Conformational changes induced in lens alpha- and gamma-crystallins by modification with glucose 6-phosphate. Implications for cataract.
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ABSTRACT:
SUBMITTER: Beswick HT
PROVIDER: S-EPMC1148342 | biostudies-other | 1987 Sep
REPOSITORIES: biostudies-other
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Project description: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 gamma-crystallins that are un likely to adopt their native fold. In three different inherited murine cataracts involving this type of gamma-crystallin mutation, large inclusions containing the altered gamma-crystallins were found in the nuclei of the primary lens fibre cells. Their formation preceded not only the first gross morphological changes in the lens, but also the first signs of cataract. The inclusions contained filamentous material that could be stained with the amyloid-detecting dye, Congo red. In vitro, recombinant mutant gammaB-crystallin readily formed amyloid fibrils 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 gamma-crystallins initially disrupt nuclear function, but then this progresses to a full cataract phenotype.
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Project description:We analyze the experimentally determined phase diagram of a γD-βB1 crystallin mixture. Proteins are described as dumbbells decorated with attractive sites to allow inter-particle interaction. We use thermodynamic perturbation theory to calculate the free energy of such mixtures and, by applying equilibrium conditions, also the compositions and concentrations of the co-existing phases. Initially we fit the Tcloudversus packing fraction η measurements for a pure (x2 = 0) γD solution in 0.1 M phosphate buffer at pH = 7.0. Another piece of experimental data, used to fix the model parameters, is the isotherm x2vs. η at T = 268.5 K, at the same pH and salt content. We use the conventional Lorentz-Berthelot mixing rules to describe cross interactions. This enables us to determine: (i) model parameters for pure βB1 crystallin protein and to calculate; (ii) complete equilibrium surface (Tcloud-x2-η) for the crystallin mixtures. (iii) We present the results for several isotherms, including the tie-lines, as also the temperature-packing fraction curves. Good agreement with the available experimental data is obtained. An interesting result of these calculations is evidence of the coexistence of three phases. This domain appears for the region of temperatures just out of the experimental range studied so far. The input parameters, leading good description of experimental data, revealed a large difference between the numbers of the attractive sites for γD and βB1 proteins. This interesting result may be related to the fact that γD has a more than nine times smaller quadrupole moment than its partner in the mixture.
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Project description:To test the hypothesis that α-crystallin chaperone activity plays a central role in maintenance of lens transparency, we investigated its interactions with γ-crystallin mutants that cause congenital cataract in mouse models. Although the two substitutions, I4F and V76D, stabilize a partially unfolded γD-crystallin intermediate, their affinities to α-crystallin are marginal even at relatively high concentrations. Detectable binding required further reduction of γD-crystallin stability which was achieved by combining the two mutations. Our results demonstrate that mutants and possibly age-damaged γ-crystallin can escape quality control by lens chaperones rationalizing the observation that they nucleate protein aggregation and lead to cataract.
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Project description:Carbamylation of lens proteins may contribute to cataractogenesis in certain medical conditions where blood urea is elevated for prolonged periods. This paper reports on the effects of carbamylation on the physicochemical properties of one of the major lens structural proteins, alpha-crystallin. In particular it is shown that carbamylation alters the tertiary and secondary structure of the protein, leading to an increased reactivity of protein thiols, resulting in interchain disulphide bonding.
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Project description:Crystallins are the major proteins in the lens of the eye and function to maintain transparency of the lens. Of the human crystallins, α, β, and γ, the β-crystallins remain the most elusive in their structural significance due to their greater number of subunits and possible oligomer formations. The β-crystallins are also heavily modified during aging. This review focuses on the functional significance of deamidation and the related modifications of racemization and isomerization, the major modifications in β-crystallins of the aged human lens. Elucidating the role of these modifications in cataract formation has been slow, because they are analytically among the most difficult post-translational modifications to study. Recent results suggest that many amides deamidate to similar extent in normal aged and cataractous lenses, while others may undergo greater deamidation in cataract. Mimicking deamidation at critical structural regions induces structural changes that disrupt the stability of the β-crystallins and lead to their aggregation in vitro. Deamidations at the surface disrupt interactions with other crystallins. Additionally, the α-crystallin chaperone is unable to completely prevent deamidated β-crystallins from insolubilization. Therefore, deamidation of β-crystallins may enhance their precipitation and light scattering in vivo contributing to cataract formation. Future experiments are needed to quantify differences in deamidation rates at all Asn and Gln residues within crystallins from aged and cataractous lenses, as well as racemization and isomerization which potentially perturb protein structure greater than deamidation alone. Quantitative data is greatly needed to investigate the importance of these major age-related modifications in cataract formation.
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Project description:Lens crystallin proteins make up 90% of expressed proteins in the ocular lens and are primarily responsible for maintaining lens transparency and establishing the gradient of refractive index necessary for proper focusing of images onto the retina. Age-related modifications to lens crystallins have been linked to insolubilization and cataractogenesis in human lenses. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) has been shown to provide spatial maps of such age-related modifications. Previous work demonstrated that, under standard protein IMS conditions, α-crystallin signals dominated the mass spectrum and age-related modifications to α-crystallins could be mapped. In the current study, a new sample preparation method was optimized to allow imaging of β- and γ-crystallins in ocular lens tissue. Acquired images showed that γ-crystallins were localized predominately in the lens nucleus whereas β-crystallins were primarily localized to the lens cortex. Age-related modifications such as truncation, acetylation, and carbamylation were identified and spatially mapped. Protein identifications were determined by top-down proteomics analysis of lens proteins extracted from tissue sections and analyzed by LC-MS/MS with electron transfer dissociation. This new sample preparation method combined with the standard method allows the major lens crystallins to be mapped by MALDI IMS.
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Project description:PurposeHow corneal transparency is formed/maintained remains largely unclear. A group of enzymes which are referred to as enzymatic crystallins were proposed to contribute to corneal transparency in various animals. This study investigated whether the three classical lens crystallins, namely α-, β-, and γ-crystallins, exist in mouse and human corneas.MethodsMice, human tissues, and cultured corneal cells were studied. The expression of lens crystallins in corneas or in cultured corneal cells were detected at the mRNA level by quantitative reverse transcription-PCR (QRT-PCR) and at the protein level by immunohistochemistry or western blotting. To check the effect of exogenous factor on expression of lens crystallins, cultured corneal cells were challenged with lipopolysaccharide or hydrogen peroxide and the expression of lens crystallins was monitored.ResultsQRT-PCR revealed that the relative expression level of lens crystallins in C57BL/6 corneas were higher than in Balb/c corneas. Immunohistochemistry study showed that expression of αA-crystallin started from the embryonic stage, lasted untill old age, and was largely restricted to the epithelium or endothelium of the corneas. β- and γ-crystallins also were found in murine corneal epithelium. Upon treatment with lipopolysaccharide or hydrogen peroxide of cultured corneal epithelial cells, lens crystallins expression was significantly increased as detected by QRT-PCR or western blot assay. Further, both fetal corneal epithelial cultures and limbal stem cell cultures from adult human tissues were positive for lens crystallin immunofluorescence or immunohistochemistry staining.ConclusionsLens crystallins are expressed in mammalian corneas and cultured corneal cells. The expression levels depended on the animal strains or cell status. The physiologic and pathological significance of lens crystallins in corneas deserves more investigation.
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Project description:Lens γ crystallins are found at the highest protein concentration of any tissue, ranging from 300 mg/mL in some mammals to over 1000 mg/mL in fish. Such high concentrations are necessary for the refraction of light, but impose extreme requirements for protein stability and solubility. γ-crystallins, small stable monomeric proteins, are particularly associated with the lowest hydration regions of the lens. Here, we examine the solvation of selected γ-crystallins from mammals (human γD and mouse γS) and fish (zebrafish γM2b and γM7). The thermodynamic water binding coefficient B₁ could be probed by sucrose expulsion, and the hydrodynamic hydration shell of tightly bound water was probed by translational diffusion and structure-based hydrodynamic boundary element modeling. While the amount of tightly bound water of human γD was consistent with that of average proteins, the water binding of mouse γS was found to be relatively low. γM2b and γM7 crystallins were found to exhibit extremely low degrees hydration, consistent with their role in the fish lens. γM crystallins have a very high methionine content, in some species up to 15%. Structure-based modeling of hydration in γM7 crystallin suggests low hydration is associated with the large number of surface methionine residues, likely in adaptation to the extremely high concentration and low hydration environment in fish lenses. Overall, the degree of hydration appears to balance stability and tissue density requirements required to produce and maintain the optical properties of the lens in different vertebrate species.
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