Further studies on the polypeptide chains of beta-crystallin.
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Amino Acid Sequence↗
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Lens transglutaminase selects specific beta-crystallin sequences as substrate.
A Ca2+-dependent transglutaminase (EC 2.3.2.13) has been demonstrated in the eye lenses of several mammalian species [Lorand, L., Hsu, L. K. M., Siefring, G. E., Jr., & Rafferty, N. S. (1981) Proc. Natl. Acad. Sci. USA 78, 1356-1360]. Using [3H]methylamine as a convenient probe for transglutaminase activity, we have explored the action of this enzyme in the bovine eye lens. We could characterize the glutamine residues acting as acyl-donor sites in three beta-crystallin chains, which are the only substrates for lens transglutaminase among the various lens-specific structural proteins, the crystallins. A single glutamine was found to bind [3H]methylamine in each of these three chains: glutamine -9 in beta Bp (beta B2), glutamine -21 in beta B3, and glutamine -23 or -24 in beta A3. The four glutamines are all located in the NH2-terminal regions, which presumably extend from the compact two-domain structure of the beta-crystallin chains. It was, moreover, established that several components of the lens cytoskeleton are substrates for transglutaminase.
Acyltransferases↗
beta-Crystallin: endogenous substrate of lens transglutaminase. Characterization of the acyl-donor site in the beta Bp chain.
Incubation of calf lens cortex homogenate with [14C]putrescine or dansylcadaverine, followed by two-dimensional gel electrophoresis and fluorography, enabled the identification of three different beta-crystallin chains as the endogenous substrates of Ca2+-dependent lens transglutaminase (R-glutaminyl-peptide:amine-gamma-glutamylyltransferase, EC 2.3.2.13). One of these is beta Bp, the predominant subunit of beta-crystallin, of which the amino acid sequence is known. The site of amine-labeling in beta Bp could be located, by limited proteolysis, in the N-terminal domain of this chain. Tryptic digestion of the N-terminal domain and subdigestion with elastase of the N-terminal tryptic peptide identified glutamine-7 as the single residue to which the amines are bound. This is the first example of an endogenous substrate of intracellular transglutaminase in which the site of the acyl-donor glutamine residue has been established. Tryptic digestion of the putrescine-labeled beta-crystallin aggregate, followed by high-voltage paper electrophoresis, provided a preliminary characterization of the labeled peptides originating from the other two labeled beta subunits.
Acylation↗
Eye lens regeneration and the crystallins in the adult newt, Notophthalmus viridescens.
Upon lens removal, the adult Eastern Spotted newt, Notophthalmus viridescens, has the capacity to regenerate an ocular lens. Crystallins, proteins characteristic of the vertebrate lens, were studied from normal and 3-month regenerated adult newt lenses. When separated by high-performance liquid chromatography (HPLC) or Sephadex G-200SF column chromatography, the crystallins from normal and regenerated lenses were fractionated into what appear to be the classical four groups: alpha, beta High, beta Low, and gamma. Upon further examination by immunoelectrophoresis, the first peak contains both alpha and beta crystallins. This study provides evidence that most of the crystallins from the regenerated lenses share biochemical properties with those of the normal lens crystallins based on their native molecular weight, isoelectric point, and the molecular wt of their constituent polypeptides, indicating that the fidelity of gene expression in reactivated iris tissue is high. Some differences are found between normal and regenerated lens crystallins and are most obvious in the beta-crystallin region: the proportion of beta crystallins is decreased in regenerated lenses when the total proteins are fractionated by column chromatography and some of the beta-crystallin polypeptide chains found in normal lenses are missing from regenerated lenses. Iris epithelial cells are normally withdrawn from the cell cycle and are synthesizing a tissue-specific product, melanin. After lentectomy these cells dedifferentiate, redifferentiate into lens cells, and their progeny then synthesize different tissue-specific proteins, crystallins. Little is known about the specific mechanism(s) for the activation of gene expression in eukaryotes, but the regenerating lens suggests itself as a good model in which to study this biological problem.
Animals↗
Evolutionary and functional relationships between the basic and acidic beta-crystallins.
beta-Crystallins are complex oligomers composed of many related subunits. In order to understand their interactions we have built molecular models of several bovine beta-crystallins, based on their sequence similarity to the well-defined gamma-II crystallin structure, using interactive computer graphics techniques. Their common origin with gamma-crystallin is displayed in both the retention of four-fold sequence repeats of critical residues involved with stabilizing a folded beta-hairpin and the conservation of core-filling hydrophobic side-chains. The beta-crystallins have been built as bilobal molecules with each domain composed of two 'Greek key' motifs which associate about an approximate two-fold axis to form beta-sheets. The beta-crystallin sequences have previously been shown to comprise two families, the basic and acidic subunits, which have extensions of sequence. The three-dimensional models show how the two families appear to stabilize the folded beta-hairpin in the N- and C-terminal domains in ways which suggest that they have diverged from a common ancestor in different ways. Acidic beta-crystallins, like gamma-crystallins, have a regular array of charges on their N-terminal domain which has been interrupted in basic beta-crystallins by hydrophobic residues which may be related to the presence of a C-terminal extension. beta-Crystallins are more highly charged than gamma-crystallins although their charge density is higher in certain regions of the N-terminal domain, particularly in beta B1-crystallin. beta-crystallins also differ from gamma-crystallins in the virtual absence of core-filling sulphydryl groups whereas they have numerous sulphur-containing side-chains together with tryptophan and histidine rings protruding from the globular domains, particularly in the acidic subunits. The burial of these residues in subunit contacts is consistent with their spectroscopic and electrostatic properties. Protein subunit aggregation commonly occurs through hydrophobic interaction or beta-sheet extension. Analysis of the subunit surfaces has identified an N-terminal hydrophobic region common to beta B1 and beta B2 whereas a C-terminal hydrophobic loop region is common to beta B1 and beta A1 and may be correlated with their association properties. It is suggested that the polar C-terminal domain of beta B2 contributes towards the solubility of higher aggregates by interactions involving beta-sheet structure.
Amino Acid Sequence↗
Lens differentiation. Crystallin synthesis in isolated epithelia from calf lenses.
In the calf eye lens, four morphologically distinct cell types can be detected: three in the epithelial monolayer and one in the cortical part. During differentiation, there is a quantitative change in the synthesis of crystallin subunits. A marked increase in alphaA-chains and several beta-crystallin polypeptides accompanies the transition from epithelial to fiberlike lens cells while synthesis of the non-crystallin proteins diminishes significantly.
Animals↗
Preferential conservation of the globular domains of the beta A3/A1-crystallin polypeptide of the chicken eye lens.
The primary structure of the beta 19/26-crystallin polypeptide of the chicken lens has been determined by cDNA sequencing and primer extension experiments. In addition, a primer extension experiment has corrected the sequence for the N-terminal arm of the murine beta 23 polypeptide, which is the homologue of the chicken beta 19/26 polypeptide. We also show that, in the chicken and mouse, the N-terminal arm of the polypeptide is encoded on two separate exons. For simplicity, we have changed the names of both chicken beta 19/26 and murine beta 23 to beta A3/A1, which is the name of the homologous bovine polypeptide. The deduced sequence of the chicken beta A3/A1 polypeptide fits the predicted three-dimensional structure involving two homologous domains, each folded into two 'Greek key' motifs, common to the beta gamma-crystallin superfamily of proteins. Comparison of the amino acid sequence of the chicken and mammalian beta A3/A1 polypeptides indicates that different regions of the protein, which are encoded on different exons, are diverging at different rates. The N-terminal extension is the fastest evolving region of the beta A3/A1 polypeptide. Hybrid-selected translation coupled with primer extension experiments suggest that a single chicken beta A3/A1 mRNA synthesizes two polypeptides, beta A3 (25 kDa) and beta A1 (23 kDa) by utilization of different translation initiation sites.
Amino Acid Sequence↗
Structure and lens expression of the gene encoding chicken beta A3/A1-crystallin.
The beta A1- and beta A3-crystallins are major polypeptides in the lenses of vertebrates. We present evidence that a single beta A3/A1 gene encodes these two proteins in the chicken. The beta A3/A1 gene has been sequenced and its functional promoter identified in transfection experiments. The chicken beta A3/A1 gene has the same structure as the human orthologue: six exons with standard splice sites and two alternative start codons from which the protein products are apparently translated. Northern analysis revealed an abundant 0.9-kb transcript in the lenses of 1-2-day-old chickens and no detectable transcripts in the rest of the eye, brain, heart, kidney, liver or skeletal muscle. The 5'-flanking sequence of the chicken beta A3/A1 gene is very similar to that of the human and mouse genes, suggesting conservation of important putative regulatory sequences in addition to the TATA box. A thymidine-rich element (bp -218 to -163) and a potential AP-1-binding site (bp -264 to -258) are present within the chicken 5'-flanking region. A DNA fragment from -382 to +22 of the chicken beta A3/A1 gene is sufficient to promote expression of the bacterial cat gene in transfected chicken primary lens epithelial cells, but not in transfected dermal fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Messenger RNA for cataractous lens proteins are also present on normal lens polyribosomes.
We have previously described an experimental model in vivo where cataract is induced by injection of the antimitotic bleomycin in the newborn rat. The first opacity of the lens appears 12--15 days after the injection of the drug concomitantly with a group of precise biochemical modifications among the soluble crystallins. These modifications are mainly the accumulation of two additional low-molecular-weight beta crystallin subunits (beta L subunits) and of several smaller alpha-crystallin polypeptides. Messenger RNA isolated from normal and cataractous lenses was assayed for translation in a cell-free wheat germ extract. Analysis of the translation products by one-dimensional and two-dimensional gel electrophoresis indicated that the messenger RNAs coding for the two beta L subunits are also present on normal lens polyribosomes. Purification and subsequent analysis by peptide mapping of the cataractous Beta L subunits suggest that they are precursor polypeptides of the normal low-molecular-weight beta-crystallins, which are no longer processed after translation and therefore accumulate in the pathological lens cell. On the other hand, in the translation of the cataractous mRNA in vitro, only the normal alpha chains are detected. These cataractous alpha-crystallins are therefore post-translational degradation products of the normal alpha polypeptides. This phenomenon seems similar to the one observed in the senescent lens. The possible involvement of these modifications in the etiology of this experimental cataract is discussed further.
Animals↗
Studies on beta-crystallin from primate lens.
The major beta-crystallin fractions from the human lens and the lenses of other selected primates have been isolated and partially characterized. Primate beta-crystallins, like those of most other mammals, consist of two heterogenous protein fractions (betaH and beta L) of quite different molecular size. Most of the polypeptide chains comprising the betaH and beta L heteropolymers are common to both fractions. Evidence is presented suggesting that primate betaH-crystallin may be smaller than betaH from other vertebrate species. Additionally, human betaH is found to contain a major component on sodium dodecyl sulfate (SDS) electrophoresis which is much larger (about 60,000 daltons) than other beta-crystallin polypeptides. Immunochemical evidence inidcates that some components of primate beta-crystallin have evolved rapidly, although at least one antigenic component is very conservative and gives a reaction of identity with all other vertebrate beta-crystallins studied.
Amino Acids↗
Phosphorylation of beta-crystallin B2 (beta Bp) in the bovine lens.
Three major 32P-labeled polypeptides were found in the soluble fraction of bovine lenses cultured in a medium containing [32P]orthophosphate. Two of the polypeptides corresponded to the phosphorylated A and B chains of alpha-crystallin. In this communication, the third polypeptide is now identified. This polypeptide is characterized by a molecular weight of 27,000 and a pI of 6.6, eluted exclusively in the beta Low fraction of a CL-6B gel filtration separation of lens soluble material, and could be further purified by DE52 anion exchange chromatography. The only 32P-labeled amino acid detected was phosphoserine. A single 32P-labeled peptide was observed after tryptic digestion and two-dimensional mapping. The amino acid sequence of the purified peptide is Gly-Ala-Phe-His-Pro-Ser-Ser. This sequence exactly matches the expected C-terminal tryptic fragment, residues 198-204, of the bovine beta-crystallin B2. The results of carboxypeptidase A digestion of the 32P-labeled peptide suggest that only Ser203 is phosphorylated. By using the catalytic subunit of the cAMP-dependent protein kinase, purified beta B2 was phosphorylated in vitro, generating a single 32P-labeled polypeptide with the identical pI as the phosphorylated polypeptide obtained from lens culture. On the basis of these data, the Mr 27,000 32P-labeled polypeptide is identified as the phosphorylated form of the beta-crystallin B2.
Animals↗
Lens crystallin changes associated with amphibian metamorphosis: involvement of a beta-crystallin polypeptide.
Lens crystallins isolated from the tadpole and frog lenses were compared with regard to the developmental changes of crystallin compositions. The major changes during the process of metamorphosis were (1) the total contents of alpha- and gamma-crystallins decrease from more than 70% to less than 60% and (2) one of the major beta-crystallin polypeptides increases from less than 1% to about 6% and (3) an amphibian-specific rho-crystallin also increases from about 6% to more than 10% of total soluble proteins of the lens. We have characterized the metamorphosis-dependent beta-crystallin polypeptide by peptide mapping and sequence determination of the protease-digested fragments. This polypeptide showed very high sequence homology to that of the major beta Bp-crystallin chain reported for the mammalian lenses. The changes of the relative abundance of various crystallins and the gradually-elevated levels of the expression of this beta Bp-like crystallin in the developing lens during metamorphosis may also have some bearing on the maintenance of lens stability in the adult frog lenses.
Amino Acid Sequence↗
Beta B1 crystallin is an amine-donor substrate for tissue transglutaminase.
The effects of tissue transglutaminase on the water-soluble proteins in bovine lens homogenates are described. Addition of liver transglutaminase and Ca2+ to calf lens homogenates resulted not only in the appearance of 50- and 57-kDa dimers, but also in a decrease in the amount of beta B1 crystallin and the almost complete disappearance of beta B3 and beta A3. This is not the result of Ca2+-induced proteolysis, since histamine completely inhibits this phenomenon. It may be concluded that these polypeptides are involved in beta-crystallin crosslinking by transglutaminase. This notion was confirmed by using beta B1- and beta Bp-specific antisera. Both sera reacted with the 57-kDa dimer; the beta Bp-specific antiserum also reacted with the 50-kDa dimer. No reaction in the region 50-57 kDa was detectable when EDTA was used instead of Ca2+. Using reconstituted mixtures of beta B1- and beta Bp-crystallin chains, and N-terminally truncated derivatives thereof, it was shown that in the beta B1/beta Bp dimer, glutamine residue -9 of beta Bp crosslinks to one of the lysine residues in the N-terminal extension of beta B1.
Animals↗
Differential effects of galactose-induced cataractogenesis on the soluble crystallins of rat lens.
Soluble lens crystallins from 6-10-week-old, galactose-fed, male Sprague-Dawley rats were analyzed by two-dimensional polyacrylamide gel electrophoresis at each of the five Sippel stages of cataractogenesis. Electrophoretograms were compared with similarly analyzed crystallins from comparably aged, chow-fed controls. Polypeptides were assigned to crystallin families and subfamilies on the basis of chromatographic fractionations with Sephadex G-200, superfine. Staining intensities of polypeptides from control lenses remained essentially unchanged throughout the experimental period, while those of the polypeptides from cataractous lenses showed non-uniform changes. Staining of the genomic gamma-crystallins increases up to at least stage 3; by stage 4, staining of gamma-chains, with perhaps those of gamma 5 and gamma 6 excepted, diminishes and in the total cataract, staining of all chains is further reduced. With possibly the addition of one chain, the total number of postsynthetically modified gamma-crystallins in cataractous lenses does not exceed that in the comparably aged normal lens. The genomic alpha- and beta-crystallin polypeptides are sustained close to normal levels up to stages 3 or 2, respectively, after which their gradually falling levels are accompanied by the generation of new species or elevated levels of existing post-translational species. An exception to this behavior is the rapid and total loss of beta B1a, a genomic subunit implicated in the aggregation of beta H-crystallins. Charge heterogeneity and variable pI displayed by beta B1a and other highly cationic beta- and gamma-crystallin polypeptides can be induced during isoelectric focusing and may be due to thiol group oxidation.
Animals↗
Biochemical characterization of crystallins from pigeon lenses: structural and sequence analysis of pigeon delta-crystallin.
Crystallins from pigeon eye lenses were isolated and purified by gel-permeation chromatography and characterized by gel electrophoresis, amino-acid composition and sequence analysis. Alpha- and beta-crystallins could be obtained in relatively pure forms by single-step size-exclusion chromatography whereas an extra step of ion-exchange chromatography was needed for the separation of delta-crystallin from the beta-crystallin fraction. In contrast to most characterized vertebrate species, a large amount of glycogen is eluted as a high molecular form in the first peak of the gel filtration column. Pigeon delta-crystallin, similar to duck and reptilian delta-crystallins, exists as a tetrameric structure of about 200 kDa in the native form and is composed of one major subunit of 50 kDa with heterogeneous isoelectric points spreading in a range of 4.7 to 6.8. In contrast to those obtained from duck, goose and caiman, delta-crystallin isolated from the pigeon lens possessed very little argininosuccinate lyase activity. However, pigeon delta-crystallin can still cross-react with the antibody against enzymically active duck delta-crystallin as revealed by the sensitive immunoblotting technique. It was also shown that the delta-crystallin content of the total pigeon soluble proteins decreased with the age of the animal. Structural analysis of purified delta-crystallin fraction was made with respect to its amino-acid composition and protein primary sequence. N-terminal sequence analysis indicated the presence of blocked amino-termini in all crystallin fractions of pigeon lenses. Therefore, a sequence analysis of PCR (polymerase chain reaction) amplified delta-crystallin cDNA was employed to deduce the protein sequence of this crystallin. Structural comparison of delta-crystallin sequences from pigeon, chicken and duck lenses casts some doubts on the recent claim that His-89-->Gln mutation in the chicken delta-crystallin may account for the loss of argininosuccinate lyase activity in this avian species, as compared to high enzymic activity in the duck crystallin (Barbosa et al. (1991) J. Biol. Chem. 266, 5286-5290).
Amino Acid Sequence↗
Isolation and characterization of cDNAs encoding beta A2- and beta A4-crystallins: heterologous interactions in the predicted beta A4-beta B2 heterodimer.
Except for the two acidic chains, beta A2 and beta A4, the primary structures of all bovine beta-crystallins have previously been elucidated, either by direct protein sequencing or prediction from cDNA sequencing. Both beta A2 and beta A4 were found to be synthesized in half-year-old calf lenses and are therefore likely to be present in a cDNA bovine library constructed from mRNA isolated from lenses of that age. A large number of cDNA clones was screened with all available crystallin, actin, vimentin and lens membrane protein MP26 probes and finally with a randomly primed mRNA probe. Clones positive for the latter, but negative for known lens proteins, were isolated and sequenced. beta A2, comprising 197 aa, and beta A4, comprising 209 aa, were identified. Both proteins have a conserved two-domain structure and an N-terminal extension which is variable. A three-dimensional model of the structure of beta A4 was made based on the coordinates of one subunit from the beta B2 dimer which has recently been solved using x-ray diffraction techniques. The resulting heterodimer structure, together with the compiled bovine beta-crystallin sequences, was used to indicate those regions of the sequences which distinguish acidic from basic beta-crystallins with a view to defining structural features necessary for subunit recognition in beta-crystallin aggregates. With the aid of the present data, the complete evolutionary tree of the bovine beta-crystallin family has been constructed, which confirms the early separation of the genes encoding the three acidic and the three basic beta-crystallins.
Amino Acid Sequence↗
Interactions of lens proteins. Concentration dependence of beta-crystallin aggregation.
The concentration-dependence of beta-crystallin aggregation was studied by both high- and low-pressure size exclusion chromatography of calf lens cortical extract (0.5-249 mg ml-1), nuclear extract (0.2-304 mg ml-1) and purified beta-crystallins (0.4-52 mg ml-1). A reversible equilibrium exists between beta H(igh)-crystallins (predominantly hexamers) and a portion of the beta L(ow)-crystallins (predominantly dimers). Association to beta H-crystallin is more extensive in the nucleus than in the cortex. Moreover, at physiological protein concentrations, the weight percentage of beta H-crystallins is greater than that of beta L-crystallins, in both the cortex and the nucleus. beta H-Crystallins can be fully dissociated to beta L-crystallin at low protein concentration. On the other hand, not all of the beta L species are competent to associate to beta H at high concentrations. This association appears to be directly dependent on the presence of beta B1 chains. We therefore propose that the concentration and spatial distribution of beta H-crystallin in vivo is actually regulated by differential synthesis of beta B1 polypeptides.
Animals↗
Covalent changes at the N- and C-terminal regions of gamma crystallin during aging of the normal human lens.
Polyclonal antisera have been made to synthetic peptides corresponding to the N- and C-terminal regions of the putative gamma 1-2 gene from human lens. These antisera are specific for gamma crystallin, showing no cross-reactivity with any polypeptides of the alpha- and beta-crystallin fractions. Western blot analysis demonstrates a dramatic decrease in the binding of these antisera to gamma crystallin during aging of the normal human lens, while identical analysis with polyclonal antisera to the major cyanogen bromide fragment (19,000 MW) of human gamma crystallin shows little, if any, change during aging. Together, these demonstrate that antisera to synthetic peptides of the N- and C-terminal regions of the gamma 1-2 gene are very specific probes that can demonstrate extensive covalent modification from both ends of the gamma crystallin molecule during aging of the normal human lens.
Adolescent↗