual Maturation.

Adequate genetic information is essential for sustainable crustacean fisheries and aquaculture management. The commercially important orange mud crab, Scylla olivacea, is prevalent in Southeast Asia region and is highly sought after. Although it is a suitable aquaculture candidate, full domestication of this species is hampered by the lack of knowledge about the sexual maturation process and the molecular mechanisms behind it, especially in males. To date, data on its whole genome is yet to be reported for S. olivacea. The available transcriptome data published previously on this species focus primarily on females and the role of central nervous system in reproductive development. De novo transcriptome sequencing for the testes of S. olivacea from immature, maturing and mature stages were performed. A total of approximately 144 million high-quality reads were generated and de novo assembled into 160,569 transcripts with a total length of 142.2 Mb. Approximately 15-23% of the total assembled transcripts were annotated when compared to public protein sequence databases (i.e. UniProt database, Interpro database, Pfam database and Drosophila melanogaster protein database), and GO-categorised with GO Ontology terms. A total of 156,181 high-quality Single-Nucleotide Polymorphisms (SNPs) were mined from the transcriptome data of present study. Transcriptome comparison among the testes of different maturation stages revealed one gene (beta crystallin like gene) with the most significant differential expression-up-regulated in immature stage and down-regulated in maturing and mature stages. This was further validated by qRT-PCR. In conclusion, a comprehensive transcriptome of the testis of orange mud crabs from different maturation stages were obtained. This report provides an invaluable resource for enhancing our understanding of this species' genome structure and biology, as expressed and controlled by their gonads.

Effect of Asp 96 isomerization on the properties of a lens αB-crystallin-derived short peptide.

One of the major reasons for age-related cataract formation is an accumulation of insoluble lens proteins. In particular, higher-order α-crystallin aggregates, comprising αA and αB subunits, are insolubilized by the build up of various post-translational modifications over time. Although we previously found an exceptional amount of Asp96 isomerization in αB-crystallin from aged human lens, the biological effect remains unknown. To approximate the effect of Asp 96 isomerization in αB-crystallin, here residues 93-103 of αB-crystallin were chemically synthesized as peptides in which l-α-Asp was replaced with l-ß-Asp, D-α-Asp, or D-ß-Asp. The resulting peptides were then compared in a biological assay. The results showed that isomerization of Asp 96 altered both the local structure of peptide and its stability against enzymatic digestion. In addition, the synthesized peptides decreased the insoluble fraction of heated α-crystallin. The D-ß-Asp-containing peptide further decreased heat-induced precipitation of α-crystallin, and a chaperone assay based on heated alcohol dehydrogenase implied differential interaction of the peptides with substrate depending on the Asp isomer present in each. Our results suggest that the formation of Asp isomers is likely to affect the higher-order oligomer structure of α-crystallin and thereby its chaperone functions in aged lens.

Ca2+-binding motif of ßγ-crystallins.

ßγ-Crystallin-type double clamp (N/D)(N/D)XX(S/T)S motif is an established but sparsely investigated motif for Ca(2+) binding. A ßγ-crystallin domain is formed of two Greek key motifs, accommodating two Ca(2+)-binding sites. ßγ-Crystallins make a separate class of Ca(2+)-binding proteins (CaBP), apparently a major group of CaBP in bacteria. Paralleling the diversity in ßγ-crystallin domains, these motifs also show great diversity, both in structure and in function. Although the expression of some of them has been associated with stress, virulence, and adhesion, the functional implications of Ca(2+) binding to ßγ-crystallins in mediating biological processes are yet to be elucidated.

Strain-independent increases of crystallin proteins in the retina of type 1 diabetic rats.

Diabetic retinopathy is the leading cause of vision loss in working-age individuals in the United States and is expected to continue growing with the increased prevalence of diabetes. Streptozotocin-induced hyperglycemia in rats is the most commonly used model for diabetic retinopathy. Previous studies have shown that this model can lead to different inflammatory changes in the retina depending on the strain of rat. Our previous work has shown that crystallin proteins, including members of the alpha- and beta/gamma-crystallin subfamilies, are upregulated in the STZ rat retina. Crystallin proteins have been implicated in a number of cellular processes, such as neuroprotection, non-native protein folding and vascular remodeling. In this current study, we have demonstrated that unlike other strain-dependent changes, such as inflammatory cytokines and growth factor levels, in the STZ rat, the protein upregulation of crystallins is consistent across the Brown Norway, Long-Evans and Sprague-Dawley rat strains in the context of diabetes. Taken together, these data illustrate the potential critical role played by crystallins, and especially alpha-crystallins, in the retina in the context of diabetes.

Structural integrity of the Greek key motif in ßγ-crystallins is vital for central eye lens transparency.

BACKGROUND: We highlight an unrecognized physiological role for the Greek key motif, an evolutionarily conserved super-secondary structural topology of the ßγ-crystallins. These proteins constitute the bulk of the human eye lens, packed at very high concentrations in a compact, globular, short-range order, generating transparency. Congenital cataract (affecting 400,000 newborns yearly worldwide), associated with 54 mutations in ßγ-crystallins, occurs in two major phenotypes nuclear cataract, which blocks the central visual axis, hampering the development of the growing eye and demanding earliest intervention, and the milder peripheral progressive cataract where surgery can wait. In order to understand this phenotypic dichotomy at the molecular level, we have studied the structural and aggregation features of representative mutations. METHODS: Wild type and several representative mutant proteins were cloned, expressed and purified and their secondary and tertiary structural details, as well as structural stability, were compared in solution, using spectroscopy. Their tendencies to aggregate in vitro and in cellulo were also compared. In addition, we analyzed their structural differences by molecular modeling in silico. RESULTS: Based on their properties, mutants are seen to fall into two classes. Mutants A36P, L45PL54P, R140X, and G165fs display lowered solubility and structural stability, expose several buried residues to the surface, aggregate in vitro and in cellulo, and disturb/distort the Greek key motif. And they are associated with nuclear cataract. In contrast, mutants P24T and R77S, associated with peripheral cataract, behave quite similar to the wild type molecule, and do not affect the Greek key topology. CONCLUSION: When a mutation distorts even one of the four Greek key motifs, the protein readily self-aggregates and precipitates, consistent with the phenotype of nuclear cataract, while mutations not affecting the motif display 'native state aggregation', leading to peripheral cataract, thus offering a protein structural rationale for the cataract phenotypic dichotomy "distort motif, lose central vision".

Vitamin E reduces TGF-beta2-induced changes in human trabecular meshwork cells.

AIM: To determine the effects of vitamin E on transforming growth factor-beta2 (TGF-ß2)-induced cellular changes in cultured human trabecular meshwork (TM) cells. MATERIALS AND METHODS: Human TM cells were pre-treated with different concentrations of vitamin E. Afterwards, cells were exposed to 1.0 ng/ml TGF-ß2 for 24 h. Expressions of the heat shock protein αB-crystallin, the extracellular matrix (ECM) component fibronectin and the ECM-degrading enzyme matrix metalloproteinase-2 (MMP-2) were examined by real-time polymerase chain reaction (PCR) analyses. The cytoskeleton was investigated by phalloidin staining. RESULTS: TGF-ß2 increased the expressions of αB-crystallin and fibronectin and reduced the levels of MMP-2. TGF-ß2 induced the formation of actin stress fibers and cross-linked actin networks. Pre-treatment with different concentrations of vitamin E reversed the TGF-ß2-induced cellular changes in cultured human TM cells. CONCLUSIONS: TGF-ß2-mediated changes in human TM cells could be reduced by pre-treatment with vitamin E. Therefore, it may be speculated that increasing the antioxidative capacity may help to lower the incidence of characteristic glaucomatous changes in the TM.

Changes in zebrafish (Danio rerio) lens crystallin content during development.

PURPOSE: The roles that crystallin proteins play during lens development are not well understood. Similarities in the adult crystallin composition of mammalian and zebrafish lenses have made the latter a valuable model for examining lens function. In this study, we describe the changing zebrafish lens proteome during development to identify ontogenetic shifts in crystallin expression that may provide insights into age-specific functions. METHODS: Two-dimensional gel electrophoresis and size exclusion chromatography were used to characterize the lens crystallin content of 4.5-day to 27-month-old zebrafish. Protein spots were identified with mass spectrometry and comparisons with previously published proteomic maps, and quantified with densitometry. Constituents of size exclusion chromatography elution peaks were identified with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. RESULTS: Zebrafish lens crystallins were expressed in three ontogenetic patterns, with some crystallins produced at relatively constant levels throughout development, others expressed primarily before 10 weeks of age (ßB1-, ßA1-, and γN2-crystallins), and a third group primarily after 10 weeks (α-, ßB3-, and γS-crystallins). Alpha-crystallins comprised less than 1% of total lens protein in 4.5-day lenses and increased to less than 7% in adult lenses. The developmental period between 6 weeks and 4 months contained the most dramatic shifts in lens crystallin expression. CONCLUSIONS: These data provide the first two-dimensional gel electrophoresis maps of the developing zebrafish lens, with quantification of changing crystallin abundance and visualization of post-translational modification. Results suggest that some crystallins may play stage specific roles during lens development. The low levels of zebrafish lens α-crystallin relative to mammals may be due to the high concentrations of γ-crystallins in this aquatic lens. Similarities with mammalian crystallin expression continue to support the use of the zebrafish as a model for lens crystallin function.

Modeling human protein aggregation cardiomyopathy using murine induced pluripotent stem cells.

Several mutations in αB-crystallin (CryAB), a heat shock protein with chaperone-like activities, are causally linked to skeletal and cardiac myopathies in humans. To better understand the underlying pathogenic mechanisms, we had previously generated transgenic (TG) mice expressing R120GCryAB, which recapitulated distinguishing features of the myopathic disorder (e.g., protein aggregates, hypertrophic cardiomyopathy). To determine whether induced pluripotent stem cell (iPSC)-derived cardiomyocytes, a new experimental approach for human disease modeling, would be relevant to aggregation-prone disorders, we decided to exploit the existing transgenic mouse model to derive iPSCs from tail tip fibroblasts. Several iPSC lines were generated from TG and non-TG mice and validated for pluripotency. TG iPSC-derived cardiomyocytes contained perinuclear aggregates positive for CryAB staining, whereas CryAB protein accumulated in both detergent-soluble and insoluble fractions. iPSC-derived cardiomyocytes identified by cardiac troponin T staining were significantly larger when expressing R120GCryAB at a high level in comparison with TG low expressor or non-TG cells. Expression of fetal genes such as atrial natriuretic factor, B-type natriuretic peptide, and α-skeletal α-actin, assessed by quantitative reverse transcription-polymerase chain reaction, were increased in TG cardiomyocytes compared with non-TG, indicating the activation of the hypertrophic genetic program in vitro. Our study demonstrates for the first time that differentiation of R120G iPSCs into cardiomyocytes causes protein aggregation and cellular hypertrophy, recapitulating in vitro key pathognomonic hallmarks found in both animal models and patients. Our findings pave the way for further studies exploiting this cell model system for mechanistic and therapeutic investigations.

The congenital cataract-linked A2V mutation impairs tetramer formation and promotes aggregation of ßB2-crystallin.

ß/γ-Crystallins, the major structural proteins in human lens, are highly conserved in their tertiary structures but distinct in the quaternary structures. The N- and C-terminal extensions have been proposed to play a crucial role in mediating the size of ß-crystallin assembly. In this research, we investigated the molecular mechanism underlying the congenital hereditary cataract caused by the recently characterized A2V mutation in ßB2-crystallin. Spectroscopic experiments indicated that the mutation did not affect the secondary and tertiary structures of ßB2-crystallin. The mutation did not affect the formation of ßB2/ßA3-crystallin heteromer as well as the stability and folding of the heteromer, suggesting that the mutation might not interfere with the protein interacting network in the lens. However, the tetramerization of ßB2-crystallin at high protein concentrations was retarded by the A2V mutation. The mutation slightly decreased the thermal stability and promoted the thermal aggregation of ßB2-crystallin. Although it did not influence the stability of ßB2-crystallin against denaturation induced by chemical denaturants and UV irradiation, the A2V mutant was more prone to be trapped in the off-pathway aggregation process during kinetic refolding. Our results suggested that the A2V mutation might lead to injury of lens optical properties by decreasing ßB2-crystallin stability against heat treatment and by impairing ßB2-crystallin assembly into high-order homo-oligomers.

The p53-Bak apoptotic signaling axis plays an essential role in regulating differentiation of the ocular lens.

The tumor suppressor p53 is a master regulator of apoptosis and also plays a key role in cell cycle checking. In our previous studies, we demonstrated that p53 directly regulates Bak in mouse JB6 cells (Qin et al. 2008. Cancer Research. 68(11):4150) and that p53-Bak signaling axis plays an important role in mediating EGCG-induced apoptosis. Here, we demonstrate that the same p53-Bak apoptotic signaling axis executes an essential role in regulating lens cell differentiation. First, during mouse lens development, p53 is expressed and differentially phosphorylated at different residues. Associated with p53 expression, Bak is also significantly expressed during mouse lens development. Second, human p53 directly regulates Bak promoter and Bak expression in p53 knockout mice (p53-/-) was significantly downregulated. Third, during in vitro bFGF-induced lens cell differentiation, knockdown of p53 or Bak leads to significant inhibition of lens cell differentiation. Fourth, besides the major distribution of Bak in cytoplasm, it is also localized in the nucleus in normal lens or bFGF-induced differentiating lens cells. Finally, p53 and Bak are co-localized in both cytoplasm and nucleus, and their interaction regulates the stability of p53. Together, these results demonstrate for the first time that the p53-Bak apoptotic signaling axis plays an essential role in regulating lens differentiation.

Iterative cloning, overexpression, purification and isotopic labeling of an engineered dimer of a Ca(2+)-binding protein of the ßγ-crystallin superfamily from Methanosarcina acetivorans.

ßγ-Crystallins are a large superfamily of proteins found in vertebrate eye lens. They are hetero-dimers (linked in tandem by a specific peptide) and are shown to bind calcium. The monomers possess two ß-strand rich greek-key motifs. Recently, a structurally closest member to the family of lens ßγ-crystallins has been described, for the first time, from the archaea Methanosarcina acetivorans, which is named as M-crystallin. Unlike lens ßγ-crystallins, M-crystallin exits as a monomer. Here, we synthesized a dimeric gene of M-crystallin in which two monomers are linked by a 10-amino acid residue coding sequence. The linker sequence in the target protein is long and flexible enough to reduce the proximity between the individual crystallins in the dimer. This methodology would be highly beneficial in designing polyproteins (two or more proteins linked in tandem to aid mechanical stretching studies) that are regularly used in single-molecule force spectroscopy. The dimer of M-crystallin was overexpressed in Escherichia coli BLR(DE3) strain. The overexpressed protein containing an N-terminal hexa-histidine tag was purified using nickel affinity chromatography and then by size-exclusion chromatography. Further, a method to purify isotopically ((15)N) labeled protein with high yield for NMR studies is reported. The uniformly (15)N-labeled M-crystallin dimer thus produced has been characterized by recording sensitivity enhanced 2D [(15)N-(1)H] HSQC and other optical spectroscopy techniques. Observation of only one set of peaks in the HSQC, along with the structural characterization using optical spectroscopy, suggests that the domains in the dimer possess similar structure as that of the monomer.

Lens growth and protein changes in the eastern grey kangaroo.

PURPOSE: Development in marsupials takes place predominantly ex utero while the young is attached to a nipple in the mother's pouch, very different from that in other species. This study was undertaken to examine whether this affects lens growth and the production of lens proteins in kangaroos. METHODS: Fresh lenses were obtained at official culls from eastern gray kangaroos (Macropus giganteus). Wet weights were recorded for all and protein contents were determined for one lens from each animal. Dry weights, after fixation were obtained for 20 lenses. Ages were determined using both molar progression and total lens protein content. Lenses were divided into concentric layers by controlled dissolution using phosphate buffered saline. Samples were taken for determination of protein contents and dry weights, which were then used to determine the age of the layer removed. Soluble crystallin distributions were determined by fractionation of the centrifuged extracts using HPLC-GPC and the polypeptide contents of both soluble and insoluble proteins were assessed by SDS-PAGE. RESULTS: Lens growth is continuous from birth throughout adulthood and the increases in wet weight and fixed dry weight can be described with a single logistic growth functions for the whole life span. Three major crystallin classes, α-, ß-, and γ-crystallins, were identified in the immature pouch-young animals aged around 60 days after birth. Adult lenses contain, in addition, the taxon-specific µ-crystallin. The proportions of these vary with the age of the lens tissue due to age related insolubilization as well as changes in the synthesis patterns. During early lactation (birth to 190 days), the α-, ß-, and γ-crystallins represent 25, 53, and 20% of the total protein, respectively. After the pouch-young first releases the nipple (190 days), there is a rapid decrease in the production of γ-crystallins to around 5% of the total and a corresponding increase in µ-crystallin, from 0.5% to 15%. These changes were complete by the time the animal was fully weaned, around 1.5 years, and the final proportions of the 4 protein classes were maintained for the rest of life. The solubilities of α- and ß-crystallins in the center of the lens decreased after age 5 years. CONCLUSIONS: Kangaroo lens growth is asymptotic, similar to that in most other species, even though most development of the young animal takes place ex utero. Changes in the patterns of lens protein synthesis in the kangaroo are similar to those observed in other species except for the large decrease in γ-crystallin and the matching increase in the marsupial-specific µ-crystallin, during late lactation.

Evolutionary remodeling of ßγ-crystallins for domain stability at cost of Ca2+ binding.

The topologically similar ßγ-crystallins that are prevalent in all kingdoms of life have evolved for high innate domain stability to perform their specialized functions. The evolution of stability and its control in ßγ-crystallins that possess either a canonical (mostly from microorganisms) or degenerate (principally found in vertebrate homologues) Ca2+-binding motif is not known. Using equilibrium unfolding of ßγ-crystallin domains (26 wild-type domains and their mutants) in apo- and holo-forms, we demonstrate the presence of a stability gradient across these members, which is attained by the choice of residues in the (N/D)(N/D)XX(S/T)S Ca2+-binding motif. The occurrence of a polar, hydrophobic, or Ser residue at the 1st, 3rd, or 5th position of the motif is likely linked to a higher domain stability. Partial conversion of a microbe-type domain (with a canonical Ca2+-binding motif) to a vertebrate-type domain (with a degenerate Ca2+-binding motif) by mutating serine to arginine/lysine disables the Ca2+-binding but significantly augments its stability. Conversely, stability is compromised when arginine (in a vertebrate-type disabled domain) is replaced by serine (as a microbe type). Our results suggest that such conversions were acquired as a strategy for desired stability in vertebrate members at the cost of Ca2+-binding. In a physiological context, we demonstrate that a mutation such as an arginine to serine (R77S) mutation in this motif of γ-crystallin (partial conversion to microbe-type), implicated in cataracts, decreases the domain stability. Thus, this motif acts as a "central tuning knob" for innate as well as Ca2+-induced gain in stability, incorporating a stability gradient across ßγ-crystallin members critical for their specialized functions.

Temperature-dependent coaggregation of eye lens αB- and ß-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 ß- 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 ß-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.

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.