The Experts below are selected from a list of 25026 Experts worldwide ranked by ideXlab platform
David L. Smith - One of the best experts on this subject based on the ideXlab platform.
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human β Crystallins modified by backbone cleavage deamidation and oxidation are prone to associate
Experimental Eye Research, 2003Co-Authors: Zhongli Zhang, David L. Smith, Jean B. SmithAbstract:Abstract Information about β-Crystallins and their post-translational modifications has been scarce because of difficulties in isolating the individual β-Crystallins. These difficulties arise because the β-crystallin sequences are highly homologous and because β-Crystallins undergo many age-related modifications that lead to a variety of molecular masses and a range of acidities for each crystallin. In this study, human β-Crystallins were isolated using several steps of chromatography both before and after two-dimensional gel electrophoresis. Many previously unidentified in vivo modifications, including deamidations among all β-Crystallins except βB3, truncation of βA3, βB1 and βA4, and oxidation of some methionines and tryptophans were located among the isolated β-Crystallins. Many modifications occurred before age 20 with modest increases in modification for β-Crystallins from lenses 20–87 years old. The tendency of the modified β-Crystallins to form non-covalent complexes was evident from their chromatographic behaviour. The presence in these complexes of βB2-crystallin, the least modified and most soluble of the β-Crystallins, points to a possible role for βB2 in solubilizing the more heavily modified β-Crystallins. The greater solubility of β-Crystallins compared with α- and γ-Crystallins in aging lenses may be due to β-crystallin modifications and their non-covalent associations.
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Methylation and carbamylation of human γ-Crystallins
Protein science : a publication of the Protein Society, 2003Co-Authors: Veniamin N. Lapko, David L. Smith, Jean B. SmithAbstract:Accessible sulfhydryls of cysteine residues are likely sites of reaction in long-lived proteins such as human lens Crystallins. Disulfide bonding between cysteines is a major contributor to intermolecular cross-linking and aggregation of Crystallins. A recently reported modification of gammaS-Crystallins, S-methylation of cysteine residues, can prevent disulfide formation. The aim of this study was to determine whether cysteines in gammaC-, gammaD-, and gammaB-Crystallins are also S-methylated. Our data show that all the gamma-Crystallins are S-methylated, but only at specific cysteines. In gammaD-crystallin, methylation is exclusively at Cys 110, whereas in gammaC- and gammaB-Crystallins, the principal methylation site is Cys 22 with minor methylation at Cys 79. gammaD-crystallin is the most heavily methylated gamma-crystallin. gammaD-Crystallins from adult lenses are 37%-70% methylated, whereas gammaC and gammaB are approximately 12% methylated. The specificity of gamma-crystallin methylation and its occurrence in young clear lenses supports the idea that inhibition of disulfide bonding by S-methylation may play a protective role against cataract. Another modification, not reported previously, is carbamylation of the N termini of gammaB-, gammaC-, gammaD-Crystallins. N-terminal carbamylation is likely a developmentally related modification that does not negatively impact crystallin function.
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Resistance of Human βB2-crystallin to in vivo Modification
Experimental eye research, 2001Co-Authors: Zhongli Zhang, Larry L. David, David L. Smith, Jean B. SmithAbstract:Post-translational modifications and/or structural changes induced by modifications are likely causes of the decrease in crystallin solubility associated with aging and the development of cataract. Characterization of human lens Crystallins by mass spectrometry has demonstrated that βB2-crystallin undergoes less modification than any of the other Crystallins. As the lens ages, βB2-crystallin retains its hydrophilic N-terminus while the hydrophilic C-termini of α-Crystallins and large portions of the N-termini of βA3/A1 and βB1 are truncated. The hydrophilic terminal regions of Crystallins contribute to their solubility. Furthermore, deamidation and disulfide bond formation, other modifications that may affect solubility by altering conformation, are less extensive in βB2 than in the other Crystallins. This resistance to modification results in higher levels of βB2 compared with the other Crystallins in the water-soluble fraction of older lenses. The solubility of βB2 and its propensity to form non-covalent associations with less soluble β-Crystallins may contribute to the solubility of the other β-Crystallins. A current hypothesis is that the chaperone-like properties of α-Crystallins contribute to lens crystallin solubility, particularly in younger lenses. In older lenses, where most of the α-Crystallins have become water-insoluble, βB2-Crystallins may play a dominant role in lens crystallin solubility.
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Human Lens β-Crystallin Solubility
The Journal of biological chemistry, 2000Co-Authors: Jinhua Feng, David L. Smith, Jean B. SmithAbstract:Abstract The human lens is composed primarily of water and proteins called Crystallins. Insolubility of these Crystallins is correlated with aging and cataractogenesis. The α-Crystallins have chaperone-like activity in maintaining the solubility of denatured β- and γ-Crystallins. One established test of this chaperone activity is the ability of α-crystallin to prevent thermal destabilization of β-Crystallins. Several studies have addressed the effects of structural modifications of α-crystallin on chaperone activity, but little is known about the solubilities of the various β-Crystallins or the effects of post-translational modifications. Understanding the solubilities of different forms of β-Crystallins is important to elucidating the mechanism of chaperone activity. In this study, the solubilities of β-Crystallins were examined. The β-Crystallins included the gene products of βB2, βA1/A3, βA4, and βB1 as well as forms modified in vivo. Analysis of the β-Crystallins by high performance liquid chromatography and mass spectrometry before and after heating revealed large differences in the relative solubilities of the β-Crystallins. These results demonstrate a decreased solubility of specific β-Crystallins and post-translational modifications that may play a role in the crystallin insolubility associated with aging and cataract.
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modifications of the water insoluble human lens α Crystallins
Experimental Eye Research, 1996Co-Authors: Anders L Lund, Jean B. Smith, David L. SmithAbstract:Since the water-insoluble Crystallins of the lens may be the precursors of cataract, identifying the modifications that differentiate the water-insoluble from the water-soluble Crystallins may provide the basis for understanding the chemistry leading to cataract. This investigation of the alpha-Crystallins of the water-insoluble urea-soluble portion of 45-year-old normal clear lenses, isolated using gel filtration, ion exchange and reversed phase chromatography, has employed state-of-the-art mass spectrometric techniques to identify and locate the modifications of the water-insoluble alpha-Crystallins. Modifications present in the isolated alpha-Crystallins were identified by the molecular weights of the modified proteins, by the molecular weights of peptides produced by enzymatic digestion of the proteins, and by the fragmentation patterns produced by collisional activation of the peptides. Modifications that are either unique to the water-insoluble alpha-Crystallins or are more prevalent in the water-soluble portion than in the water-soluble part include complete oxidation of the two Cys residues of alpha A-crystallin to form an intra-molecular disulfide bond, partial truncation at both the C-termini and N-termini of alpha A- and alpha B-Crystallins, partial oxidation of Met residues to methionine sulfoxide, partial deamidation of several Asn and Gln residues, and evidence of peptide bond cleavage at some of the deamidated residues. Although many reactions have been proposed to contribute to the insolubility of Crystallins, this compilation of in vivo post-translational modifications of water-insoluble alpha-Crystallins delineates products that are actually present at levels of 5% or more. From these results, it is hypothesized that alpha-crystallin becomes water-insoluble following deamidation of various Asn and Gln residues which cause conformational changes leading to formation of an intra-molecular disulfide bond between the Cys residues of alpha A-crystallin.
Joseph Horwitz - One of the best experts on this subject based on the ideXlab platform.
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Altered Chaperone-like Activity of α-Crystallins Promotes Cataractogenesis
Journal of Biological Chemistry, 2010Co-Authors: Catherine Cheng, Linlin Ding, Joseph Horwitz, Qingling Huang, Xiaohua GongAbstract:Abstract 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.
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vertebrate like βγ Crystallins in the ocular lenses of a copepod
Journal of Comparative Physiology A-neuroethology Sensory Neural and Behavioral Physiology, 2005Co-Authors: Jonathan H Cohen, Linlin Ding, Joram Piatigorsky, Nansi Jo Colley, Rebecca Ward, Joseph HorwitzAbstract:The diverse Crystallins are water-soluble proteins that are responsible for the optical properties of cellular lenses of animal eyes. While all vertebrate lenses contain physiological stress-related α- and βγ-Crystallins, some also contain taxon-specific, often enzyme-related Crystallins. To date, the α- and βγ-Crystallins have been found only in vertebrate lenses. Here we report lenses from an invertebrate, the pontellid copepod Anomalocera ornata, accumulate βγ-crystallin family members as judged by immunocytochemistry, western immunoblotting and microsequencing. Our data provide the first example of βγ-crystallin members in an invertebrate lens, establishing that the use of this protein family as lens Crystallins is not confined to vertebrates.
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Alpha-crystallin.
Experimental Eye Research, 2003Co-Authors: Joseph HorwitzAbstract:Alpha A and alpha B-Crystallins are a major protein component of the mammalian eye lens. Being a member of the small heat-shock protein family they possess chaperone-like function. The alpha-Crystallins and especially alpha B is also found outside the lens having an extensive tissue distribution. Alpha B-crystallin is found to be over-expressed in many neurological diseases, and mutations in alpha A or B-crystallin can cause cataract and myopathy. This review deals with some of the unique properties of the alpha-Crystallins emphasizing especially what we don't know about its function and structure.
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Ω-Crystallin of the Scallop Lens A DIMERIC ALDEHYDE DEHYDROGENASE CLASS 1/2 ENZYME-CRYSTALLIN
The Journal of biological chemistry, 2000Co-Authors: Joram Piatigorsky, Linlin Ding, Joseph Horwitz, Zbynek Kozmik, Eleonora Carosa, W. Gerald Robison, Peter J. Steinbach, Ernst R. TammAbstract:Abstract While many of the diverse Crystallins of the transparent lens of vertebrates are related or identical to metabolic enzymes, much less is known about the lens Crystallins of invertebrates. Here we investigate the complex eye of scallops. Electron microscopic inspection revealed that the anterior, single layered corneal epithelium overlying the cellular lens contains a regular array of microvilli that we propose might contribute to its optical properties. The sole crystallin of the scallop eye lens was found to be homologous to Ω-crystallin, a minor crystallin in cephalopods related to aldehyde dehydrogenase (ALDH) class 1/2. Scallop Ω-crystallin (officially designated ALDH1A9) is 55–56% identical to its cephalopod homologues, while it is 67 and 64% identical to human ALDH 2 and 1, respectively, and 61% identical to retinaldehyde dehydrogenase/η-crystallin of elephant shrews. Like other enzyme-Crystallins, scallop Ω-crystallin appears to be present in low amounts in non-ocular tissues. Within the scallop eye, immunofluorescence tests indicated that Ω-crystallin expression is confined to the lens and cornea. Although it has conserved the critical residues required for activity in other ALDHs and appears by homology modeling to have a structure very similar to human ALDH2, scallop Ω-crystallin was enzymatically inactive with diverse substrates and did not bind NAD or NADP. In contrast to mammalian ALDH1 and -2 and other cephalopod Ω-Crystallins, which are tetrameric proteins, scallop Ω-crystallin is a dimeric protein. Thus, ALDH is the most diverse lens enzyme-crystallin identified so far, having been used as a lens crystallin in at least two classes of molluscs as well as elephant shrews.
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alpha crystallin can function as a molecular chaperone
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: Joseph HorwitzAbstract:Abstract The alpha-Crystallins (alpha A and alpha B) are major lens structural proteins of the vertebrate eye that are related to the small heat shock protein family. In addition, Crystallins (especially alpha B) are found in many cells and organs outside the lens, and alpha B is overexpressed in several neurological disorders and in cell lines under stress conditions. Here I show that alpha-crystallin can function as a molecular chaperone. Stoichiometric amounts of alpha A and alpha B suppress thermally induced aggregation of various enzymes. In particular, alpha-crystallin is very efficient in suppressing the thermally induced aggregation of beta- and gamma-Crystallins, the two other major mammalian structural lens proteins. alpha-Crystallin was also effective in preventing aggregation and in refolding guanidine hydrochloride-denatured gamma-crystallin, as judged by circular dichroism spectroscopy. My results thus indicate that alpha-crystallin refracts light and protects proteins from aggregation in the transparent eye lens and that in nonlens cells alpha-crystallin may have other functions in addition to its capacity to suppress aggregation of proteins.
Jean B. Smith - One of the best experts on this subject based on the ideXlab platform.
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human β Crystallins modified by backbone cleavage deamidation and oxidation are prone to associate
Experimental Eye Research, 2003Co-Authors: Zhongli Zhang, David L. Smith, Jean B. SmithAbstract:Abstract Information about β-Crystallins and their post-translational modifications has been scarce because of difficulties in isolating the individual β-Crystallins. These difficulties arise because the β-crystallin sequences are highly homologous and because β-Crystallins undergo many age-related modifications that lead to a variety of molecular masses and a range of acidities for each crystallin. In this study, human β-Crystallins were isolated using several steps of chromatography both before and after two-dimensional gel electrophoresis. Many previously unidentified in vivo modifications, including deamidations among all β-Crystallins except βB3, truncation of βA3, βB1 and βA4, and oxidation of some methionines and tryptophans were located among the isolated β-Crystallins. Many modifications occurred before age 20 with modest increases in modification for β-Crystallins from lenses 20–87 years old. The tendency of the modified β-Crystallins to form non-covalent complexes was evident from their chromatographic behaviour. The presence in these complexes of βB2-crystallin, the least modified and most soluble of the β-Crystallins, points to a possible role for βB2 in solubilizing the more heavily modified β-Crystallins. The greater solubility of β-Crystallins compared with α- and γ-Crystallins in aging lenses may be due to β-crystallin modifications and their non-covalent associations.
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Methylation and carbamylation of human γ-Crystallins
Protein science : a publication of the Protein Society, 2003Co-Authors: Veniamin N. Lapko, David L. Smith, Jean B. SmithAbstract:Accessible sulfhydryls of cysteine residues are likely sites of reaction in long-lived proteins such as human lens Crystallins. Disulfide bonding between cysteines is a major contributor to intermolecular cross-linking and aggregation of Crystallins. A recently reported modification of gammaS-Crystallins, S-methylation of cysteine residues, can prevent disulfide formation. The aim of this study was to determine whether cysteines in gammaC-, gammaD-, and gammaB-Crystallins are also S-methylated. Our data show that all the gamma-Crystallins are S-methylated, but only at specific cysteines. In gammaD-crystallin, methylation is exclusively at Cys 110, whereas in gammaC- and gammaB-Crystallins, the principal methylation site is Cys 22 with minor methylation at Cys 79. gammaD-crystallin is the most heavily methylated gamma-crystallin. gammaD-Crystallins from adult lenses are 37%-70% methylated, whereas gammaC and gammaB are approximately 12% methylated. The specificity of gamma-crystallin methylation and its occurrence in young clear lenses supports the idea that inhibition of disulfide bonding by S-methylation may play a protective role against cataract. Another modification, not reported previously, is carbamylation of the N termini of gammaB-, gammaC-, gammaD-Crystallins. N-terminal carbamylation is likely a developmentally related modification that does not negatively impact crystallin function.
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Resistance of Human βB2-crystallin to in vivo Modification
Experimental eye research, 2001Co-Authors: Zhongli Zhang, Larry L. David, David L. Smith, Jean B. SmithAbstract:Post-translational modifications and/or structural changes induced by modifications are likely causes of the decrease in crystallin solubility associated with aging and the development of cataract. Characterization of human lens Crystallins by mass spectrometry has demonstrated that βB2-crystallin undergoes less modification than any of the other Crystallins. As the lens ages, βB2-crystallin retains its hydrophilic N-terminus while the hydrophilic C-termini of α-Crystallins and large portions of the N-termini of βA3/A1 and βB1 are truncated. The hydrophilic terminal regions of Crystallins contribute to their solubility. Furthermore, deamidation and disulfide bond formation, other modifications that may affect solubility by altering conformation, are less extensive in βB2 than in the other Crystallins. This resistance to modification results in higher levels of βB2 compared with the other Crystallins in the water-soluble fraction of older lenses. The solubility of βB2 and its propensity to form non-covalent associations with less soluble β-Crystallins may contribute to the solubility of the other β-Crystallins. A current hypothesis is that the chaperone-like properties of α-Crystallins contribute to lens crystallin solubility, particularly in younger lenses. In older lenses, where most of the α-Crystallins have become water-insoluble, βB2-Crystallins may play a dominant role in lens crystallin solubility.
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Human Lens β-Crystallin Solubility
The Journal of biological chemistry, 2000Co-Authors: Jinhua Feng, David L. Smith, Jean B. SmithAbstract:Abstract The human lens is composed primarily of water and proteins called Crystallins. Insolubility of these Crystallins is correlated with aging and cataractogenesis. The α-Crystallins have chaperone-like activity in maintaining the solubility of denatured β- and γ-Crystallins. One established test of this chaperone activity is the ability of α-crystallin to prevent thermal destabilization of β-Crystallins. Several studies have addressed the effects of structural modifications of α-crystallin on chaperone activity, but little is known about the solubilities of the various β-Crystallins or the effects of post-translational modifications. Understanding the solubilities of different forms of β-Crystallins is important to elucidating the mechanism of chaperone activity. In this study, the solubilities of β-Crystallins were examined. The β-Crystallins included the gene products of βB2, βA1/A3, βA4, and βB1 as well as forms modified in vivo. Analysis of the β-Crystallins by high performance liquid chromatography and mass spectrometry before and after heating revealed large differences in the relative solubilities of the β-Crystallins. These results demonstrate a decreased solubility of specific β-Crystallins and post-translational modifications that may play a role in the crystallin insolubility associated with aging and cataract.
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modifications of the water insoluble human lens α Crystallins
Experimental Eye Research, 1996Co-Authors: Anders L Lund, Jean B. Smith, David L. SmithAbstract:Since the water-insoluble Crystallins of the lens may be the precursors of cataract, identifying the modifications that differentiate the water-insoluble from the water-soluble Crystallins may provide the basis for understanding the chemistry leading to cataract. This investigation of the alpha-Crystallins of the water-insoluble urea-soluble portion of 45-year-old normal clear lenses, isolated using gel filtration, ion exchange and reversed phase chromatography, has employed state-of-the-art mass spectrometric techniques to identify and locate the modifications of the water-insoluble alpha-Crystallins. Modifications present in the isolated alpha-Crystallins were identified by the molecular weights of the modified proteins, by the molecular weights of peptides produced by enzymatic digestion of the proteins, and by the fragmentation patterns produced by collisional activation of the peptides. Modifications that are either unique to the water-insoluble alpha-Crystallins or are more prevalent in the water-soluble portion than in the water-soluble part include complete oxidation of the two Cys residues of alpha A-crystallin to form an intra-molecular disulfide bond, partial truncation at both the C-termini and N-termini of alpha A- and alpha B-Crystallins, partial oxidation of Met residues to methionine sulfoxide, partial deamidation of several Asn and Gln residues, and evidence of peptide bond cleavage at some of the deamidated residues. Although many reactions have been proposed to contribute to the insolubility of Crystallins, this compilation of in vivo post-translational modifications of water-insoluble alpha-Crystallins delineates products that are actually present at levels of 5% or more. From these results, it is hypothesized that alpha-crystallin becomes water-insoluble following deamidation of various Asn and Gln residues which cause conformational changes leading to formation of an intra-molecular disulfide bond between the Cys residues of alpha A-crystallin.
Yogendra Sharma - One of the best experts on this subject based on the ideXlab platform.
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ca2 binding motif of βγ Crystallins
Journal of Biological Chemistry, 2014Co-Authors: Shanti Swaroop Srivastava, Amita Mishra, Bal Krishnan, Yogendra SharmaAbstract:Abstract βγ-Crystallin-type double clamp N/D-N/D-X-X-S/T-S motif is an established but sparsely investigated motif for Ca2+-binding. A βγ-crystallin domain is formed of two Greek key motifs, accommodating two Ca2+-binding sites. βγ-Crystallins make a separate class of Ca2+-binding proteins (CaBP), apparently a major group of CaBP in bacteria. Paralleling the diversity in βγ-crystallin domains, these motifs too show great diversity, both in structure and function. Though the expression of some of them has been associated with stress, virulency and adhesion, the functional implications of Ca2+-binding to βγ-Crystallins in mediating biological processes are yet to be elucidated.
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Solution Structure and Calcium-Binding Properties of M-Crystallin, A Primordial βγ-Crystallin from Archaea
Journal of molecular biology, 2008Co-Authors: Ravi Pratap Barnwal, Maroor K. Jobby, Yogendra Sharma, K. Manjula Devi, Kandala V. R. CharyAbstract:The lens betagamma-crystallin superfamily has many diverse but topologically related members belonging to various taxa. Based on structural topology, these proteins are considered to be evolutionarily related to lens Crystallins, suggesting their origin from a common ancestor. Proteins with betagamma-crystallin domains, although found in some eukaryotes and eubacteria, have not yet been reported in archaea. Sequence searches in the genome of the archaebacterium Methanosarcina acetivorans revealed the presence of a protein annotated as a betagamma-crystallin family protein, named M-crystallin. Solution structure of this protein indicates a typical betagamma-crystallin fold with a paired Greek-key motif. Among the known structures of betagamma-crystallin members, M-crystallin was found to be structurally similar to the vertebrate lens betagamma-Crystallins. The Ca(2+)-binding properties of this primordial protein are somewhat more similar to those of vertebrate betagamma-Crystallins than to those of bacterial homologues. These observations, taken together, suggest that amphibian and vertebrate betagamma-crystallin domains are evolutionarily more related to archaeal homologues than to bacterial homologues. Additionally, identification of a betagamma-crystallin homologue in archaea allows us to demonstrate the presence of this domain in all the three domains of life.
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Calcium-binding to lens βB2- and βA3-Crystallins suggests that all β-Crystallins are calcium-binding proteins
FEBS Journal, 2007Co-Authors: Maroor K. Jobby, Yogendra SharmaAbstract:Crystallins are the major proteins of a mammalian eye lens. The topologically similar eye lens proteins, β- and γ-Crystallins, are the prototype and founding members of the βγ-crystallin superfamily. βγ-Crystallins have until recently been regarded as structural proteins. However, the calcium-binding properties of a few members and the potential role of βγ-Crystallins in fertility are being investigated. Because the calcium-binding elements of other member proteins, such as spherulin 3a, are not present in βB2-crystallin and other βγ-Crystallins from fish and mammalian genomes, it was argued that lens βγ-Crystallins should not bind calcium. In order to probe whether β-Crystallins can bind calcium, we selected one basic (βB2) and one acidic (βA3) β-crystallin for calcium-binding studies. Using calcium-binding assays such as 45Ca overlay, terbium binding, Stains-All and isothermal titration calorimetry, we established that both βB2- and βA3-crystallin bind calcium with moderate affinity. There was no significant change in their conformation upon binding calcium as monitored by fluorescence and circular dichroism spectroscopy. However, 15N-1H heteronuclear single quantum correlation NMR spectroscopy revealed that amide environment of several residues underwent changes indicating calcium ligation. With the corroboration of calcium-binding to βB2- and βA3-Crystallins, we suggest that all β-Crystallins bind calcium. Our results have important implications for understanding the calcium-related cataractogenesis and maintenance of ionic homeostasis in the lens.
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Calcium‐binding to lens βB2‐ and βA3‐Crystallins suggests that all β‐Crystallins are calcium‐binding proteins
The FEBS journal, 2007Co-Authors: Maroor K. Jobby, Yogendra SharmaAbstract:Crystallins are the major proteins of a mammalian eye lens. The topologically similar eye lens proteins, β- and γ-Crystallins, are the prototype and founding members of the βγ-crystallin superfamily. βγ-Crystallins have until recently been regarded as structural proteins. However, the calcium-binding properties of a few members and the potential role of βγ-Crystallins in fertility are being investigated. Because the calcium-binding elements of other member proteins, such as spherulin 3a, are not present in βB2-crystallin and other βγ-Crystallins from fish and mammalian genomes, it was argued that lens βγ-Crystallins should not bind calcium. In order to probe whether β-Crystallins can bind calcium, we selected one basic (βB2) and one acidic (βA3) β-crystallin for calcium-binding studies. Using calcium-binding assays such as 45Ca overlay, terbium binding, Stains-All and isothermal titration calorimetry, we established that both βB2- and βA3-crystallin bind calcium with moderate affinity. There was no significant change in their conformation upon binding calcium as monitored by fluorescence and circular dichroism spectroscopy. However, 15N-1H heteronuclear single quantum correlation NMR spectroscopy revealed that amide environment of several residues underwent changes indicating calcium ligation. With the corroboration of calcium-binding to βB2- and βA3-Crystallins, we suggest that all β-Crystallins bind calcium. Our results have important implications for understanding the calcium-related cataractogenesis and maintenance of ionic homeostasis in the lens.
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Triose phosphate isomerase, a novel enzyme‐crystallin, and τ‐crystallin in crocodile cornea
The FEBS journal, 2006Co-Authors: Thandavarayan Kathiresan, Kannan M. Krishnan, Vaithilingam Krishnakumar, Raman Agrawal, Amit Anand, Dasari Muralidhar, Anurag Kumar Mishra, Vishnu M. Dhople, Ramesh K. Aggrawal, Yogendra SharmaAbstract:Several enzymes are known to accumulate in the cornea in unusually high concentrations. Based on the analogy with lens Crystallins, these enzymes are called corneal Crystallins, which are diverse and species-specific. Examining Crystallins in lens and cornea in multiple species provides great insight into their evolution. We report data on major proteins present in the crocodile cornea, an evolutionarily distant taxon. We demonstrate that τ-crystallin/α-enolase and triose phosphate isomerase (TIM) are among the major proteins expressed in the crocodile cornea as resolved by 2D gel electrophoresis and identified by MALDI-TOF. These proteins might be classified as putative corneal Crystallins. τ-Crystallin, known to be present in turtle and crocodile lens, has earlier been identified in chicken and bovine cornea, whereas TIM has not been identified in the cornea of any species. Immunostaining showed that τ-crystallin and TIM are concentrated largely in the corneal epithelium. Using western blot, immunofluorescence and enzymatic activity, we demonstrate that high accumulation of τ-crystallin and TIM starts in the late embryonic development (after the 24th stage of embryonic development) with maximum expression in a two-week posthatched animal. The crocodile corneal extract exhibits significant α-enolase and TIM activities, which increases in the corneal extract with development. Our results establishing the presence of τ-crystallin in crocodile, in conjunction with similar reports for other species, suggest that it is a widely prevalent corneal crystallin. Identification of TIM in the crocodile cornea reported here adds to the growing list of corneal Crystallins.
Kirsten J Lampi - One of the best experts on this subject based on the ideXlab platform.
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New focus on alpha-Crystallins in retinal neurodegenerative diseases
Experimental Eye Research, 2010Co-Authors: Patrice Fort, Kirsten J LampiAbstract:The crystallin proteins were initially identified as structural proteins of the ocular lens and have been recently demonstrated to be expressed in normal retina. They are dramatically upregulated by a large range of retinal diseases including diabetic retinopathy, age-related macular degeneration, uveitis, trauma and ischemia. The crystallin family of proteins is composed of alpha-, beta- and gamma-crystallin. Alpha-Crystallins, which are small heat shock proteins, have received substantial attention recently. This review summarizes the current knowledge of alpha-Crystallins in retinal diseases, their roles in retinal neuron cell survival and retinal inflammation, and the regulation of their expression and activity. Their potential role in the development of new treatments for neurodegenerative diseases is also discussed.
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age related changes of alpha crystallin aggregate in human lens
Amino Acids, 2007Co-Authors: Noriko Fujii, Yoshiari Shimmyo, Miyo Sakai, Yutaka Sadakane, Tadatoshi Kinouchi, Yukio Morimoto, Yuji Goto, T. Nakamura, Kirsten J LampiAbstract:Lens alpha-crystallin, composed of two subunits alpha A- and alpha B-crystallin, forms large aggregates in the lens of the eye. The present study investigated the aggregate of human lens alpha-crystallin from elderly and young donors. Recombinant alpha A- and alpha B-Crystallins in molar ratios of alpha A to alpha B at 1:1, corresponding to the aged sample, were also studied in detail. We found by ultra-centrifugation analysis that the alpha-crystallin aggregate from elderly donors was large and heterogeneous with an average sedimentation coefficient of 30 S and a range of 20–60 S at 37 °C. This was higher compared to the young samples that had an average sedimentation coefficient of 17 S. The sedimentation coefficients of recombinant alpha A- and alpha B-Crystallins were approximately 12 S and 15 S, respectively. Even when recombinant alpha-Crystallins were mixed in molar ratios equivalent to those found in vivo, similar S values as the native aged alpha-crystallin aggregates were not obtained.