Mutation Causing Self-Aggregation in Human C-Crystallin Leading to Congenital Cataract (original) (raw)
Related papers
PLoS ONE, 2012
Background: Human cS-crystallin is an important component of the human eye lens nucleus and cortex. The mutation V42M in the molecule causes severe congenital cataract in children. We compare the structure of the mutant protein with that of the wild type in order to understand how structural changes in the mutant relate to the mechanism of opacification. Methods: Both proteins were made using conventional cloning and expression procedures. Secondary and tertiary structural features of the proteins were analyzed using spectral methods. Structural stabilities of the proteins were analyzed using chemical and thermal denaturation methods. Self-aggregation was monitored using extrinsic spectral probes. Molecular modeling was used to compare the structural features of the two proteins. Results: While the wild type and mutant have the same secondary structure, molecular modeling and fluorescence analysis suggest the mutant to have a more open tertiary structure, with a larger hydrophobic surface. Experiments using extrinsic probes reveal that the mutant readily self-aggregates, with the suggestion that the aggregates might be similar to amyloidogenic fibrils. Chemical denaturation indicates that while the wild type exhibits the classic two-state transition, V42M goes through an intermediate state, and has a distinctly lower stability than the wild type. The temperature of thermal unfolding of the mutant is also distinctly lower. Further, the mutant readily precipitates and scatters light more easily than the wild type. Conclusion: The replacement of valine in position 42 by the longer and bulkier methionine in human cS-crystallin perturbs the compact b-sheet core packing topology in the N-terminal domain of the molecule, exposes nonpolar residues thereby increasing the surface hydrophobicity and weakens the stability of the protein, thus promoting self-aggregation leading to light scattering particles. This set of changes in the properties of the mutant offers a molecular insight into the mechanism of opacification.
Investigative Ophthalmology & Visual Science, 2008
To understand the molecular features underlying autosomal dominant congenital cataracts caused by the deletion mutations W156X in human ␥D-crystallin and W157X in human ␥C-crystallin. METHODS. Normal and mutant cDNAs (with the enhanced green fluorescent protein [EGFP] tag in the front) were cloned into the pEGFP-C1 vector, transfected into various cell lines, and observed under a confocal microscope for EGFP fluorescence. Normal and W156X ␥D cDNAs were also cloned into the pET21a(ϩ) vector, and the recombinant proteins were overexpressed in the BL-21(DE3)pLysS strain of Escherichia coli, purified, and isolated. The conformational features, structural stability, and solubility in aqueous solution of the mutant protein were compared with those of the wild type using spectroscopic methods. Comparative molecular modeling was performed to provide additional structural information. RESULTS. Transfection of the EGFP-tagged mutant cDNAs into several cell lines led to the visualization of aggregates, whereas that of wild-type cDNAs did not. Turning to the properties of the expressed proteins, the mutant molecules show remarkable reduction in solubility. They also seem to have a greater degree of surface hydrophobicity than the wild-type molecules, most likely accounting for self-aggregation. Molecular modeling studies support these features. CONCLUSIONS. The deletion of C-terminal 18 residues of human ␥Cand ␥D-crystallins exposes the side chains of several hydrophobic residues in the sequence to the solvent, causing the molecule to self-aggregate. This feature appears to be reflected in situ on the introduction of the mutants in human lens epithelial cells. (Invest Ophthalmol Vis Sci. 2008;49:3483-3490)
Protein Science, 2003
Human ␥D crystallin (H␥D-Crys), a major protein of the human eye lens, is a primary component of cataracts. This 174-residue primarily -sheet protein is made up of four Greek keys separated into two domains. Mutations in the human gene sequence encoding H␥D-Crys are implicated in early-onset cataracts in children, and the mutant protein expressed in Escherichia coli exhibits properties that reflect the in vivo pathology. We have characterized the unfolding, refolding, and competing aggregation of human wild-type H␥D-Crys as a function of guanidinium hydrochloride (GuHCl) concentration at neutral pH and 37°C, using intrinsic tryptophan fluorescence to monitor in vitro folding. Wild-type H␥D-Crys exhibited reversible refolding above 1.0 M GuHCl. The GuHCl unfolded protein was more fluorescent than its native counterpart despite the absence of metal or ion-tryptophan interactions. Aggregation of refolding intermediates of H␥D-Crys was observed in both equilibrium and kinetic refolding processes. The aggregation pathway competed with productive refolding at denaturant concentrations below 1.0 M GuHCl, beyond the major conformational transition region. Atomic force microscopy of samples under aggregating conditions revealed the sequential appearance of small nuclei, thin protofibrils, and fiber bundles. The H␥D-Crys fibrous aggregate species bound bisANS appreciably, indicating the presence of exposed hydrophobic pockets. The mechanism of H␥D-Crys aggregation may provide clues to understanding age-onset cataract formation in vivo.
Mechanism of Cataract Formation in αA-crystallin Y118D Mutation
Investigative Opthalmology & Visual Science, 2009
Purpose-The aim of this study was to elucidate the molecular mechanisms that lead to a dominant nuclear cataract in a mouse harboring the Y118D mutation in the αA-crystallin gene. Methods-The physicochemical properties of α-crystallin obtained from mouse lenses with the Y118D mutation as well as a recombinant Y118D αA-crystallin were studied using gel filtration, two-dimensional (2D) gel electrophoresis, multi-angle light scattering, circular dichroism, fluorescence, and chaperone activities. Results-Both native α-crystallin from mutant lens and recombinant αA-Y118D displayed higher molecular mass distribution than the wild-type. Circular dichroism spectra indicated changes in the secondary structures of αA-Y118D. The αA-Y118D protein prevented nonspecific protein aggregation more effectively than wild-type αA-crystallin. The gel filtration and 2D gel electrophoresis analysis showed a significant reduction of Y118D mutant protein in comparison with wild-type αA protein of heterozygous mutant lenses. Quantitative RT-PCR results confirmed a decrease in αA and αB transcripts in the homozygous mutant α A(Y118D/Y118D) lenses. Conclusions-The αA-Y118D mutant protein itself displays an increased chaperone-like activity. However, the dominant nuclear cataract is associated with a significant decrease in the amount of αA-crystallin, leading to a reduction in total chaperone capacity needed for maintaining lens transparency. Alpha-crystallin is the major protein of vertebrate eye lens, and plays a structural role in maintaining the lens transparency and proper refractive index. It is composed of two highly homologous subunits, αA and αB. 1,2 Although the molecular weight of each individual subunit is approximately 20 kDa, the α-crystallins exist as multimers, with mass ranging from ~300 to more than 1000 kDa. The size of the oligomer varies depending on the temperature, pH, and ionic strength. 3 The two subunits assemble in vivo to form heteromeric oligomers by means of subunit exchange. 4,5
Altered aggregation properties of mutant γ-crystallins cause inherited cataract
The EMBO Journal, 2002
Protein inclusions are associated with a diverse group of human diseases ranging from localized neurological disorders through to systemic non-neuropathic diseases. Here, we present evidence that the formation of intranuclear inclusions is a key event in cataract formation involving altered g-crystallins that are unlikely to adopt their native fold. In three different inherited murine cataracts involving this type of g-crystallin mutation, large inclusions containing the altered g-crystallins were found in the nuclei of the primary lens ®bre cells. Their formation preceded not only the ®rst gross morphological changes in the lens, but also the ®rst signs of cataract. The inclusions contained ®lamentous material that could be stained with the amyloid-detecting dye, Congo red. In vitro, recombinant mutant gB-crystallin readily formed amyloid ®brils under physiological buffer conditions, unlike wild-type protein. These data suggest that this type of cataract is caused by a mechanism involving the nuclear targeting and deposition of amyloid-like inclusions. The mutant g-crystallins initially disrupt nuclear function, but then this progresses to a full cataract phenotype.
The γ-Crystallins and Human Cataracts: A Puzzle Made Clearer
The American Journal of Human Genetics, 1999
Despite the fact that cataracts constitute the leading cause of blindness worldwide, the mechanisms of lens opacification remain unclear. We recently mapped the aculeiform cataract to the g-crystallin locus (CRYG) on chromosome 2q33-35, and mutational analysis of the CRYG-genes cluster identified the aculeiform-cataract mutation in exon 2 of g-crystallin D (CRYGD). This mutation occurred in a highly conserved amino acid and could be associated with an impaired folding of CRYGD. During our study, we observed that the previously reported Coppock-like-cataract mutation, the first human cataract mutation, in the pseudogene CRYGE represented a polymorphism seen in 23% of our control population. Further analysis of the original Coppocklike-cataract family identified a missense mutation in a highly conserved segment of exon 2 of CRYGC. These mutations were not seen in a large control population. There is no direct evidence, to date, that up-regulation of a pseudogene causes cataracts. To our knowledge, these findings are the first evidence of an involvement of CRYGC and support the role of CRYGD in human cataract formation.
The P23T Cataract Mutation Causes Loss of Solubility of Folded γD-Crystallin
Journal of Molecular Biology, 2004
Mutations in the human gD-crystallin gene have been linked to several types of congenital cataracts. In particular, the Pro23 to Thr (P23T) mutation of human gD crystallin has been linked to cerulean, lamellar, coralliform, and fasciculiform congenital cataracts. We have expressed and purified wild-type human gD, P23T, and the Pro23 to Ser23 (P23S) mutant. Our measurements show that P23T is significantly less soluble than wild-type human gD, with P23S having an intermediate solubility. Using synchrotron radiation circular dichroism spectroscopy, we have determined that the P23T mutant has a slightly increased content of b-sheet, which may be attributed to the extension of an edge b-strand due to the substitution of Pro23 with a residue able to form hydrogen bonds. Neither of the point mutations appears to have reduced the thermal stability of the protein significantly, nor its resistance to guanidine hydrochloride-induced unfolding. These results suggest that insolubility, rather than loss of stability, is the primary basis for P23T congenital cataracts.
Altered aggregation properties of mutant gamma-crystallins cause inherited cataract
Embo Journal 2002 Vol 21 Pp 6005 6014 Peer Reviewed Journal, 2002
Protein inclusions are associated with a diverse group of human diseases ranging from localized neurological disorders through to systemic non-neuropathic diseases. Here, we present evidence that the formation of intranuclear inclusions is a key event in cataract formation involving altered g-crystallins that are unlikely to adopt their native fold. In three different inherited murine cataracts involving this type of g-crystallin mutation, large inclusions containing the altered g-crystallins were found in the nuclei of the primary lens ®bre cells. Their formation preceded not only the ®rst gross morphological changes in the lens, but also the ®rst signs of cataract. The inclusions contained ®lamentous material that could be stained with the amyloid-detecting dye, Congo red. In vitro, recombinant mutant gB-crystallin readily formed amyloid ®brils under physiological buffer conditions, unlike wild-type protein. These data suggest that this type of cataract is caused by a mechanism involving the nuclear targeting and deposition of amyloid-like inclusions. The mutant g-crystallins initially disrupt nuclear function, but then this progresses to a full cataract phenotype.
Proceedings of the National Academy of Sciences, 2011
The prevalent eye disease age-onset cataract is associated with aggregation of human γD-crystallins, one of the longest-lived proteins. Identification of the γ-crystallin precursors to aggregates is crucial for developing strategies to prevent and reverse cataract. Our microseconds of atomistic molecular dynamics simulations uncover the molecular structure of the experimentally detected aggregation-prone folding intermediate species of monomeric native γD-crystallin with a largely folded C-terminal domain and a mostly unfolded N-terminal domain. About 30 residues including a, b, and c strands from the Greek Key motif 4 of the C-terminal domain experience strong solvent exposure of hydrophobic residues as well as partial unstructuring upon N-terminal domain unfolding. Those strands comprise the domain-domain interface crucial for unusually high stability of γD-crystallin. We further simulate the intermolecular linkage of these monomeric aggregation precursors, which reveals domain-swapped dimeric structures. In the simulated dimeric structures, the N-terminal domain of one monomer is frequently found in contact with residues 135-164 encompassing the a, b, and c strands of the Greek Key motif 4 of the second molecule. The present results suggest that γD-crystallin may polymerize through successive domain swapping of those three C-terminal β-strands leading to age-onset cataract, as an evolutionary cost of its very high stability. Alanine substitutions of the hydrophobic residues in those aggregation-prone β-strands, such as L145 and M147, hinder domain swapping as a pathway toward dimerization. These findings thus provide critical molecular insights onto the initial stages of age-onset cataract, which is important for understanding protein aggregation diseases.
Journal of Biological Chemistry, 1999
Two unique polypeptides, 22.4 and 16.4 kDa, were prominent in some human cataracts. Both proteins were identified as modified forms of the small heat shock protein, ␣B-crystallin. The concentration of total ␣Bcrystallin in most of these cataracts was significantly increased. The 22.4-kDa protein was subsequently designated as ␣B g. Mass spectrometric analyses of tryptic and Asp-N digests showed ␣B g is ␣B-crystallin minus the C-terminal lysine. ␣B g constituted 10-90% of the total ␣B-crystallin in these cataracts and was preferentially phosphorylated over the typical form of ␣B-crystallin. Human ␣B g and ␣B-crystallin were cloned and expressed in Escherichia coli. The differences in electrophoretic mobility and the large difference in native pI values suggest some structural differences exist. The chaperone-like activity of recombinant human ␣B g was comparable to that of recombinant human ␣B-crystallin in preventing the aggregation of lactalbumin induced by dithiothreitol. The mechanism involved in generating ␣B g is not known, but a premature termination of the ␣B-crystallin gene was ruled out by sequencing the polymerase chain reaction products of the last exon for the ␣B-crystallin gene from lenses containing ␣B g. The 16.4-kDa protein was an N-terminally truncated fragment of ␣B g. The high concentration of ␣B-crystallin in these cataracts is the first observation of this kind in human lenses.