Total Lens Protein Research Articles

Differential Phosphorylation of Alpha-A Crystallin in Human Lens of Different Age

Previous studies have demonstrated that a major site of in vivo phosphorylation of alpha-A crystallin from human lens is serine-122. To determine the relative degree of this phosphorylation in alpha-A crystallin from human lenses of different age, alpha-A crystallin was purified from total lens proteins, followed by sequential digestion with lys-C and asp-N endoproteases. Mass spectral analysis of the asp-N peptide fragments that contained serine-122 demonstrated undetectable levels of phosphorylation from infant human lenses (41 days, 2 months and 4 months of age). Identical analysis of alpha-A crystallin from older lenses (12, 15, 40 and 73 years of age) indicated significant phosphorylation of serine-122, demonstrating that phosphorylation of the serine-122 residue of alpha-A crystallin does not occur during the aging process, but is rather a developmentally regulated event in the human lens.

Vitamin A2 Bound to Cellular Retinol-binding Protein as Ultraviolet Filter in the Eye Lens of the Gecko Lygodactylus picturatus

The yellow eye lenses of the diurnal gecko Lygodactylus picturatus contain, in addition to the usual crystallins, a monomeric protein with a molecular mass of 16kDa. It comprises 6-8% of the total water-soluble lens proteins. We here identify it as a novel type of crystallin, most closely related with cellular retinol-binding protein I (CRBP I). Because of its tiny size, we designate it as iota-crystallin. The typical endogenous ligand of CRBP is all-trans-retinol. In the gecko lens, however, the ligand of iota-crystallin turns out to be 3-dehydroretinol (vitamin A2), which causes the yellow color of this lens. The iota-crystallin.3-dehydroretinol complex absorbs shortwave radiation, supposedly improving the optical quality of the dioptric apparatus and protecting the retina against ultraviolet damage. Whereas other crystallins have been recruited from stress proteins and metabolic enzymes, iota-crystallin represents a completely new class of taxon-specific lens proteins. Also, its ligand 3-dehydroretinol represents a novel type of lens pigment.

Evaluation of the relationship between total lens protein deposition and patient-rated comfort of hydrophilic (soft) contact lenses

The relationship between soft contact lens comfort and total protein deposit level was studied, and the correlation of total lens protein and comfort perception with lens replacement rationales was also evaluated. Worn soft lenses (N = 977) representing all U.S. Food and Drug Administration (FDA) lens groups and varied care systems were collected from multiple clinical sites, provided that they required replacement as the result of discomfort, unacceptable visual acuity (VA), surface deposits, positive slit-lamp findings, or discoloration. Patients rated the wearing comfort of replaced lenses. When only one lens required replacement, the companion lens was also collected. For each lens, deposits were rated visually and total protein was quantitated by chemical assay. Almost half of the lenses in each lens group were replaced for discomfort; other major reasons were unacceptable VA and visually detected deposits. For all lens groups, there was no significant difference in protein levels between lenses replaced for discomfort and lenses not requiring replacement (companion lenses). For all FDA lens groups, no significant differences in total protein were found for any replacement categories except for those Group 1 lenses which were replaced because of discoloration. Strikingly, there was no correlation between protein levels and patient comfort ratings for the entire sample or for any individual FDA lens group. This demonstrates that total accumulated lens-bound protein is not the sole or primary factor in determining lens comfort and intolerance.

Enzyme Activities and Crystallin Profiles of Clear and Cataractous Lenses of the RCS Rat

Royal College of Surgeons (RCS) rats have hereditary retinal degeneration in association with posterior subcapsular opacities. Cataract formation is thought to be correlated with an increase in lipid peroxidation products in the vitreous (Zigler and Hess, 1985). In order to examine the possibility that parallel changes in enzyme activity are occurring within the lens, we analysed the activity of four key enzymes and the crystallin protein profile. We compared RCS rat lenses at three different stages of cataract formation to clear lenses of the nonpigmented RCS rat, lenses from pigmented RCS rat and to normal (Fisher) rat. Our data shows that concomitant with the appearance of the RCS cataract, the ratio of the crystallins β1, β H and γ to the total lens protein was reduced. The crystallin profile of a clear RCS lens was similar to that of a normal (Fisher) lens. No significant difference in the activity of the enzymes hexokinase and glucose-6-phosphate dehydrogenase (G6PD) was found among the lenses, however the activity of glutathione reductase and aldolase was reduced in the cataractous lenses.

Identification and characterization of the enzymatic activity of zeta-crystallin from guinea pig lens. A novel NADPH:quinone oxidoreductase.

zeta-Crystallin is a major protein in the lens of certain mammals. In guinea pigs it comprises 10% of the total lens protein, and it has been shown that a mutation in the zeta-crystallin gene is associated with autosomal dominant congenital cataract. As with several other lens crystallins of limited phylogenetic distribution, zeta-crystallin has been characterized as an "enzyme/crystallin" based on its ability to reduce catalytically the electron acceptor 2,6-dichlorophenolindophenol. We report here that certain naturally occurring quinones are good substrates for the enzymatic activity of zeta-crystallin. Among the various quinones tested, the orthoquinones 1,2-naphthoquinone and 9,10-phenanthrenequinone were the best substrates whereas menadione, ubiquinone, 9,10-anthraquinone, vitamins K1 and K2 were inactive as substrates. This quinone reductase activity was NADPH specific and exhibited typical Michaelis-Menten kinetics. Activity was sensitive to heat and sulfhydryl reagents but was very stable on freezing. Dicumarol (Ki = 1.3 x 10(-5) M) and nitrofurantoin (Ki = 1.4 x 10(-5) M) inhibited the activity competitively with respect to the electron acceptor, quinone. NADPH protected the enzyme against inactivation caused by heat, N-ethylmaleimide, or H2O2. Electron paramagnetic resonance spectroscopy of the reaction products showed formation of a semiquinone radical. The enzyme activity was associated with O2 consumption, generation of O2- and H2O2, and reduction of ferricytochrome c. These properties indicate that the enzyme acts through a one-electron transfer process. The substrate specificity, reaction characteristics, and physicochemical properties of zeta-crystallin demonstrate that it is an active NADPH:quinone oxidoreductase distinct from quinone reductases described previously.

Lambda-crystallin, a major rabbit lens protein, is related to hydroxyacyl-coenzyme A dehydrogenases.

It has recently been discovered that several lens proteins in birds and lower vertebrates are active enzymes or enzyme-related proteins (Wistow, G., Mulders, J. W. M., and de Jong, W. W. (1987) Nature 326, 622-624; Wistow, G., and Piatigorsky, J. (1987) Science 236, 1554-1556). We report here a novel lens protein, designated as lambda-crystallin, that occurs in rabbit and hare. It constitutes 7-8% of the total lens protein and has a subunit molecular mass of 35 kDa. Sequencing of cDNA clones encoding rabbit lambda-crystallin revealed 30% homology (at the amino acid sequence level) with L-3-hydroxyacyl-CoA dehydrogenase from pig mitochondria and 26% homology with enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase from rat peroxisomes. Also, the presence of a putative beta-alpha-beta nucleotide-binding fold and low levels of non-lens expression are indicative of some enzymatic function for lambda-crystallin (or highly related sequences) in non-lens tissues. lambda-Crystallin thus represents the first example of an enzyme-related crystallin in lenses from mammalian species. The recruitment of enzymes as lens structural proteins apparently is an evolutionary strategy which has been applied independently in different lineages.

Heterogeneity of γ-crystallins from spiny dogfish ( Squalus acanthias) eye lens

Mammalian lenses contain multiple gamma-crystallin gene products, which are differentially synthesized during lens development. We now report the isolation and characterization of multiple gamma-crystallins from lenses of adult spiny dogfish (Squalus acanthias) aged about 20-30 years. About 50% of total lens protein solubilized in 50 mM phosphate, pH 7.0; about 25% of this soluble fraction consists of gamma-crystallins as determined by gel filtration. These gamma-crystallins appear homogeneous with respect to molecular weight (approximately equal to 20,000) on SDS-polyacrylamide gels, but their isoelectric points range from below pH 6 to above 10. Preparative cation-exchange chromatography on SP-Sephadex at pH 4.8 resolves four major subfractions, while anion-exchange on DEAE-cellulose at pH 9.5 resolves seven subfractions. Although these procedures separate basic from acidic polypeptides, most of these gamma-crystallin subfractions still consist of polypeptide mixtures, as determined by ion-exchange HPLC and isoelectric focusing. Analytical cation-exchange HPLC on SynChropak CM300 at pH 6.0 resolves at least 10 different gamma-crystallin components. Amino acid compositions of all the subfractions are similar, yet distinct in the sense that three subclasses can be distinguished. Sulfhydryl residues range from three to six per chain, most of which are buried. The large heterogeneity of gamma-crystallins in adult lens may result from different gene products in combination with post-translational modification.

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.

Immunochemical characterization and quantitative distribution of crystallins in the epithelium and differentiating fibre cell populations of chick embryonic lens

Lenses from 19-day chick embryos are fractionated by a double punch method to obtain the epithelium-annular pad complex (EP), outer fibres (OF), middle fibres (MF) and central fibres (CF). Water-soluble crystallins are characterized by SDS PAGE, isoelectric focusing (IEF) and two-dimensional IEF-SDS PAGE. Crystallins are also characterized by immunoelectrophoresis (IE), rocket IE, IEF-immunoblotting, and quantified by two-dimensional antigen-antibody crossed electrophoresis using antibodies to total 19-day embryonic as well as adult crystallins. In the adult lens, α-, β- and δ-crystallins are 19%, 67% and 14%, respectively, while these are present at concentrations of 9%, 27% and 64%, respectively, in 19-day embryonic lens. In absolute amounts, δ-crystallin increases only by 1·23-fold between 19-day embryonic age and 6 months post-hatching, while total lens protein increases 12·5-fold. The predominance of δ-crystallin in central fibres, located along the optical axis, suggests that this protein is of embryonic origin. δ-Crystallin from fibres is electrofocused as 12 distinct molecular classes (pI 5·2–5·42) which react against anti-δ-crystallin on an immunoblot. Of these, the three most anodal species are not detected in EP. Fibres contain 50 000, 48 000 and 45 000 dalton δ-crystallin subunits while only 50 000 and 48 000 dalton subunits are present in EP.

Studies on the water soluble lens proteins of the lizard, Calotes versicolor. I. Fractionation and molecular weight determination

The water soluble lens proteins of the lizard, Calotes versicolor are characterized by Sephadex G-200 gel filtration, isoelectric precipitation and paper electrophoresis. Gel filtration resolves the total soluble lens proteins into four fractions, F 1 to F 4. The molecular weights of these are around 410 000, 160 000, 70 000 and 6500; and they constitute 0·16, 0·08, 0·57 and 0·18 of the total soluble lens proteins respectively. From the molecular weights, F 1 and F 3 appear to be α- and β-crystallins respectively. F 2, a minor fraction, may be either δ-crystallin or a high molecular weight β-crystallin. F 4, the most retained fraction on Sephadex gel column is interesting, since it is the first report of such a low molecular weight crystallin from vertebrate lens. Results of isoelectric precipitation of proteins at pH 5·0 and subsequent gel filtration confirm that F 1 of the gel filtration is α-crystallin. Paper electrophoresis also reveals three bands, possibly corresponding to F 1, F 3 and F 4 gel filtration fractions.

Isofocusing and immunological investigations on cephalopod lens proteins

Soluble lens proteins from Octopus vulgaris, Sepia officinalis, and Loligo vulgaris were analyzed by thin-layer isoelectric focusing and compared by various immunochemical methods using antibodies directed against total soluble lens protein antigens from the said three species. The results show close similarity between Sepia and Loligo lens proteins. Antibodies made against Sepia and Loligo lens proteins did not react against Octopus total lens soluble protein antigens. Our results suggest a common ancestry for Sepia and Loligo which is different from that of Octopus.

Changes in the distribution of proteins in the aging human lens

The rate of extraction of lens proteins was found to vary with age. Therefore, a method involving exhaustive extraction of the lens, at room temperature, was used to separate the lens proteins into water-soluble, urea-soluble, solubilized urea-insoluble and insoluble proteins. In 28 normal lenses of ages from 0 to 90, the level of water-soluble proteins decroased linearly from over 96% of the total lens proteins at birth to about 89% at age 90. The urea-soluble proteins increased from 2% to about 10% while the levels of the solubilized urea-soluble and insoluble proteins remained constant at 1·3 and 0·2% respectively. The water-soluble proteins were fractionated on DEAE-cellulose scaled down so that as little as 500 μg of protein could be fractionated. The proteins so obtained were characterized by amino acid analysis, isoelectric focusing and SDS gel electrophoresis. The levels of all the soluble crystallin in the lens were found to decrease linearly with age while the protein eluted with NaOH increased. This fraction most closely resembled the α-crystallins. Large changes were found in the amino acid compositions of the α-crystallins while the compositions of the other fractions appeared to remain unchanged. The reasons for the differences between some of the results presented in this paper and those published by other workers are discussed.

Preparation of specific antiserum against Rana esculenta pre-alpha lens crystallin.

Specific antiserum against Rana esculenta lens pre-alpha crystallin was prepared in a rabbit by injecting antigen-antibody precipitate of this crystallin obtained from immunoelectrophoresis of esculenta total soluble lens proteins against homologous antiserum.

Ontogeny and localization of the lens crystallins in Xenopus laevis lens regeneration

ABSTRACT Ontogeny and localization of the lens crystallins, especially the γ-crystallins were investigated in Xenopus laevis lens regenerating system by the ‘indirect’ immunofluorescence staining method. Antibodies directed against Rana pipiens γ-crystallin antigen were used for the detection of this crystallin; the validity of such an experiment has been shown in a previous report. To detect total lens proteins we used X. laevis anti-total lens protein antibody. The regenerates were staged according to Freeman (1963) and the first positive reaction with both the two antisera was observed in an early stage-4 regenerate. The site of the immunofluorescence reaction was nearly identical in both, suggesting that γ-crystallins are one of the first, if not the first of the lens crystallins to appear during lens regeneration. The secondary fibres, when developed, showed less immunofluorescence than the primary fibres with R. pipiens anti-γ crystallin antibody, though the reaction was intense in the secondary fibres with X. laevis anti-total lens protein antibody. The intensity and distribution of immunofluorescence increased with the growth of the lens. With the R. pipiens anti-γ crystallin antibody, the lens epithelium did not show any immunofluorescence reaction at any stage of lens regeneration. With X. laevis anti-total lens protein antibody, the epithelium showed an immunofluorescence reaction earlier than in the normal lens development. With the two antisera we used, we did not observe any immunofluorescence outside the lens tissue.

Direct evidence for transformation of differentiated iris epithelial cells into lens cells.

Although it is generally assumed that the lens regenerated in the newt eye after complete lentectomy is formed by cells derived from the dorsal iris epithelium, experimental evidence so far obtained for this transformation does not rule out participation of cells from the dorsal iris stroma. When the normal dorsal iris epithelium of adult Notophthalmus (Triturus) viridescens was isolated and cultured in the presence of frog retinal complex, newt lens tissue was produced in 88% of cultures. These lens tissues were positive for immunofluorescence for lens-fiber-specific gamma crystallins as well as for total lens protein. On the basis of a study of stromal cells contaminating the samples of dorsal iris epithelium and a test for the lens-forming capacity in vitro of the dorsal iris stroma in the presence of frog retinal complex, it is concluded that lens formation observed in the above experiment is not dependent on the contaminating stromal cells. This implies that, in Wolffian lens regeneration, fully differentiated adult cells completely withdrawn from the cell cycle are transformed into another cell type. An additional culture experiment demonstrated that lens-forming capacity is not restricted to the dorsal half of the iris epithelium, but extends into its ventral half.

Ontogeny and localization of the crystallins during embryonic lens development in Xenopus laevis.

AbstractThe time of appearance and histological localization of the crystallins, especially the lens‐fiber specific γ crystallins, in the normally developing lens of Xenopus laevis were determined by immunofluorescence techniques. Antibodies to total soluble lens proteins as well as to purified γ crystallins were applied to sections through the eye region of X. laevis embryos, Nieuwkoop‐Faber Stages 23–45. Seven stages of lens development, the earliest highly atypical, were recognized in this species. The first positive immunofluorescence reaction for both the anti‐total and anti‐γ crystallin antibodies occurred at Lens Developmental Stage 3. At this time the lens rudiment is irregular and flattened, with a slightly‐condensed central mass of cells. These centrally located cells, probably prospective primary lens fiber cells, are positive for γ crystallins, while both they and surrounding retinally‐oriented lens cells are positive for total lens crystallins. This general immunofluorescence profile continues during lens development, with only the lens fiber areas positive for γ crystallins. No reaction with either antibody type, however, could be detected in the corneally‐oriented external layer or, later, the lens epithelium, until very late in the development of the lens (Nieuwkoop‐Faber Stage 45). The lack of crystallins in this cell type, together with the transitory appearance of a lens vesicle (lens cavity), delayed until after fiber cells have been formed, sets X. laevis morphological and biochemical lens development apart from that of other vertebrates.

Ontogeny of γ-crystallin in the chick lens

Lyophilized extracts of embryonic, post-embryonic and adult chick total lens proteins were tested against pigeon lens γ-crystallin antiserum by different immunochemical methods in order to determine the time of appearance of this crystallin antigen in chick lens. Results obtained from the experiments agree in showing that the immunoprecipitin line against pigeon lens γ-crystallin antiserum first begins to appear from the 1-week-old lens. This delay in appearance could possibly be one of the reasons why the amount of γ-crystallin is so low in adult chick lens.

Thin layer isoelectric focusing of the soluble lens extracts from larval stages and adult Xenopus laevis

Soluble lens extracts from different larval stages and also from adult Xenopus laevis were studied by thin layer isoelectric focusing. These lens extracts were also tested against antiserum to adult X. laevis total lens proteins by two-dimensional antigen-antibody crossed electrophoresis. By isoelectric focusing the lens crystallins were resolved into 13 major components. In the adult lens some components of the γ-crystallins were lost, the β-crystallins formed the major fraction and the α-crystallin appeared to be the weakest of the crystallin fractions.

Changes in Weight and in Protein-Water Ratio of the Lens in Human Senile Cataracts

Changes in wet weight, dry weight and water content have been studied in different types of senile cataract and compared With transparent control lenses. While nuclear brunescent cataractous lens show normal weight, protein and water content, cortical opacification is characterized by a progressive decrease of total lens proteins associated with important terminal alterations of the percentage of water content. Posterior subcapsular cataracts in very early stages of the disease have a significantly lower wet weight than control lenses, but a normal protein-water ratio; this is interpreted as a decreased lens growth starting far back in time and producing apparently normal lens substance. The question is raised whether this could be interpreted as a manifestation of the same pathogenetic cause which determines the appearance of this type of lens opacification.

The relationship between the ontogeny of antigens and of the polypeptide chains of the crystallins during chick lens development

Lenses from 19-day chick embryos are fractionated by a double punch method to obtain the epithelium-annular pad complex (EP), outer fibres (OF), middle fibres (MF) and central fibres (CF). Water-soluble crystallins are characterized by SDS PAGE, isoelectric focusing (IEF) and two-dimensional IEF-SDS PAGE. Crystallins are also characterized by immunoelectrophoresis (IE), rocket IE, IEF-immunoblotting, and quantified by two-dimensional antigen-antibody crossed electrophoresis using antibodies to total 19-day embryonic as well as adult crystallins. In the adult lens, α-, β- and δ-crystallins are 19%, 67% and 14%, respectively, while these are present at concentrations of 9%, 27% and 64%, respectively, in 19-day embryonic lens. In absolute amounts, δ-crystallin increases only by 1·23-fold between 19-day embryonic age and 6 months post-hatching, while total lens protein increases 12·5-fold. The predominance of δ-crystallin in central fibres, located along the optical axis, suggests that this protein is of embryonic origin.δ-Crystallin from fibres is electrofocused as 12 distinct molecular classes (pI 5·2–5·42) which react against anti-δ-crystallin on an immunoblot. Of these, the three most anodal species are not detected in EP. Fibres contain 50 000, 48 000 and 45 000 dalton δ-crystallin subunits while only 50 000 and 48 000 dalton subunits are present in EP.