The lens proteins in adult and embryos of the periodic albino mutant ofXenopus laevis

Summary The crystallins of normal and a^p mutants ofX. laevis have been studied using biochemical (electrophoresis in agar and polyacrylamide gels, isoelectric focusing) and immunochemical methods (immunoelectrophoresis, immunodiffusion, immunoabsorption, immunofluorescence, isoelectrofocusing with immunoidentification). The immunochemical analysis was carried out with rabbit antisera prepared against electrophoretic fractions of the mutant lens.Crystallins of adultX. laevis (a^p/a^p; ++/++) are heterogenous as judged by electrophoretic mobility, isoelectric point, antigenic and species specificity.No qualitative nor quantitative differences were found between crystallins of normal and mutant animals at the level of the protein subunits. These conclusions, however, are valid only for those crystallins, which are solubilized at pH 9.0.Immunofluorescence studies showed that crystallins appear in the normal and mutant embryos at practically the same time. No significant differences in the appearance of specific immunofluorescence between the normal and mutant embryos were found.Some of the gamma and, perhaps, beta-crystallins appear first; alpha-crystallins appear later. It has been shown for the first time that some gamma-crystallins are formed at advanced developmental stages.The periodic albino mutation does not affect the function of genes coding for crystallins either in embryos or in the adultX. laevis.     

Immunochemical markers of embryonic lens differentiation in Rana temporaria. I. Composition and properties of water-soluble lens antigens]

Antisera were obtained to the total extract and individual electrophoretic fractions of lens proteins: alpha-, beta-, gamma1- and gamma2-crystallins. The crystallins under study are immunochemically heterogenous: each class of lens proteins contains 2--4 antigens. Using the indirect method of fluorescent antibodies, it was established that the appearance of crystallins during development coincided with the onset of formation of the presumptive lens fibers. No crystallins were found in the lens placode and early lens vesicle. gamma-Crystallins appear later than the other lens proteins and are characteristic, mainly, for the lens fibers; at the advanced stages of organogenesis gamma-crystallins are regularly found in the epithelial cells of the developing lens as well.     

Embryonic Appearance of α, β, and γ Crystallins in the Periodic Albinism (ap) Mutant of Xenopus laevis

The appearance of the crystallins during lens development in the periodic albinism (a^p/a^p) mutant of Xenopus laevis has been studied. Using antibodies specific for total crystallins, α+β crystallins, and γ crystallins in the immunofluorescence technique, the first positive reaction for all could be demonstrated in the Nieuwkoop-Faber Stage 31 lens rudiment. The antibody to α+β crystallins exhibited differences in intensity from cell to cell in the early rudiment, while the reaction to the other antibodies was uniform throughout the rudiment. As lens differentiation progressed, immunofluorescence was restricted in all cases to the lens fiber area, up to and including Nieuwkoop-Faber Stage 45. The lens epithelium of the one-year-old adult a^p/a^p was positive, however, for total lens crystallins.
These results are at variance with earlier studies on lens development and the crystallins in wild-type (+/+) X. laevis , where a positive reaction for y and total crystallins could be detected earlier, and in the lens epithelium of Nieuwkoop-Faber Stage 41 embryos for total lens crystallins. That this divergence in the mutant is due to a pleiotropic effect or directly to the inductive failure of the endomesoderm to initiate melanogenesis, is discussed.     

Immunochemical markers of embryonic lens differentiation in Rana temporaria. II. Immunohistochemical analysis of the manifestation and localization of individual classes of lens proteins]

Individual lens proteins were studied during development of Rana temporaria. Antisera to alpha-, beta-crystallins of chicks and gamma-crystallins of Rana ridibunda were used as immunochemical markers. Besides the main crystallins, a new antigen was found in the R. temporaria lens tentatively called alphabeta-crystallin. It appears to be characteristic only for the amphibian lens. Using the indirect method of fluorescent antibodies, it was shown that all the antigens under study appeared in the lens of the R. temporaria tadpoles within 1--2 days (at 20 degrees). The crystallins are found initially only in the developing lens fibers and later in the lens epithelium. It was established that the lens epithelium contained gamma-crystallins which appeared somewhat earlier than alpha- and beta-crystallins, but simultaneously with alphabeta-crystallin.     

Experimental and biochemical aspects of crystalline lens induction during embryogenesis]

The lens induction is a two-step process and involves morphogenetic influences from the archencepalic endoderm and optic vesicle. One can suggest that the lens induction is primed by specific proteins which are synthesized and secreted by the optic vesicle cells. The proteins-inductors appear to penetrate in the cells and, while interacting (directly or via the cytoplasm) with the nuclei, "programme" the ectodermal cells towards the lens differentiation. The contact interactions and extracellular matrix are of substantial, but not crucial value for the lens induction. The synthesis of specific proteins (crystallins) is to be considered as the most objective criterion of lens differentiation. In vertebrates, there is a lag-period between the moment of lens induction and synthesis of crystallins which is the most long-term in amphibians. The chick embryos constitute an exception and the synthesis of crystallin mRNA occurs in them a few hours after the lens induction. The developing retina loses its capacity to induce lens but stimulates the processes of fiber formation and synthesis of crystallins. A factor was found in the definitive lens epithelium which may be considered as a possible regulator of lens differentiation. On the basis of experiments with heterogenous and native lens inductors, a suggestion is put forward to the effect that the activity of inducing substances is determined by a definite determinant group of the molecule, rather than by the whole molecule.     

Embryonic appearance of alpha, beta, and gamma crystallins in the periodic albinism (ap) mutant of Xenopus laevis.

The appearance of the crystallins during lens development in the periodic albinism (ap/ap) mutant of Xenopus laevis has been studied. Using antibodies specific for total crystallins, alpha + beta crystallins, and gamma crystallins in the immunofluorescence technique, the first positive reaction for all could be demonstrated in the Nieuwkoop-Faber Stage 31 lens rudiment. The antibody to alpha + beta crystallins exhibited differences in intensity from cell to cell in the early rudiment, while the reaction to the other antibodies was uniform throughout the rudiment. As lens differentiation progressed, immunofluorescence was restricted in all cases to the lens fiber area, up to and including Nieuwkas positive, however, for total lens crystallins. These results are at variance with earlier studies on lens development and the crystallins in wildtype (+/+) X. laevis, where a positive reaction for gamma and total crystallins could be detector total lens crystallins. That this divergence in the mutant is due to a pleiotropic effect or directly to the inductive failure of the endomesoderm to initiate melanogenesis, is discussed.     

The appearance of alpha-crystallin in relation to cell cycle phase in the embryonic mouse lens

Specific protein synthesis in the embryonic mouse lens was studied by immunofluorescence with antisera to adult mouse lens or crystallin fractions. Positive reactions were first detected in a few cells of the lens cup 18-24 hr after contact between optic vesicle and presumptive lens ectoderm had been established. During formation of the lens vesicle a rapidly increasing fraction of cells produced crystallins. At the time of detachment of the vesicle from the surface all cells of its posterior wall showed immunofluorescence. After fiber elongation became distinct cells of the anterior epithelium began to fluoresce and shortly afterwards the entire rudiment produced crystallins. The early reactions were due entirely to the presence of alpha-crystallin. Reactions were restricted to the lens. Thus, in the mouse as in other species crystallins were detectable by immunofluorescence in vivo only after lens morphogenesis was well underway and only in the lens rudiment itself. Cells first synthesizing crystallins always had an elongated shape and their nuclei were in a basal position. A few hours later mitotic cells displayed fluorescence. Taking into account earlier found relations between cell morphology and cell cycle phase, this indicates that alpha-crystallin is first demonstrable in the S-or early G-2 phase of the cell cycle, and that the start of its synthesis does not preclude continued cell replication. It is interesting that the cellular location, cell cycle phase, and developmental stage, in which crystallins first appear, are comparable in mouse and chick embryo. Yet, entirely different proteins are involved: alpha-crystallin in the first, delta-crystallin in the latter. Implications of this for our understanding of lens induction are discussed.     

Evolution of crystallins: expression of lens-specific proteins in the blind mammals mole (Talpa europaea) and mole rat (Spalax ehrenbergi)

The mole (Talpa europaea; Insectivora) and the mole rat (Spalax ehrenbergi; Rodentia) both have degenerated eyes as a convergent adaptation to subterranean life. The rudimentary eye lenses of these blind mammals no longer function in a visual process. The crystallin genes, which display a lens-specific expression pattern, were studied in these blind mammals and in related species with normal eyes by hybridizing their genomic DNAs with probes obtained from cDNA clones for alpha A-, alpha B-, and beta Bp-crystallins from calf and gamma 3- crystallin from the rat. For all crystallin genes examined, the hybridization signals of mole and mole rat genomic DNA were comparable, respectively, with those of shrew and of rat and mouse, normal-vision representatives of the orders Insectivora and Rodentia. The expression of the crystallins at the protein level was tested by using antiserum specific for alpha-crystallin in immunofluorescence reactions on lens sections of mole and mole rat eyes and by using antisera against the beta- and gamma-crystallins on sections of the mole eye. All antisera gave positive fluorescence reactions exclusively with lens tissue of these blind mammals, indicating that the crystallins are still normally expressed despite the fact that these lenses have had no function in a visual process in these mammals for at least many million years. These findings apparently imply that some unknown selective advantage has conserved the crystallin genes and their expression after the loss of normal function of the lenses.      

Homology models of human gamma-crystallins: structural study of the extensive charge network in gamma-crystallins

The lens is composed of highly stable and long-lived proteins, the crystallins which are divided into alpha-, beta-, and gamma-crystallins. Human gamma-crystallins belong to the betagamma superfamily. A large number of gamma-crystallins have been sequenced and have been found to share remarkable sequence homology with each other. Some of the gamma-crystallins from various sources have also been elucidated structurally by X-ray crystallographic or NMR spectroscopic experiments. Their three-dimensional structures are also similar having consisted of two domains each possessing two Greek key motifs. In this study we have constructed the comparative or homology models of the four major human gamma-crystallins, gammaA-,gammaB-, gammaC-, and gammaD-crystallins and studied the charge network in these crystallins. Despite an overall structural similarity between these crystallins, differences in the ion pair formation do exist which is partly due to the differences in their primary sequence and partly due to the structural orientation of the neighboring amino acids. In this study, we present an elaborate analysis of these charged interactions and their formation or loss with respect to the structural changes.     

Rat lens gamma-crystallins. Characterization of the six gene products and their spatial and temporal distribution resulting from differential synthesis

We have isolated, purified and characterized six individual gamma-crystallin polypeptides present in the rat lens. Comparison of their amino acid compositions with the known structure of the six gamma-crystallin genes permits a one-to-one correspondence to be made between each protein synthesized and the encoding gene. This demonstrates that each of the six genes is actually expressed in vivo. Two classes of three gamma-crystallins each, which we have designated classes gamma ABC and gamma DEF, are known to exist, on the basis of internal sequence homology. We have measured the temperature-dependent phase-separation characteristics of solutions of the six purified gamma-crystallins, and find that the three members of the gamma DEF class (gamma 2-2, gamma 3-1 and gamma 4-1) are all cryo-proteins with relatively high phase-separation temperatures, whereas the three gamma ABC crystallins (gamma 1-1, gamma 1-2 and gamma 2-1) do not show phase separation above -7 degrees C. We have measured the spatial distribution in rat lens of each of the alpha-, beta- and gamma-crystallins as a function of age from 1 to 420 days, using size-exclusion and ion-exchange high-pressure liquid chromatography (HPLC). Our findings in the cortical layer permit us to establish the differential synthesis of each of the crystallins during lens development. Particular attention has been devoted to the spatial and temporal distribution of the six individual gamma-crystallins. Up to birth, synthesis of the three components of the gamma DEF class predominates, and in particular that of gamma 2-2. In subsequent development the three components of the gamma ABC class assume a greater proportion of monomeric crystallins synthesized, while beta s-crystallin synthesis predominates in late development. Our analysis of different layers within single lenses provides novel information on spatial gradients of the water-soluble and water-insoluble protein fractions as a function of age. We consider the consequences of these findings for lens transparency and opacity in both rat and mouse lens. We show that the high concentrations of gamma DEF-crystallins appear to be responsible for the opacity known to occur in young rat lenses. We conclude from these observations that close control of the differential synthesis of gamma-crystallins plays an important role in maintaining lens transparency during development.     

Surface interactions of gamma-crystallins in the crystal medium in relation to their association in the eye lens

A comparative study of intermolecular interactions in crystals of two homologous low molecular weight proteins, gamma-II and gamma-IIIb crystallins, from calf eye lens was carried out. Crystal packings for these proteins are very different: intermolecular contact areas compose about 33% of the total accessible surface area of gamma-II as compared with 13% in gamma-III. Two key residues seem to be mainly responsible for the differences in protein association in the crystal medium. These are Ser 103 and Leu 155 in gamma-II, which are replaced by Met 103 and His 155 in gamma-IIb. A similar substitution of these residues is observed in different gene products of gamma-crystallins from a number of vertebrates. This is consistent with the existence of a genetically controlled mechanism for determining intermolecular association of gamma-crystallins in the native medium of the lens.     

Ontogeny and localization of the α, β, and γ crystallins in newt eye lens development

The α-, β-, and γ-crystallins, proteins characteristic for the vertebrate eye lens, have been localized in the developing lens of Notophthalmus viridescens, the eastern spotted newt. Using the immunofluorescence technique, antibodies to the α-, β-, and γ-crystallin classes were applied to tissue sections through the eye region of developing N. viridescens embryos, Harrison (external) Stages 30 to 46+. β-Crystallins were the first of the crystallins to appear in a few cells of the lens vesicle even before the lengthening of the prospective primary fiber cells. γ-Crystallins were first detectable at a slightly more advanced stage in the prospective primary fibers, and α-crystallins in a few cells of the beginning primary fiber area. The external layer/epithelium was negative for β-crystallins until late in lens morphogenesis, and α- and γ-crystallins could not be detected in these cells at any time. This, the first use in amphibia of homologous antibodies specific for the crystallin classes, makes clear that phylogenetic differences exist as to the primacy and relevance of specific crystallins to events during morphogenesis of the eye lens.     

Cell differentiation of the head ectoderm in mouse embryos in vitro

Cell differentiation has been studied in the explants of head ectoderm of 8, 9 and 10 day old mouse (CBA) embryos and of head epidermis of 13 day old embryos. Pieces of ectoderm were taken from the temporal region. It was established by indirect immunofluorescence that within 10, 15 and 20 days of cultivation spheroids with keratins or crystallins in some groups of fibres formed in the head ectoderm explants from 9 and 10 day old embryos. When cultivating the regions of head epidermis from 13 day old embryos, spheroids formed with keratin only in their cells. The data obtained suggest that there appear to be two clones of cells determined to the synthesis of keratins or crystallins in the head ectoderm of early mouse embryos. During embryogenesis, the number of cells determined to the synthesis of keratins appears to increase in the regions not related to the eye area. At the same time, the clone of cells determined to the synthesis of crystallins appears to be eliminated.     

Sequence comparison of gamma-crystallins from the reptilian and other vertebrate species

Lens crystallins were isolated from homogenates of reptilian eye lenses (Caiman crocodylus apaporiensis) by gel-permeation chromatography and characterized by gel electrophoresis, and amino acid and N-terminal sequence analyses. Four fractions corresponding to alpha-, delta/epsilon/beta-, beta- and gamma-crystallins were identified on the basis of their electrophoretic patterns as revealed by SDS gel electrophoresis. Comparison of the amino acid contents of reptilian crystallins with those of mammals suggests that each orthologous class of crystallins from the evolutionarily distant species still exhibits similarity in their amino acid compositions and probably sequence homology as well. All fractions except that of gamma-crystallin were found to be N-terminally blocked. N-terminal sequence analysis of the purified gamma-crystallin subfractions showed extensive homology between the reptilian gamma-crystallin polypeptides themselves and also those from other vertebrate species, suggesting the existence of a multigene family and their close relatedness to gamma-crystallins of other vertebrates.     

Lens crystallins and oxidation: the special case of gammaS

Among lens crystallins, gamma-crystallins are particularly sensitive to oxidation, because of their high amount of Cys and Met residues. They have the reputation to induce, upon ageing, lens structural modifications leading to opacities. A combination of small angle X-ray scattering and chromatography was used to study the oxidation of gamma-crystallins. At pH 7.0, all the gamma-crystallins under study were checked to have the same structure in solution. Under gentle oxidation conditions at pH 8.0, human gammaS (hgammaS) and bovine gammaS (bgammaS) formed disulfide-linked dimers, whereas the other bgamma-crystallins did not. Cys20 was shown to be responsible for dimer formation since the C20S mutant only formed monomers. The hgammaS dimers were stable for weeks and did not form higher oligomers. In contrast, monomeric gammaS-crystallins freshly prepared at pH 8.0, and submitted to more drastic oxidation by X-ray induced free radicals, were rapidly transformed into higher oligomers. So, only extensive oxidation causing partial unfolding could be detrimental to the lens and linked to cataract formation. The gammaS-crystallins lack the temperature-induced opacification observed with the other gamma-crystallins and known as cold cataract. The oxidation-induced associative behaviour and cold cataract are therefore demonstrated to be uncoupled.     

The appearance of α, β and γ crystallins in an anophthalmic strain of mice

Abstract. A certain percentage of congenitally anophthalmic mouse embryos have the ability to generate small lens vesicles that have previously been shown to produce alpha crystallin at 13-day gestation. Further immunohistological analysis of 13- and 15-day-gestation anophthalmia embryos indicates that beta crystallin is present in those 13-day embryos which have lens vesicles with lens-fiber formation. Also, 15-day embryos with lenses demonstrating fiber elongation can produce both beta and gamma crystallins. The conclusion is drawn that the genetic potential to produce at least three characteristic biochemical markers of normal lens differentiation is present in the anophthalmia mutant. The spatial distribution patterns of the crystallins in normal and anophthalmia embryos were similar. However, there appeared to be a transposition in the temporal appearance of beta and gamma crystallins in the anophthalmia mutant. Optic cups and associated lenses in 15-day anophthalmia specimens were much smaller than those in controls. The optic and lens rudiments in these anophthalmia embryos were fairly proportional in size, which indicates that some degree of allometric growth compensation had occurred during the course of development. This ability for differential growth compensation in the mouse eye appears to be restricted to the predifferentiative stages of eye formation.     

The synthesis and localization of crystallins in different cell compartments of the crystalline lens in adult frogs: immunoautoradiographic and immunofluorescent research

The purpose of this study was to analyze immunochemically the synthesis and distribution of tissue-specific proteins, i.e., alpha-, beta- gamma- and rho-crystallins, in morphologically distinct regions of the frog (Rana temporaria L.) lens which consist of cells at various stages of differentiation, maturation and aging. Five such cell compartments can be distinguished in the lens: (1) central zone of lens epithelium (stem/clonogenic cells); (2) equatorial epithelial cells (differentiating cells); (3) lens fibers of the outer cortex (post-mitotic differentiated cells); (4) lens fibers of the deep cortex (cells without nuclei at terminal stage of differentiation); and (5) cells of the lens "nucleus" (cells formed during embryogenesis). Intact lenses and isolated lens epithelium were cultured in vitro in the presence of 35S-methionine. Then lens epithelium, outer and deep cortex, and lens nucleus were extracted with buffered saline and extracts used for immunoautoradiography. Distribution of crystallins in paraffin sections of the whole lens or isolated lens epithelium was studied using indirect immunofluorescence. Synthesis of alpha-crystallins was observed in lens epithelium and cortex, but not in lens nucleus. According to immunohistochemistry, these proteins were absent from central part of the lens epithelium: positive fluorescence was observed only in elongating cells at its periphery and in lens fibers. The data on beta-crystallins are similar except that synthesis of these proteins (traces) was detected also in lens nucleus. Synthesis of gamma-crystallins was detected in lens cortex and nucleus (traces) but not in epithelium. Immunohistochemistry showed that these proteins are absent from all regions of lens epithelium and found only in fiber cells of cortex and nucleus. Rho-crystallin was synthesized in all cell compartments of the adult lens, and all lens cells contained this protein. Our results show that cells of central lens epithelium do not contain alpha- beta- or gamma-crystallins (or the rate of their synthesis is insignificant). While cells are moving towards lens equator and elongating, synthesis of alpha- and beta-crystallins is activated. Gamma-crystallins are synthesized later, first in young lens fibers near lens equator. During embryonic development in amphibia, in contrast, gamma- and beta-crystallins are detected at earlier stages than alpha- and rho-crystallins (Mikha?lov et al., 1988). These data suggest that different mechanisms are involved in differentiation on lens fibers from embryonic precursor cells, on one hand, and from epithelial stem cells of adult lens, on the other.     

Immunofluorescence studies of developmental changes in sea urchin eggs and embryos

By means of indirect immunofluorescence techniques, distinct sea urchin antigens were localized in eggs and embryos (Paracentrotus lividus). The specificity of the method was ascertained from controls in which the specific rabbit anti-sea-urchin sera were substituted by rabbit antiserum to an unrelated antigen (human serum albumin), by normal rabbit serum or by phosphate-buffered saline. The specificity of staining was also evaluated by comparing the different staining patterns obtained either with antisera to whole homogenates of eggs and embryos or with antisera to distinct antigens.     

Modifications of human betaA1/betaA3-crystallins include S-methylation, glutathiolation, and truncation

Disulfide bonding of lens crystallins contributes to the aggregation and insolubilization of these proteins that leads to cataract. A high concentration of reduced glutathione is believed to be key in preventing oxidation of crystallin sulfhydryls to form disulfide bonds. This protective role is decreased in aged lenses because of lower glutathione levels, especially in the nucleus. We recently found that human gamma-crystallins undergo S-methylation at exposed cysteine residues, a reaction that may prevent disulfide bonding. We report here that betaA1/A3-crystallins are also methylated at specific cysteine residues and are the most heavily methylated of the human lens crystallins. Among the methylated sites, Cys 64, Cys 99, and Cys 167 of betaA1-crystallin, methylation at Cys 99 is highest. Cys 64 and Cys 99 are also glutathiolated, even in a newborn lens. These post-translational modifications of the exposed cysteines may be important for maintaining the crystallin structure required for lens transparency. Previously unreported N-terminal truncations were also found.     

Immunofluorescent studies on aphakia,a mutation of a gene involved in the control of lens differentiation in the mouse embryo

Aphakia, an autosomal recessive single gene mutation in the mouse, seriously affects the development of the ocular lens. Up to advanced stages of lens invagination morphogenesis proceeds normally. In the late lens cup and early lens vesicle stage, however, the epithelium of the lens rudiment becomes disorganized and the lumen of the vesicle fills up with rounded cells, apparently released from the epithelium. The lens stalk persists frequently. Probably as a consequence of the aphakic state other parts of the eye secondarily become abnormal.Immunofluorescent studies were done on embryonic normal and aphakia eyes with antisera against adult mouse crystallins. In the normal embryo the first positive reactions were found in the late lens cup stage ($\text{10}\text{3}\text{4}\text{–11}$ days of gestation). By Day 12 all cells of the lens vesicle were brightly fluorescent. A day later the cells of the posterior wall, now lens fibers, had elongated sufficiently to obliterate the lumen of the vesicle. The entire organ was highly fluorescent, indicating that all of its cells contained large amounts of crystallins. The mutant lens, studied over the same time span, showed no reaction at all. The most likely explanation is, that the multiple structural genes, which normally must be involved in the production of the crystallins, are not expressed up to this time in the mutant.The combination of morphological and biochemical defects suggests that the gene involved in the mutation somehow functions in the control of lens differentiation.