Fluorescence quenching studies of the structures of calf gamma-II, III, and IV crystallins

The structures of the calf lens crystallin fractions gamma-II, gamma-III, and gamma-IV have been investigated using the fluorescence quenching method. The three crystallin fractions showed very large differences in the quenching rates of their fluorescent tryptophan residues, for quenching by acrylamide or iodide in pH 7.5 phosphate buffer solutions. The experimentally measured quenching rate constants were kq(II) = 3.2 x 10(8), kq(III) = 9.9 x 10(8), and kq(IV) = 1.8 x 10(9) M-1 sec-1. Smaller rate constants were obtained for iodide quenching of the three crystallins, but the values were in approximately the same ratios as the ones found for acrylamide quenching. The conclusion is that the tryptophan residues in gamma-II crystallin are 6-10 times less easily quenched than those of gamma-IV crystallin and 3-6 times less easily quenched than those of gamma-III. These conclusions are in accord with those reached by Mandal et. al. based on fluorescence and CD data, who found the following order of Trp hydrophobicities: gamma-II greater than gamma-III greater than gamma-IV. The significance of these structural differences for lens function and stability remains to be elucidated.     

Effect of acetylation by aspirin on the thermodynamic stability of lens crystallins

To assess the effect of aspirin on cataractogenesis, we compared the stability of individual, native protein fractions alpha L, beta H, beta L, beta s, beta B2, gamma-II, gamma-III and gamma-IV with that of their acetylated counterparts. The conformational stabilities of native fractions beta B2 and beta s, which were not reported earlier, were determined first from their thermal and a thermal denaturation behaviour. Since alpha L, beta H and beta L fractions are oligomeric, no thermodynamic analysis of these fractions was attempted. The thermal stability of beta s and beta B2 is rather low; their melting temperature (T1/2) range is 58-60 degrees C compared with 67-75 degrees C for the gamma-crystallins. Furthermore, except for alpha L, which remains stable even at 100 degrees C, and beta B2, all crystallins aggregate at temperatures slightly above T1/2. The Gibbs free energy of unfolding, delta GH2OD, calculated from guanidine HCl (GdnHCl) denaturation, is surprising low (3-9 kcal mol-1) for all crystallin fractions. The low values of delta GH2OD indicate that the structural destabilization of these proteins, which may lead to cataract formation, could result from a slight disturbance of a particular kind (sugar, UV light, oxidation, and other factors). The overall effect of acetylation on the individual crystallin fractions is mixed. The thermal stability of beta B2 increased, tended to decrease in the case of gamma-crystallins, but remained virtually unchanged for other proteins. Delta GH2OD values of the native crystallin fractions do not differ significantly from those of their acetylated counterparts.     

Spectroscopic studies on the mixed disulfide formation of lens crystallin with glutathione

Mixed disulfide was formed through thiol-disulfide exchange reaction of lens crystallin with oxidized glutathione (GSSG). The reaction was monitored by isoelectric focusing (IEF) and DTNB [5,5'-dithiobis(2-nitrobenzoic acid)] assay. The effects on protein conformation were studied by circular dichroism (CD) and fluorescence. The DTNB shows 22% and 49% decrease of SH groups after the exchange reaction in alpha-crystallin and gamma-crystallin, respectively. The exchange reaction was further shown by an acidic shifting in IEF pattern. The near ultraviolet CD shows a slight decrease in the GSSG-treated crystallins. The fluorescence measurements of the SH specific probe IANBD, 4-(N-iodoacetoxy)ethyl-N-methylamino-7-nitrobenz-2-oxa-1,3-diazole, indicate that the surface SH groups were oxidized in the GSSG-treated samples. The labeling with amine selectively reactive probe FITC, fluorescein-5-isothiocyanate, indicates an increase of amine reactivity with mixed disulfide formation. Polarization measurements show that bound FITC probes are in a less rigid structure in the mixed disulfide rich crystallin. All these results point out that the formation of mixed disulfide partially unfolds protein.     

A new HPLC method to determine glutathione-protein mixed disulfide

A sensitive method for measuring glutathione-protein mixed disulfides is described. The method is based on cleavage of the protein disulfides with performic acid followed by reaction with dinitrofluorobenzene and HPLC analysis with a Bondapak-amine column. Samples containing 0.1 nmoles or more of glutathione-protein mixed disulfide can be detected. The method has been used to demonstrate (a) the presence of low levels of glutathione mixed disulfide in gamma crystallin isolated from bovine lenses, (b) a dramatic increase in such mixed disulfides after exposure of denatured gamma crystallin to O2 in the presence of glutathione, and (c) the formation of glutathione-protein mixed disulfide in lens epithelial cells exposed to 0.6 mM H2O2 for one hour.     

Raman spectroscopy of calf lens gamma-II crystallin: direct evidence for the formation of mixed disulfide bonds with 2-mercaptoethanol and glutathione

This study presents Raman spectra of calf lens gamma-II crystallin and its reaction products with reduced glutathione, 2-mercaptoethanol and p-hydroxymercuribenzoate. The absence of a disulfide vibration in gamma-III crystallin (both in aqueous solution and in lyophilized state) indicates that the seven thiol groups in this protein are resistant to air oxidation, and are capable of maintaining their reduced state in the absence of added reducing agents during isolation. However, treatment of the protein with low molecular weight thiols such as glutathione and 2-mercaptoethanol results in mixed disulfide bonds. We have detected, for the first time, the S--S bond stretching vibration from the mixed disulfides at 510 cm-1, which is very similar to the 508 cm-1 reported for the inter/intramolecular disulfide bonds in intact mouse lenses (Yu, N.-T., DeNagel, D.C., Pruett, P.L. and Kuck, J.F.R., Jr. (1985). Proc. Natl. Acad. Sci. U.S.A., 82, 7965-8). Upon titration with five equivalents of p-hydroxymercuribenzoate, a strong Raman line was detected at 345 cm-1, which is tentatively attributed to the Hg--S stretching vibration of the mercaptide complex. The S--H vibration region (2500-2700 cm-1) exhibits two resolved peaks at 2562 and 2580 cm-1 with an intensity ratio of 2:5. Both reactive surface thiol groups and buried cysteines give rise to the S--H vibration at 2580 cm-1.     

High-molecular-weight crystallin aggregate formation resulting from non-enzymic carbamylation of lens crystallins: relevance to cataract formation

Incubation of mixed bovine lens crystallins with 100 mM potassium cyanate causes almost all the protein to form large aggregates. These aggregates are not dispersed by powerful chaotropic agents and are held together by disulphide bonds. Experiments with beta L-crystallin show that carbamylation of this one protein class can bring about the aggregation of other unmodified crystallins. The carbamylated crystallin served as a nucleus for aggregation of other crystallins. These changes are related to the chemical modification of crystallins and the ensuing conformational changes in cataractogenesis.     

Changes in crystallin concentration in rat aqueous and vitreous humors after selenium-induced reversible cortical cataract

Five months after selenite injection, 58% of the rats that had developed cataract earlier underwent a reversal of the cortical opacity. The purpose of this study was to determine if lenses undergoing recovery from cortical opacity reestablish their ability to retain crystallins. By direct ELISA method, the aqueous humor (AH) of control rats was found to contain 18, 39 and 10 ng/ml alpha-, beta- and gamma-crystallin, respectively, while vitreous humor (VH) contained 43, 98 and 23 ng/ml of alpha-, beta- and gamma-crystallin, respectively. In rats with mature cataracts which did not recover by 5 months after selenite injection, there was an approximately 10-fold greater crystallin concentration in the AH and about 20 times greater crystallin concentration in the VH than in the controls. In contrast, rats undergoing recovery from cortical cataract showed almost normal concentration of crystallins in the AH. While crystallins were still elevated in the VH of the rats undergoing recovery from cortical cataract, the crystallin content was lower than in the rats which did not recover. Higher crystallin concentrations in the VH could be explained by either a greater loss through the posterior capsule, or a slower turnover of VH. Decreased crystallin loss from lenses undergoing recovery suggested that the recovered lens at least partly reestablishes its ability to retain crystallins. These data may demonstrate that the lens fiber permeability is lowered while lens repair is occurring.     

Characterization of disulfide-linked crystallins associated with human cataractous lens membranes

In order to characterize possible disulfide-linked interactions between lens fiber cell membranes and crystallins, two-dimensional diagonal electrophoresis has been used in combination with Western blot analysis. When these blots were probed with monospecific antisera against alpha, beta and gamma crystallins, membrane from five individual normal lenses showed no disulfide-bonded components. Membrane from 13 individual cataractous human lenses showed no disulfide-bonded alpha crystallin, but did show significant amounts of disulfide-bonded beta crystallin in four out of the 13 lenses studied, and significant amounts of disulfide-bonded gamma crystallin in 10 out of the 13 lenses studied. Together, these studies demonstrate that intermolecular disulfide bonding of crystallins to purified fiber cell membranes is found only in cataractous lenses, and that the predominant polypeptide species involved in this interaction is gamma crystallin.     

Light scattering and photocrosslinking in the calf lens crystallins gamma-II, III and IV

The calf lens proteins gamma-II, -III and -IV crystallin have been photolyzed in pH 7.5 phosphate buffer solution at 25 degrees C. The photolysis light source was either a xenon arc lamp/monochromator system set to pass 290 +/- 5 nm or a nitrogen laser operating at 337.1 nm. Photolysis experiments at 337.1 nm were done both in the presence and absence of added 1.0 x 10(-4) M N-formylkynurenine (NFK). In addition, 1 x 10(-5) M riboflavin was added as a photosensitizer in a few of the experiments. All solutions were 1.0 mg ml-1 protein, and 1.0 ml of solution was irradiated for periods ranging from 10 min to 3 hr. During the 337.1 nm irradiations, the turbidity of the protein solutions was continuously monitored using a He-Ne laser at 632.8 nm. Progress of the 290 nm irradiations was monitored by observing the loss of tryptophan fluorescence for each of the gamma crystallin proteins. The rate of growth of light scattering, upon 337.1 nm irradiation, was greatest for gamma-IV. Addition of NFK caused the rates of growth of UV-induced light scattering of all three gamma crystallins to increase significantly. These rates were in the order: gamma-IV much greater than gamma-III greater than gamma-II. Following UV exposure, the protein solutions were analyzed using UV-visible absorption spectroscopy and SDS-PAGE. Irradiated gamma crystallin solutions showed increased optical density throughout the visible region, resulting from solution turbidity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical characterization of lens crystallins from three mammalian species

Lens crystallins were isolated from the homogenates of mammalian eye lenses derived from three different species by gel permeation chromatography and characterized by SDS-gel electrophoresis, isoelectric focusing, amino acid analysis and N-terminal sequence analysis. Five fractions corresponding to HM alpha-, alpha-, beta H-, beta L- and gamma-crystallins were obtained for the crystallins from these phylogenetically distant species. The native molecular masses for these purified fractions and their polypeptide compositions were determined by gel filtration and SDS-gel electrophoresis respectively, revealing the typical subunit compositions for each classified crystallin. The gel pattern of gamma-crystallins from the marmot lens appeared to be more complex than those of gibbon and deer lenses. Comparison of the amino acid contents of each orthologous class of mammalian crystallins with those of evolutionarily distant species still exhibited similarity in their amino acid compositions. The charge heterogeneity of each crystallin fraction can be detected by isoelectric focusing under denaturing conditions. N-terminal sequence analysis of the crystallin fractions revealed that all fractions except that of gamma-crystallin are N-terminally blocked. Extensive sequence similarity between mammalian gamma-crystallin polypeptides were found, which suggested the close relatedness of gamma-crystallins amongst different species of mammals and also established the heterogeneous nature of this multigene family.     

Fluorescence polarization studies of fluorescein isothiocyanate conjugates of bovine lens crystallins

Bovine lens alpha-, beta H- and gamma-crystallin were labeled with the amine-specific fluorescent probe, fluorescein isothiocyanate (FITC) and studied with steady-state polarization measurements. Rotational relaxation times (rho) were estimated for various crystallins and were compared with calculated values. The observed rho value is considerably faster for alpha- and beta H-crystallin conjugate than the calculated value, indicating existence of a segmental motion of the probe on these two crystallins. The segmental flexibility may result from a less tightly folded structure in these crystallins. alpha-Crystallin isolated from the cow lens nucleus shows a smaller rho value than the young cortical alpha-crystallin. The protein partial unfolding process appears to be age-related, and a possible consequence is that crystallin becomes more susceptible to chemical modifications.     

Phosphorescence measurements of calf gamma-II, III, and IV crystallins at 77 and 293 K

Structural and dynamical features of bovine gamma-crystallin tryptophan residues were investigated by phosphorescence measurements at 77 and 293 K. The low temperature phosphorescence spectra and lifetimes of calf gamma-II, III, and IV crystallins did not reflect heterogeneity among the gamma-crystallins. The 0-0 bands were all at 414 +/- 1 nm and the emission lifetimes were all single-exponential with lifetimes of 5.1, 5.3 and 5.3 +/- 0.3 sec, respectively. In contrast, phosphorescence measurements at room temperature were sensitive to subtle differences in exposure, accessibility, and flexibility of gamma-crystallin tryptophan residues. Thorough deoxygenation allowed for measurement of the normally-quenched room-temperature phosphorescence, and we report the first native phosphorescence measurements of lens crystallins at ambient temperature. The emission maxima for gamma-II, III and IV were 446, 442, and 440 +/- 2 nm, respectively. The intensity decay curves were all non-single exponential, and the decays were fit to a sum of two exponentials with lifetimes of 9.1 and 93 msec (gamma-II), 11 and 75 msec (gamma-III), and 4.2 and 68 msec (gamma-IV), +/- 10%. The components of the gamma-II emission were assigned to the four tryptophans based on X-ray structural information. Quantum yields of the phosphorescence emission were in the ratio of 20:7:1 for gamma-II, III and IV, and comparison of lifetimes and quantum yields suggests that tryptophan rigidity increases in the order gamma-IV less than III less than II. Acrylamide quenching constants for the long-lived components of gamma-II and III were roughly equal, while the short-lived tryptophans of gamma-III were an order of magnitude more accessible than those of gamma-II. The wide range of phosphorescence lifetimes and quenching constants allowed for discrimination of distinct contributions to the phosphorescence emission, and we suggest that room-temperature phosphorescence measurements will be an effective tool for studying conformational changes of lens crystallins.     

The major in vivo modifications of the human water-insoluble lens crystallins are disulfide bonds, deamidation, methionine oxidation and backbone cleavage

This investigation of the water-insoluble crystallins from human lenses has used multiple chromatographic separations to obtain proteins of sufficient purity for mass spectrometric analysis. Each fraction was analysed to determine the molecular masses of the constituent proteins as well as peptides in tryptic digests of these proteins. The major components of the water-insoluble crystallins were identified as alphaA- and alphaB-crystallins. In addition, gammaS-, betaB1-, gammaD-, betaA3/A1- and betaB2-crystallins were found, in order of decreasing abundance. Although there was evidence of some backbone cleavage, the predominant forms of alphaA-, alphaB, betaB2-, gammaS- and gammaD-crystallins were the intact polypeptide chains. The major modifications distinguishing the water-soluble crystallins were increased disulfide bonding, oxidation of Met, deamidation of Gln and Asn and backbone cleavage. Of the many reactions hypothesized to lead to crystallin insolubility and cataract, these results most strongly support metal-catalysed oxidation, deamidation and truncation as initiators of conformational changes that favor aggregation.     

Two-dimensional gel electrophoretic analysis of human lens proteins.

Human lens proteins from clear lenses were separated and identified using two-dimensional polyacrylamide electrophoresis. Isoelectric focusing, both equilibrium and non-equilibrium, was performed in the first dimension and SDS electrophoresis in the second dimension. Proteins were identified by Western blotting and sequencing techniques and by comparison with patterns obtained with purified crystallin fractions. Analyses were performed on total urea soluble proteins of lenses varying in age from fetal to 73 yr. Several hundred protein spots representing crystallins, cytoskeletal proteins and enzymes were resolved in the fetal lens. In the older lenses there was a dramatic increase in the number of protein species in the molecular weight range of the crystallins and a reduced number of discrete protein species visible at molecular weights greater than 50,000. Conversely, a number of proteins below approximately 15 kDa were visible even in the fetal lens. The number and amount of polypeptides in this molecular weight range were increased in the older lenses. Many of these low molecular weight species could be assigned to either the alpha-, beta- or gamma-crystallin fractions. An age dependent increase in the number of acidic species of both crystallins and other proteins, such as, glyceraldehyde 3-phosphate dehydrogenase was observed as well as the loss or mobility change of gamma-crystallin. Two-dimensional gel electrophoresis provides a sensitive and practical technique for characterizing all of the proteins of the human lens.     

Resistance of human betaB2-crystallin to in vivo modification.

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 betaB2-crystallin undergoes less modification than any of the other crystallins. As the lens ages, betaB2-crystallin retains its hydrophilic N-terminus while the hydrophilic C-termini of alpha-crystallins and large portions of the N-termini of betaA3/A1 and betaB1 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 betaB2 than in the other crystallins. This resistance to modification results in higher levels of betaB2 compared with the other crystallins in the water-soluble fraction of older lenses. The solubility of betaB2 and its propensity to form non-covalent associations with less soluble beta-crystallins may contribute to the solubility of the other beta-crystallins. A current hypothesis is that the chaperone-like properties of alpha-crystallins contribute to lens crystallin solubility, particularly in younger lenses. In older lenses, where most of the alpha-crystallins have become water-insoluble, betaB2-crystallins may play a dominant role in lens crystallin solubility.     

Characterization of water-insoluble proteins in normal and cataractous human lens

The mechanism of lens protein aggregation with age and/or cataract formation was investigated using the peptides resolubilized from the insoluble protein fraction of normal and cataractous human lenses. The insoluble fraction was treated with reductants for cleaving disulfide bonds, or with chelating agents for removing calcium ions from the aggregates. This study demonstrates that the insoluble protein aggregates consist of an approximately 400 Kd complex, which is formed by the peptides with lower molecular weight. Protein aggregation in the cataractous lens might be caused by disulfide bonds whereas, in aging, the aggregate might be preferentially formed by calcium ion bridges rather than by disulfide bonds. It was observed that the aggregate from the cataractous lenses involved a peptide with a molecular mass lower by 1 Kd or 2 Kd than the peptides found in the normal lens. The composition of crystallins in aggregating proteins and their secondary structures were also different in the normal and the cataractous lenses. Such changes of molecular weight, conformation, and/or crystallin species in the lens may lead to the disintegration of the orderly arrangement of crystallins, resulting in the diffused reflection and lens opacities which are seen in senile cataract.     

Review: A case for corneal crystallins.

It is established that the diverse, multifunctional crystallins are responsible for the optical properties of the cellular, transparent lens of the complex eyes of vertebrates and invertebrates. Lens crystallins often differ among species and may be enzymes or stress proteins. I present here the idea that abundant water-soluble enzymes and other proteins may also be used for cellular transparency in the epithelial cells and, possibly, stromal keratocytes of the cornea. Aldehyde dehydrogenases and transketolase are among the putative "corneal crystallins" in mammals, and gelsolin may be a corneal crystallin in the zebrafish. In invertebrates, the glutathione S-transferase-related S-crystallins of the lens appear to be used also as corneal crystallins in the squid, and an aldehyde dehydrogenase-related protein is the crystallin in the lens and, possibly, cornea of the scallop. The use of abundant, taxon-specific water-soluble proteins as crystallins for cellular transparency in the cornea would provide a new conceptual link between this tissue and the lens.     

Alkylation of rat lens crystallins with iodoacetamide

Alkylation of rat lens proteins with iodoacetamide during homogenization had no effect on protein disulfide content or on the proportions of soluble and insoluble lens protein. These results were similar for lenses, from young or older animals. With the exceptin of the β_H-crystallin fraction. alkylation yielded a normal crystallin population as determined by gel-permeation chromatography. Although each crystallin fraction reacted with iodoacetamide in proportion to its free sulfhydryl content, only the alkylated β_H-crystallin demonstrated an apparent reduction in molecular size so that it co-chromatographed with β_L-crystallin. Molecular weights of subunits for both β_H- and β_L-crystallin were modified by alkylation. Isoelectric focusing further revealed bands of higher isoelectric pH after alkylation. We conclude that subunit interactions which result in the protein assemblies, characterized as crystallins, do not depend on disulfide formation during isolation, and that β_H-crystallin has unique conformational or subunit properties which are sensitive to modification of the free-sulfhydryl group.     

Resistance of alpha-crystallin-glutathione mixed-disulfide to tryptic digestion

Protein-mixed disulfides (PSSG) were formed by interaction of glutathione disulfide (GSSG) with lens crystallins. Total water-soluble crystallins and alpha-crystallin purified on a Sephacryl S-200 column were separately incubated with 0, 2, 4, and 8 mM (final concentrations) GSSG overnight and then dialyzed to remove unbound GSSG and GSH. Either TPCK-treated trypsin or TLCK-treated alpha-chymotrypsin were added to about 200 micrograms crystallin samples and incubated for 20 min at room temperature. Reactions were terminated by boiling in SDS-mercaptoethanol-Tris (pH 6.8) solution and subjected to electrophoresis on 10% polyacrylamide slab gels. Comparison of SDS-PAGE patterns of proteolysis with or without GSSG treatment showed that GSSG at a concentration of 2 mM or higher reduced or abolished proteolysis of alpha-crystallin by trypsin but not by alpha-chymotrypsin. The protective effect of GSSG was greater with alpha-crystallin than with beta-crystallins. Addition of alpha-crystallin-mixed-disulfide to an assay system in which trypsin was hydrolyzing N-alpha-benzoyl-DL-arginine-P-anilide (BAPNA) inhibited the tryptic activity. Direct addition of GSSG or native alpha-crystallin had no significant inhibitory effect on trypsin. Based on these results, it is speculated that alpha-crystallin glutathione mixed-disulfide appears to become resistant to trypsin probably by non-competetive inhibition of the enzyme.     

Zeta-crystallin, a novel lens protein from the guinea pig

Lens proteins from the guinea pig (Cavia porcellus) were found to be similar to those of other mammals with the exception of the presence of a previously undescribed constituent comprising about 10% of the total soluble lens proteins. This oligomeric protein is composed of polypeptides with apparent molecular weight of 38,000 and elutes from gel exclusion chromatography columns in the beta H-crystallin fraction. Following purification by ion exchange chromatography an antibody was raised against the protein. Using that antibody and antibodies specific for other crystallins we could detect no cross-reactivity between the guinea pig protein and any other reported lens crystallin. This protein, which we have named zeta (zeta)-crystallin, is the first reported mammalian lens crystallin which is not part of the alpha- or beta-gamma families of crystallins. Unlike all other known mammalian crystallins, which have little or no alpha-helical structure, zeta-crystallin is estimated to be approximately 30-40% alpha-helix.