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At least 19 recordsLinked to original sources

PubMed2005

Effect of oxidized betaB3-crystallin peptide on lens betaL-crystallin: interaction with betaB2-crystallin.

PURPOSE: To investigate the interaction of oxidized betaB3-crystallin peptide (residues 152-166) with betaL-crystallin and to identify peptide-interaction sites. METHODS: Peptides were oxidized by using CuSO4 and H2O2. Aggregation and light-scattering assays of bovine betaL-crystallin were conducted at 55 degrees C and 37 degrees C, respectively. Assays were performed in the presence of oxidized and nonoxidized betaB3-crystallin peptides and in the presence of alpha-crystallin. Peptide-induced change in hydrophobicity was determined by bis-ANS (4,4'-dianilino-1,1' binaphthyl-5,5' disulfonic acid) binding study. Oxidized betaB3-peptide binding sites were identified by sulfo-SBED (sulfosuccinimidyl-2-[6-(biotinamido)-2-{p-azidobenzamido}-hexanoamido] ethyl-1-3 dithiopropionate) labeling and mass spectrometric analysis. RESULTS: Aggregation and relative light-scattering of betaL-crystallin was higher in the presence of oxidized betaB3-crystallin peptide than with betaL-crystallin, without oxidized peptide and with nonoxidized peptide. Enhanced aggregation was observed despite the presence of alpha-crystallin in the assay. Furthermore, a significant increase in aggregation and light-scattering was observed in the presence of oxidized betaB3-peptide at 37 degrees C. Bis-ANS binding to betaL-crystallin treated with oxidized betaB3-peptide was two to three times higher than in the controls at 37 degrees C. The oxidized betaB3-peptide preferentially interacted with betaB2-crystallin. The data were confirmed by mass spectrometric analysis. CONCLUSIONS: Oxidized betaB3-peptide interacts with betaB2-crystallin and enhances its aggregation and precipitation. Peptide-induced aggregation and increased hydrophobicity of the lens crystallin at 37 degrees C are relevant to crystallin aggregation in the aging lenses.

Animals↗

PubMed1996

Isoelectric focusing of crystallins in microsections of calf and adult bovine lens. Identification of water-insoluble crystallins complexing under nondenaturing conditions: demonstration of chaperone activity of alpha-crystallin.

Topographic studies of crystallin fractions from the young adult bovine lens revealed that lenses do not have a homogeneous distribution of crystallins. There are, however, gradual differences between the cortices and the nucleus. The isolated lenses were separated mechanically into lens equator and inner cylinder. The latter was then sectioned in a special sectioning machine into 11-12 morphological layers (from anterior cortex through nucleus to posterior cortex). Matters of the lens sections were separated into water-soluble (WS) and water-insoluble (WI) crystallins. The WI fractions were solubilized with 100% formamide, or dissolved into 7 M urea. Crystallin profiles were obtained for each lens layer, using thin-layer isoelectric focusing in polyacrylamide gel. WS crystallins from the lens equator revealed a separation into HM-, alpha L-, beta H-, beta L-, beta S- and gamma-crystallins. The WI fractions of the layers dissolved in urea gave a separation into the individual HM- (3 components), alpha L- (4 components), beta- (6 component groups), beta S- (2 components) and gamma- (11 components) crystallins in the different morphological layers. The results confirm that a significant age-related increase in several beta- and gamma-crystallins incorporated into alpha-crystallins exists in the patterns of WI fractions of the different layers from lenses of 2.2 and 5.9 years. The WI crystallins solubilized in formamide showed only the presence of HM weight and alpha-crystallin moieties, due to the action of chaperone activity of alpha-crystallin. The nature of the WI protein fraction in the separated lens layers reflected to the aggregated state of: alpha L-, beta L-, beta S- and gamma-crystallins in the different regions of the lens, concealed in the central cavity of the alpha-crystallin chaperone model.

Aging↗

PubMed1995

Rapid refolding studies on the chaperone-like alpha-crystallin. Effect of alpha-crystallin on refolding of beta- and gamma-crystallins.

alpha-Crystallin, a multimeric protein present in the eye lens, is shown to have chaperone-like activity in preventing thermally induced aggregation of enzymes and other crystallins. We have studied the rapid refolding of alpha-crystallin, and compared it with other calf eye lens proteins, namely beta- and gamma-crystallins. alpha-Crystallin forms a clear solution upon rapid refolding from 8 M urea. The refolded alpha-crystallin has native-like secondary, tertiary, and quaternary structures as revealed by circular dichroism and fluorescence characteristics as well as gel filtration and sedimentation velocity measurements. On rapid refolding, beta- and gamma-crystallins aggregate and form turbid solutions. The presence of alpha-crystallin in the refolding buffer marginally increases the recovery of beta- and gamma-crystallins in the soluble form. However, unfolding of these crystallins together with alpha-crystallin using 8 M urea and subsequent refolding significantly increases the recovery of these proteins in the soluble form. These results indicate that an intermediate of alpha-crystallin formed during refolding is more effective in preventing the aggregation of beta- and gamma-crystallins. This supports our earlier hypothesis (Raman, B., and Rao, C. M. (1994) J. Biol. Chem. 269, 27264-27268) that the chaperone-like activity of alpha-crystallin is more pronounced in its structurally perturbed state.

Animals↗

PubMed1996

A comparison of structural relationships among alpha-crystallin, human Hsp27, gamma-crystallins and beta B2-crystallin.

The 3D structures of alpha-crystallin, a major eye lens protein, and related small heat shock proteins are unresolved. It has been assumed that alpha-crystallin is primarily a beta-sheet globular protein similar to alpha-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 beta/gamma-crystallins. Similar analyses of alpha-crystallin subunits, alpha A and alpha B, 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 beta/gamma-crystallins, both Hsp27 and alpha-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 alpha-crystallin and small heat shock protein function. Differences in flexibility profiles and estimated secondary structure distribution in alpha-crystallin by three recent/updated algorithms from far UV-CD spectra support our predicted 3D structure and the concept that alpha-crystallin and members of beta/gamma superfamily are structurally dissimilar.

Algorithms↗

PubMed2002

Role of the C-terminal extensions of alpha-crystallins. Swapping the C-terminal extension of alpha-crystallin to alphaB-crystallin results in enhanced chaperone activity.

Several small heat shock proteins contain a well conserved alpha-crystallin domain, flanked by an N-terminal domain and a C-terminal extension, both of which vary in length and sequence. The structural and functional role of the C-terminal extension of small heat shock proteins, particularly of alphaA- and alphaB-crystallins, is not well understood. We have swapped the C-terminal extensions between alphaA- and alphaB-crystallins and generated two novel chimeric proteins, alphaABc and alphaBAc. We have investigated the domain-swapped chimeras for structural and functional alterations. We have used thermal and non-thermal models of protein aggregation and found that the chimeric alphaB with the C-terminal extension of alphaA-crystallin, alphaBAc, exhibits dramatically enhanced chaperone-like activity. Interestingly, however, the chimeric alphaA with the C-terminal extension of alphaB-crystallin, alphaABc, has almost lost its activity. Pyrene solubilization and bis-1-anilino-8-naphthalenesulfonate binding studies show that alphaBAc exhibits more solvent-exposed hydrophobic pockets than alphaA, alphaB, or alphaABc. Significant tertiary structural changes are revealed by tryptophan fluorescence and near-UV CD studies upon swapping the C-terminal extensions. The far-UV CD spectrum of alphaBAc differs from that of alphaB-crystallin whereas that of alphaABc overlaps with that of alphaA-crystallin. Gel filtration chromatography shows alteration in the size of the proteins upon swapping the C-terminal extensions. Our study demonstrates that the unstructured C-terminal extensions play a crucial role in the structure and chaperone activity, in addition to generally believed electrostatic "solubilizer" function.

Amino Acid Sequence↗

PubMed2002

Porcine eye lens crystallins: antigenic similarity with human crystallins and tool for the detection of anti-crystallin antibodies.

BACKGROUND: The usual sources of antigenic material for investigations on circulating immunoglobulins with anti-lens crystallins specificity are saline extracts of human cataract lenses. This practice has a number of drawbacks, especially the possible antigenic alterations that may have occurred in cataract lenses. The aim of this investigation was to compare the antigenic properties of porcine eye lens crystallins and human crystallins, with regard to the possibility for an alternative source of antigenic material for detection of anti-crystallin antibodies in human sera. METHODS: We produced rabbit antisera against saline extracts of human and porcine eye lenses. These sera were applied for the antigenic characterizations of the two extracts with indirect and absorption enzyme-linked immunosorbent assay. The two antigens were further compared by testing them against 30 human sera from cataract patients and 30 sera from blood donors. RESULTS: The antibodies raised against human eye lens cross-reacted with antigens of the porcine lens. This finding was supported by the absorption experiments - the antigens of the porcine eye lens strongly inhibit the reactivity of the rabbit serum raised against human eye lens and vice versa. We established a significant positive correlation (Spearman, r=0.89, P<0.0001) between the reactivity of the tested sera against human and porcine lens extracts. CONCLUSION: These data demonstrated a strong antigenic similarity between human and porcine lens crystallins, suggesting the appropriateness of the use of porcine lens extracts for the detection of humoral anti-lens autoimmune response in patients with eye diseases.

Adult↗

PubMed2000

In vitro filament-like formation upon interaction between lens alpha-crystallin and betaL-crystallin promoted by stress.

PURPOSE: To determine whether alpha-crystallin is capable of forming filament-like structures with other members of the crystallin family. METHODS: Water-soluble crystallins were isolated from calf lenses and fractionated into alpha-, betaH-, betaL-, and gamma-crystallins according to standard procedures. Chaperone-like activity of alpha-crystallin was determined in control and UV-A-irradiated lenses by the heat-induced aggregation assay of betaL-crystallin. Protein samples from this assay were analyzed by electron microscopy. In vitro filament formation was examined by transmission immunoelectron microscopy using specific antibodies directed against the crystallins. Involvement of intermediate filament constituents was excluded by the results of Western blot analysis, which were all negative. Moreover, the in vitro amyloid fibril interaction test using thioflavin T (ThT) was also performed. RESULTS: At the supramolecular level heating at 60 degrees C has no effect on the morphologic appearance of alpha-crystallin as observed by transmission electron microscopy. Moreover alpha-crystallin obtained from UV-A-irradiated lenses shows a virtually identical shape. However, heating in the presence of betaL-crystallin results in the formation of filament-like alphabeta-hybrids as demonstrated by immunoelectron microscopy using specific antibodies directed either against alpha- or betaL-crystallin. Parallel experiments with alpha-crystallin derived from UV-A-irradiated lenses showed even more pronounced filamentous structures, compared with the controls. Nonetheless, we were able to show that the UV-light treatment affected the chaperone-like capacity of alpha-crystallin, as revealed by a diminished ability to inhibit in vitro denaturation of betaL-crystallin. To exclude the presence of cytoskeletal contamination in the crystallin preparations, vimentin antibodies were also tested. These latter experiments were negative. The filamentous nature of the hybrids was further confirmed by the results obtained with the ThT assay earlier applied for the detection of amyloid fibrils. CONCLUSIONS: Crystallin hybrids have previously been detected in the water-soluble lens crystallin fraction. Our findings indicate that such endogenous hybrids, formerly called "rods," may result from stress-induced interaction between alpha-crystallin and other lens constituents such as betaL-crystallin. Because the hybrid formation is enhanced when alpha-crystallin from UV-A-irradiated lenses is used as one of the two components of the hybrid, one can only speculate that this formation may be one of the factors leading to UV-A cataract.

Animals↗

PubMed2005

Screening of crystallin-crystallin interactions using microequilibrium dialysis.

PURPOSE: It has been hypothesized that short-range, protein-protein interactions of crystallin are necessary for the maintenance of lens transparency. Because of their probable weak nature, it has been difficult to both detect and quantitate the nature of these interactions. To determine if interactions exist between alpha-crystallin and gamma-crystallin under true equilibrium conditions, we have used microequilibrium dialysis. METHODS: Total alpha-crystallin and gamma-crystallin were prepared from soluble proteins of fetal bovine lenses by HPLC and gel filtration chromatography. The proteins were added to one side of a microequilibrium dialysis cell, comprised of two chambers separated by a membrane with 100 kDa molecular weight cut-off. After reaching equilibrium, the amount of free gamma-crystallin and the amount of gamma-crystallin bound to alpha-crystallin was determined by HPLC and reverse phase analysis of both chambers. Selected gamma-crystallin that bound to alpha-crystallin was further purified by ion exchange chromatography, and then incubated with alpha-crystallin, to verify the specificity of their binding. RESULTS: Analysis of both microequilibrium dialysis chambers incubated at different times at 37 degrees C indicated that equilibrium was reached at 4 days. When total alpha-crystallin and gamma-crystallin were incubated for this time period, significant binding was observed between alpha-crystallin and the IIIA, II, and IVA species of gamma-crystallin. These interactions were confirmed by microequilibrium dialysis determinations containing alpha-crystallin and purified gamma-crystallin species. CONCLUSIONS: These results show that microequilibrium dialysis can be used to demonstrate significant noncovalent interactions of alpha-crystallin and gamma-crystallin under true equilibrium conditions.

Animals↗

PubMed2005

Effect of oxidized betaB3-crystallin peptide (152-166) on thermal aggregation of bovine lens gamma-crystallins: identification of peptide interacting sites.

We studied the effect of oxidized betaB3-crystallin peptide (residues 152-166) on the thermal aggregation of bovine gamma-crystallin and on chaperone activity of alpha-crystallin. Thermal aggregation of gamma-crystallin was higher in the presence of oxidized betaB3-crystallin peptide than without oxidized peptide. Increased aggregation was not observed in the presence of unoxidized betaB3-crystallin peptide or a control oxidized peptide. Enhanced aggregation of gamma-crystallin by oxidized betaB3-crystallin peptide was observed even at 37 degrees C. Interaction with oxidized betaB3-peptide increased the hydrophobicity in the gamma-crystallin as shown by increased 4, 4'-dianilino-1, 1'-binaphthyl-5, 5'-disulfonic acid (bis-ANS) binding. Enhanced aggregation of gamma-crystallin was observed despite the presence of alpha-crystallin (a chaperone protein) in the system. Sulfo succinimidyl-2-[6-(biotinamido)-2-{p-azidobenzamido}-hexanoamido]ethyl-1-3 dithio propionate (Sulfo-SBED) cross-linker studies further confirmed the interaction between oxidized betaB3-crystallin peptide and gamma-crystallin. Peptide interacted sites in gamma-crystallin were identified by matrix assisted laser desorption time-of-flight mass spectrometric methods and the result suggests that oxidized betaB3-crystallin peptide interacted with amino acid residues present on the outer surface of the gamma-crystallin. These results imply that oxidized betaB3-crystallin peptide interact with gamma-crystallins and enhance their aggregation and light scattering.

Amino Acid Sequence↗

PubMed1998

Higher glycation of beta L- and beta S-crystallins in the anterior lens cortex and maximum glycation of gamma-crystallins in the bovine lens nucleus, demonstrated by frozen sectioning, isoelectric focusing and lectin staining.

The aim of the current study was to demonstrate glycation of beta L-, beta S- and gamma-crystallins in the young bovine lens. To establish which of the crystallins are glycated and where they are located in the lens, we carried out microsectioning of the lens, followed by isoelectric focusing (IEF). Four bovine lenses of 1.183 +/- 0.070 years were frozen-sectioned into equator and 11 layers. Water-soluble crystallins were separated by IEF and stained: (1) with Coomassie brilliant blue for proteins; (2) with the lectin concanavalin A, followed by horseradish peroxidase and diaminobenzidine, for glycated proteins. Experiments were performed with crystallins and proteins in native form, in the absence of denaturants. The crystallins were separated by IEF into alpha-crystallins of high molecular weight (HM), alpha L-, beta H-, beta L-, beta S- and gamma-crystallins. In the lectin staining experiments, only HM, beta L-, beta S- and gamma-crystallins were positive, whereas the alpha L- and beta H-crystallins were negative. Contrary to the glycated gamma-crystallins in the lens nucleus, the beta S- and beta L-crystallins were predominantly glycated in the anterior cortex and to a somewhat lower extent also in the posterior cortical regions. The degree of glycation (total densitometric readings of lectin-stained bands/Coomassie-blue-stained bands) is as follows: total gamma-crystallins 2.44, beta S-crystallins 0.77 and beta L-crystallins 0.28. Though glycation in the bovine lens is very low, lectin staining is sufficiently sensitive to detect the various glycated crystallins. The degree of glycation of gamma-crystallins was 3 times higher than that of beta S-crystallins and 9 times higher than that of beta L-crystallins.

Animals↗

PubMed2004

AlphaA-crystallin interacting regions in the small heat shock protein, alphaB-crystallin.

Amino acid sequences of alphaB-crystallin, involved in interaction with alphaA-crystallin, were determined by using peptide scans. Positionally addressable 20-mer overlapping peptides, representing the entire sequence of alphaB-crystallin, were synthesized on a PVDF membrane. The membrane was blocked with albumin and incubated with purified alphaA-crystallin. Probing the membrane with alphaA-crystallin-specific antibodies revealed residues 42-57, 60-71, and 88-123 in alphaB-crystallin to interact with alphaA-crystallin. Residues 42-57 and 60-71 interacted more strongly with alphaA-crystallin than the 88-123 sequence of alphaB-crystallin. Binding of one of the alphaB peptides (42-57) to alphaA-crystallin was also confirmed by gel filtration studies and HPLC analysis. The alphaB-crystallin sequences involved in interaction with alphaA-crystallin were distinct from the chaperone sites reported earlier as binding of the alphaB sequence from residues 42-57 does not alter the chaperone-like function of alphaA-crystallin. To identify the critical residues involved in interaction with alphaA-crystallin, R50G and P51A mutants of alphaB-crystallin were made and tested for their ability to interact with alphaA-crystallin. The oligomeric size and hydrophobicity of the mutants were similar. Circular dichroism studies showed that the P51A mutation increased the alpha-helical content of the protein. While the alphaBR50G mutant showed chaperone-like activity similar to wild-type alphaB, alphaBP51A showed reduced chaperone function. Fluorescence resonance energy transfer studies showed that the P51A mutation decreased the rate of subunit exchange with alphaA by 63%, whereas the R50G mutation reduced the exchange rate by 23%. Similar to wild-type alphaB, alphaB-crystallin peptide (42-57) effectively competed with alphaBP51A and alphaBR50G for interaction with alphaA. Thus, our studies showed that the alphaB-crystallin sequence (42-57) is one of the interacting regions in alphaB and alphaA oligomer formation.

Amino Acid Sequence↗

PubMed1994

Supramolecular order within the lens: 1H NMR spectroscopic evidence for specific crystallin-crystallin interactions.

alpha-, beta- and gamma-crystallins from bovine lens contain flexible terminal extensions which are readily observed by NMR spectroscopy. By monitoring these resonances, NMR spectroscopy therefore offers a means of examining specific protein-protein interactions in crystallin mixtures. In this paper, a 1H NMR spectroscopic study of bovine lens nuclear and cortical homogenates and various crystallin mixtures is presented. In both homogenates, resonances from the flexible C-terminal extensions of alpha-crystallin and the N-terminal extension of beta B2-crystallin are readily observed suggesting that these regions are not involved in crystallin-crystallin interactions. In the cortical homogenate, resonances from the short N-terminal extension of gamma S-crystallin are also present. The cortical homogenate gives rise to more intense resonances than the nuclear homogenate, suggesting that the cortical region has many more mobile crystallin regions. In both homogenates, the C-terminal extension of beta B2-crystallin and the very short C-terminal extension of gamma B-crystallin are not observed. Thus, the C-terminal regions of these proteins are involved in interactions with other crystallins. Similar effects are observed upon mixing of the individual crystallins, e.g. the C-terminal extension of gamma B-crystallin is absent in spectra of mixtures of total gamma-crystallin and high-molecular-weight beta-crystallin aggregates (beta H). Overall, the results are consistent with a short-range order for the crystallins within the lens.

Animals↗

PubMed2003

Subunit exchange demonstrates a differential chaperone activity of calf alpha-crystallin toward beta LOW- and individual gamma-crystallins.

The chaperone activity of native alpha-crystallins toward beta(LOW)- and various gamma-crystallins at the onset of their denaturation, 60 and 66 degrees C, respectively, was studied at high and low crystallin concentrations using small angle x-ray scattering (SAXS) and fluorescence energy transfer (FRET). The crystallins were from calf lenses except for one recombinant human gamma S. SAXS data demonstrated an irreversible doubling in molecular weight and a corresponding increase in size of alpha-crystallins at temperatures above 60 degrees C. Further increase is observed at 66 degrees C. More subtle conformational changes accompanied the increase in size as shown by changes in environments around tryptophan and cysteine residues. These alpha-crystallin temperature-induced modifications were found necessary to allow for the association with beta(LOW)- and gamma-crystallins to occur. FRET experiments using IAEDANS (iodoacetylaminoethylaminonaphthalene sulfonic acid)- and IAF (iodoacetamidofluorescein)-labeled subunits showed that the heat-modified alpha-crystallins retained their ability to exchange subunits and that, at 37 degrees C, the rate of exchange was increased depending upon the temperature of incubation, 60 or 66 degrees C. Association with beta(LOW)- (60 degrees C) or various gamma-crystallins (66 degrees C) resulted at 37 degrees C in decreased subunit exchange in proportion to bound ligands. Therefore, beta(LOW)- and gamma-crystallins were compared for their capacity to associate with alpha-crystallins and inhibit subunit exchange. Quite unexpectedly for a highly conserved protein family, differences were observed between the individual gamma-crystallin family members. The strongest effect was observed for gamma S, followed by h gamma Srec, gamma E, gamma A-F, gamma D, gamma B. Moreover, fluorescence properties of alpha-crystallins in the presence of bound beta(LOW)-and gamma-crystallins indicated that the formation of beta(LOW)/alpha- or gamma/alpha-crystallin complexes involved various binding sites. The changes in subunit exchange associated with the chaperone properties of alpha-crystallins toward the other lens crystallins demonstrate the dynamic character of the heat-activated alpha-crystallin structure.

Animals↗

PubMed1997

Characterization of gamma S-crystallin isoforms from lip shark (Chiloscyllium colax): evolutionary comparison between gamma S and beta/gamma crystallins.

gamma S-Crystallin from shark eye lenses, formerly termed beta s crystallin in mammalian lenses, is structurally characterized in this study by cDNA cloning and sequencing. To facilitate sequence characterization of gamma S-crystallin possessing intermediate structural properties between beta- and gamma-crystallins, 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 nucleotide segments encoding multiple shark gamma S-crystallins. Sequencing several positive clones revealed that a multiplicity of isoforms exists in the gamma S-crystallin class of this cartilaginous fish, similar to authentic gamma-crystallin family characterized from the same shark species. Comparison of protein sequences encoded by two representative shark gamma S1 and gamma S2 cDNAs with those published sequences of beta-, gamma-, and gamma S crystallins from bovine, human, bullfrog and carp lenses indicated that there is about 35-64% sequence homology between shark gamma S crystallins and structurally related crystallins from different evolutionary classes, with a higher sequence similarity between shark gamma S and mammalian gamma-crystallins than that of shark gamma S and carp gamma S or bovine gamma S crystallins. A phylogenetic tree constructed on the basis of the sequence divergence among various beta-, gamma-, and gamma S crystallins corroborates the closer relatedness of shark gamma S to authentic gamma-crystallin than to mammalian and teleostean gamma S crystallins. It further strengthens the supposition that ancestral precursors of gamma S-crystallins were present in the shark lens long before the appearance of present-day teleostean and mammalian gamma S-crystallins.

Amino Acid Sequence↗

PubMed1992

Biochemical characterization of crystallins from pigeon lenses: structural and sequence analysis of pigeon delta-crystallin.

Crystallins from pigeon eye lenses were isolated and purified by gel-permeation chromatography and characterized by gel electrophoresis, amino-acid composition and sequence analysis. Alpha- and beta-crystallins could be obtained in relatively pure forms by single-step size-exclusion chromatography whereas an extra step of ion-exchange chromatography was needed for the separation of delta-crystallin from the beta-crystallin fraction. In contrast to most characterized vertebrate species, a large amount of glycogen is eluted as a high molecular form in the first peak of the gel filtration column. Pigeon delta-crystallin, similar to duck and reptilian delta-crystallins, exists as a tetrameric structure of about 200 kDa in the native form and is composed of one major subunit of 50 kDa with heterogeneous isoelectric points spreading in a range of 4.7 to 6.8. In contrast to those obtained from duck, goose and caiman, delta-crystallin isolated from the pigeon lens possessed very little argininosuccinate lyase activity. However, pigeon delta-crystallin can still cross-react with the antibody against enzymically active duck delta-crystallin as revealed by the sensitive immunoblotting technique. It was also shown that the delta-crystallin content of the total pigeon soluble proteins decreased with the age of the animal. Structural analysis of purified delta-crystallin fraction was made with respect to its amino-acid composition and protein primary sequence. N-terminal sequence analysis indicated the presence of blocked amino-termini in all crystallin fractions of pigeon lenses. Therefore, a sequence analysis of PCR (polymerase chain reaction) amplified delta-crystallin cDNA was employed to deduce the protein sequence of this crystallin. Structural comparison of delta-crystallin sequences from pigeon, chicken and duck lenses casts some doubts on the recent claim that His-89-->Gln mutation in the chicken delta-crystallin may account for the loss of argininosuccinate lyase activity in this avian species, as compared to high enzymic activity in the duck crystallin (Barbosa et al. (1991) J. Biol. Chem. 266, 5286-5290).

Amino Acid Sequence↗

PubMed2004

Interactions and chaperone function of alphaA-crystallin with T5P gammaC-crystallin mutant.

T5P gammaC-crystallin mutation is associated with Coppock-like cataract, one of the autosomal dominant congenital cataracts. It is not known why the abundant alpha-crystallin cannot prevent the mutation-related aggregation. Our previous studies indicate that the mutation changes conformation and reduces solubility and stability, but it is not known whether it is these events or the loss of interaction with other crystallins that causes the cataract. It is also not known whether the alpha-crystallin can protect T5P mutant as effectively from heat-induced aggregation as the wild-type (WT) gammaC-crystallin. To investigate the mechanism of interactions and chaperone function between alphaA- and gammaC-crystallin, human alphaA-crystallin and W9F mutant as well as WT gammaC-crystallin and T5P mutant were cloned. Interactions between alphaA- and gammaC-crystallin were studied with fluorescence resonance energy transfer (FRET), and chaperone activity was assessed by the suppression of heat-induced aggregation of substrate proteins. Conformational changes of substrate proteins were studied by spectroscopic measurements. The results indicate that the T5P mutant showed a slightly greater FRET than WT gammaC-crystallin with alphaA-crystallin, and alphaA-crystallin could effectively prevent both WT and T5P gammaC-crystallin from heat-induced aggregation. Spectroscopic measurements show that both alphaA-crystallin and gammaC-crystallin underwent only slight conformational change after chaperone binding. Together with previous results obtained with a two-hybrid system assay of interactions between alphaA- and gammaC-crystallin, the present FRET and chaperone results indicate that loss of interactions of T5P mutant with other crystallins may play a larger role than the protection afforded by chaperone-like activity in Coppock-like cataract.

Cataract↗

PubMed1998

Characterization of gammaS-crystallin isoforms from a catfish: evolutionary comparison of various gamma-, gammaS-, and beta-crystallins.

gammaS-Crystallin from catfish eye lenses, formerly designated betas-crystallin in mammalian lenses, is structurally characterized in this study by cDNA cloning and sequencing. To facilitate sequence characterization of gammaS-crystallin with structural properties lying between beta- and gamma-crystallins, a cDNA mixture was constructed from the poly(A)+ mRNA isolated from catfish eye lenses, and amplification by polymerase chain reaction (PCR) was carried out to obtain nucleotide segments encoding multiple gammaS-crystallin isoforms. Sequencing several positive clones revealed that at least two distinct isoforms exist in the gammaS-crystallin class of this teleostean fish, similar to the authentic gamma-crystallin family characterized previously in species of the piscine class. Comparison of protein sequences encoded by two representative catfish gammaS1 and gammaS2 cDNAs with the published sequences of beta-, gamma-, and gammaS-crystallins from shark, carp, bullfrog, bovine, and human lenses indicates that there is about 20-50% sequence homology between catfish gammaS-crystallins and various members of the related beta/gamma-crystallin superfamily from different evolutionary classes, with a higher sequence similarity being found between catfish gammaS- and mammalian gamma-crystallins than between catfish gammaS- and bovine or carp gammaS-crystallins. Phylogenetic trees constructed on the basis of the nucleotide and protein sequence divergence among various beta-, gamma-, and gammaS-crystallins corroborate the closer relatedness of catfish gammaS- to authentic gamma-crystallin than to bovine and carp gammaS-crystallins. The results suggest that evolution of catfish gammaS-crystallins follows a different path from that of bovine and carp gammaS-crystallins and may represent a more ancient offshoot from the ancestral gamma/gammaS coding gene than carp and bovine gammaS-crystallins.

Amino Acid Sequence↗

PubMed1991

Ostrich crystallins. Structural characterization of delta-crystallin with enzymic activity.

Lens crystallins from the African ostrich (Struthio camelus) were isolated and characterized. Four crystallin fractions corresponding to alpha-, delta/beta- and beta-crystallins similar to those of duck crystallins were isolated, but epsilon-crystallin was found to be absent. The native molecular masses and subunit structures of the purified fractions were analysed by gel filtration. SDS/PAGE and isoelectric focusing, revealing various extents of heterogeneity in each orthologous crystallin class. An ion-exchange chromatographic method was used for the large-scale preparation of delta-crystallin suitable for structural and enzymic studies. It was unexpectedly found that the purified native delta-crystallin of ostrich lens possessed high argininosuccinate lyase activity, in contrast with chicken delta-crystallin. The c.d. spectra indicated a predominant beta-sheet structure in alpha- and beta-crystallins, and a significant contribution of alpha-helical structure in the delta-crystallin fraction. The estimate of secondary structures from c.d. spectroscopy for each crystallin class bears a resemblance to that of duck crystallins, except that ostrich delta-crystallin possesses much less helical content than duck delta-crystallin. Comparison of crystallin compositions and structures from aquatic and terrestrial birds revealed distinct differences.

Amino Acids↗