Beta B2-crystallin Research Articles

Laser light-scattering evidence for an altered association of beta B1-crystallin deamidated in the connecting peptide.

Deamidation is a prevalent modification of crystallin proteins in the vertebrate lens. The effect of specific sites of deamidation on crystallin stability in vivo is not known. Using mass spectrometry, a previously unreported deamidation in beta B1-crystallin was identified at Gln146. Another deamidation was investigated at Asn157. It was determined that whole soluble beta B1 contained 13%-17% deamidation at Gln146 and Asn157. Static and quasi-elastic laser light scattering, circular dichroism, and heat aggregation studies were used to explore the structure and associative properties of recombinantly expressed wild-type (wt) beta B1 and the deamidated beta B1 mutants, Q146E and N157D. Dimer formation occurred for wt beta B1, Q146E, and N157D in a concentration-dependent manner, but only Q146E showed formation of higher ordered oligomers at the concentrations studied. Deamidation at Gln146, but not Asn157, led to an increased tendency of beta B1 to aggregate upon heating. We conclude that deamidation creates unique effects depending upon where the deamidation is introduced in the crystallin structure.

INVITED EDITORIAL The Genetics of Cataract: Our Vision Becomes Clearer

I would like to thank Drs. Joe Horwitz, Muriel Kaiser-Kupfer, Mark Kantorow, Eric Wawrousek, and Sam Zigler for a close reading of the manuscript.

Effects of controlled mutations on the N- and C-terminal extensions of chick lens beta B1 crystallin.

While gamma-crystallin exists as a monomer, beta-crystallin, which unlike gamma-crystallin contains N- and C-terminal arms, associates to form homodimers. In order to answer the question of whether the extensions are involved in dimerisation of chick lens beta B1 crystallin, we have developed a heterologous expression system for chicken beta B1 crystallin in Escherichia coli, and produced three mutations by site-directed mutagenesis. We have substituted residues in the PAPA segment of the N-terminal extension, curtailed the N-terminal extension by five residues, and deleted 16 residues from the C-terminal extension. High-resolution gel filtration chromatography and non-denaturing gel electrophoresis show that the mutations did not influence dimerisation of the beta B1 crystallin, while circular dichroism and tryptophan fluorescence indicated that the mutations did not have a major influence on beta B1 crystallin structure or its heat stability. Our experiments show that as with rat lens beta B2 crystallin, dimerisation of beta B1 crystallin is not affected by alterations to the conserved PAPA region and that the peptide linker region rather than the N- and C-terminal extensions must be important in dimerisation.

The X‐ray structure of a mutant eye lens βB2‐crystallin with truncated sequence extensions

beta-Crystallins are oligomeric eye lens proteins that are related to monomeric gamma-crystallins by domain swapping: like gamma-crystallins, they are comprised of two similar domains but they differ in having long sequence extensions. beta B2, a major component of beta-crystallin oligomers, self-associates to a homodimer in solution. In two crystal structures of native beta B2, the protein is a 222-symmetric tetramer of eight domains. It has previously been shown that a mutant of rat beta B2-crystallin, in which the bulk of the N- and C-terminal sequence extensions has been deleted, assembles into dimers and tetramers. Here we present the 3.0 A resolution X-ray structure of the tetramer, beta B2 delta NC1. The mutant tetramer has a very similar set of domain interactions to the native structure. However, the structures differ in the relative orientation of the two sets of four domains. The paired N- and C-terminal domain interface, which is at the heart of the dimer structure, is very similar to the native structure. However, the truncation of the C-terminal extension removes an important tryptophan residue, which prevents the extension from acting as a (non-covalent) linker, as it does in native beta B2. There is a knock-on structural effect that removes a contact between extension and covalent linker, and this appears to cause a small twist in the linker that is amplified into a 20 degrees rotation between sets of paired domains.

Disulfide Bond Formation of Cysteine-37 and Cysteine-66 of BetaB2 Crystallin During Cataractogenesis of the Human Lens

BetaB2 crystallin has been prepared from total protein of normal (transparent) and cataractous human lenses. After digestion with endoprotease lys-C, the crude digests were resolved on a C18reverse phase column. The lys-C digests of betaB2 crystallin from cataractous lenses consistently showed the presence of a major peptide that was not present in the lys-C digests from normal lenses. Treatment of this peptide with dithiothreitol resulted in the production of two peptides, corresponding to betaB2 crystallin sequences 18–41 and 48–67, containing cysteine-37 and cysteine-66, respectively. The results demonstrated that intramolecular disulfide bonding of these two residues had occurred in vivo. Based upon previous knowledge of the three dimensional structure of betaB2 crystallin, formation of this disulfide bond suggests that significant denaturation of the betaB2 crystallin molecule has occurred during the process of human lens opacification.

The Cooperation Between Two Silencers Creates an Enhancer Element that Controls Both the Lens‐Preferred and the Differentiation Stage‐Specific Expression of the Rat βB2‐Crystallin Gene

The rat beta B2-crystallin gene is active only during a specific stage of the differentiation of rat lens fibre cells directed by basic fibroblast growth factor. The regulatory elements that determine the transient activity of this gene are located in the -750/-123 region and in the first intron. Singly, these elements act as silencers, together they constitute an enhancer that is active only during the specific differentiation stage. An additional silencer is found between -123 and -77. The proximal promoter region contains a Pax-6 binding site at -65/-51. In vitro, binding to this site could be detected but, according to in vivo footprinting experiments, this site is not occupied in the endogenous gene. Furthermore, co-expression of Pax-6 did not enhance promoter activity. Finally, mutation or deletion of this site did not affect promoter activity: the region -37/+10 sufficed for basal promoter activity. The cooperation between the -750/ -123 region and the first intron of the beta B2-crystallin gene not only determines the differentiation stage-specific activity of the gene, but also contributes to the highly increased expression in lens cells compared with non-lens cells.

Age-dependent cleavage at the C-terminal region of lens βB2 crystallin

A previous study has demonstrated that in vivo, the peptide bonds at the C-terminal region of the alpha-A crystallins were cleaved in an age-dependent manner, between the two hydroxyl-containing amino acids in the sequence -PS(T)S (Takemoto, 1995). Bovine beta B2 crystallin also contains the sequence-PSS at its C-terminus. To determine if this specific site of cleavage occurred in other lens proteins besides alpha-A crystallin, beta B2 crystallin was prepared from total proteins of young versus adult bovine lens fiber cells. After cleavage by cyanogen bromide, the C-terminal fragments were characterized by mass spectrometry. The results showed that peptide cleavage also occurred between the two hydroxyl-containing amino acids in the sequence -PSS of beta B2 crystallin from older fiber cells, demonstrating that age-dependent cleavage of this peptide bond occurs in multiple proteins of the aging lens.

1241 Heterologous expression in E. coli and site directed mutagenesis of chick beta B1 crystallin

1241 Heterologous expression in E. coli and site directed mutagenesis of chick beta B1 crystallin

Loss of the C-terminal serine residue from bovine βB 2-crystallin

Electrospray mass spectrometric (ES-MS) examination of bovine beta-crystallins showed a significant component corresponding in mass to beta B2-crystallin less one serine residue. Tryptic digestion, followed by isolation and characterisation of the C-terminal peptide, demonstrated that this new species has arisen by the loss of the C-terminal serine residue. This phenomenon appears to be age-related since no truncation was detected in beta B2-crystallin from foetal lenses and the proportion of the truncated form, as judged by ES-MS, was lower in beta-crystallin isolated from calf lenses than that from the lenses of 3-year-old animals. This process therefore is similar to a recently reported loss of the C-terminal serine from alpha A-crystallin, which we have confirmed using ES-MS. Loss of a C-terminal serine from both crystallins may indicate the presence of carboxypeptidase-A-like activity in bovine lenses. ES-MS data provided no evidence for a significant degree of phosphorylation of beta B2-crystallin.

The role of the sequence extensions in beta-crystallin assembly.

The modular construction of the eye lens beta gamma-crystallins makes them good candidates for protein engineering to ascertain the rules of assembly of oligomers. X-ray studies have shown that although the polypeptide chains of beta B2-crystallin and gamma-crystallins fold to form similar N- and C-terminal domains, the conformation of the connecting peptides are such that the gamma-crystallins are monomers and the beta-crystallin is a dimer. Unlike gamma-crystallins, the numerous beta-crystallins have extensions of variable sequence from the globular domains. We have tested the effect of removing the N- and C-terminal extensions from rat beta B2-crystallin using a bacterial expression system. Abundant proteins were produced in Escherichia coli using the pET or pQE vectors. Full-length and truncated proteins were purified and checked for refolding using circular dichroism. Sizing of the truncated proteins using gel filtration chromatography showed that the absence of either the N- or C-terminal extension does not affect dimerization of beta B2-crystallin.

Domain interactions and connecting peptides in lens crystallins

beta B2- and gamma B-crystallin from bovine eye-lens are closely related proteins, topologically distinct mainly by virtue of the linker peptide connecting the two domains in each polypeptide chain. In homodimeric beta B2-crystallin, the extended conformation of the connecting peptide has been suggested to force the beta B2-molecule to favor intermolecular domain interactions compared with intramolecular contacts in monomeric gamma B-crystallin. From this one may postulate that the conserved interdomain contacts are essential for the overall stability of crystallins. This was clearly confirmed for gamma B-crystallin, since its isolated C-terminal domain is significantly less stable than in the context of native gamma B. Exchanging the linker peptide of gamma B- for that of beta B2-crystallin yields a monomeric protein with stability characteristics identical to gamma B-crystallin. We conclude that the domain-interface itself rather than the connecting peptide determines the mode of domain association in crystallins, as the linker in the gamma B beta-mutant is evidently twisted to a turn similar to the one in natural gamma B-crystallin.

1H-NMR spectroscopy of beta B2-crystallin from bovine eye lens. Conformation of the N- and C-terminal extensions.

1H-NMR spectroscopic studies of a 46-kDa homodimer, beta B2-crystallin, from bovine eye lens are presented. beta B2-crystallin has terminal extensions extending from globular N- and C-terminal domains that are well resolved in the NMR spectra, whereas, in the main, resonances from the bulk of the protein are not observed. Using two-dimensional NMR methods on beta B2-crystallin, its synthesised terminal extensions and a proteolysed sample of beta B2-crystallin with a portion of its C-terminus removed, it was possible to assign resonances to most of the amino acids in the terminal extensions. One-dimensional experiments at various pH values provided H-2 chemical shifts for the three terminal extension histidines from which their pKa values were measured. It is concluded that the terminal extensions appear to be of little ordered conformation, are accessible to solvent and flex freely from the main body of the protein. The results of the NMR spectroscopic studies of beta B2-crystallin are in excellent agreement with those for the X-ray crystal structure [Bax, B., Lapatto, R., Nalini, V., Driessen, H., Lindley, P. F., Mahadevan, D., Blundell, T. L. & Slingsby, C. (1990) Nature 347, 776-780]. No change in the spectrum of beta B2-crystallin was observed in the presence of calcium, suggesting that the termini are not involved in calcium binding.

1H-NMR spectroscopy of bovine lens beta-crystallin. The role of the beta B2-crystallin C-terminal extension in aggregation.

1H-NMR spectroscopic studies of bovine eye lens beta-crystallin aggregates (dimer, trimer and octomer) are presented. The NMR spectra for all three beta-crystallin aggregates are dominated by resonances from the beta B2 subunit, particularly from the N- and C-terminal extensions of this subunit. Resonances from other beta subunits, which all have terminal extensions, are, in general, absent from spectra of the beta-crystallin aggregates. Therefore, the beta B2 subunit and, in particular its terminal extensions, has enhanced flexibility compared to the other beta-crystallin subunits. Furthermore, resonances arising from the C-terminal extension of beta B2-crystallin are not present in the spectrum of the octomer, which is consistent with the C-terminal extension binding in this aggregate and hence being involved in large aggregate formation. A possible interaction between the C-terminal extension of beta B2 and the hydrophobic beta B1 subunit, which is only found in the octomer, is discussed. At higher temperatures (45 degrees C) in the octomer, partial exposure of the C-terminal extension of beta B2 occurs indicating that the octomer may be starting to break up into smaller aggregates.

Characterization of anti-crystallin autoantibodies in patients with cataract.

Anti-crystallin autoantibodies have often been demonstrated in the serum of healthy persons and, especially, patients with cataract. In no case, however, have the specific crystallin subunits been identified against which such antibodies are directed. This information would be of particular interest in view of the recent finding that several crystallin subunits occur constitutively outside the lens. To fill this gap, we analysed the sera of 15 patients with mature cataract by means of 1- and 2-dimensional immunoblotting. The circulating antibodies turned out to be directed against several beta- and gamma-crystallin subunits. The types of subunits and the intensities of the responses varied considerably between patients. No or only occasional and very weak reactions were observed against the alpha A-, alpha B- and beta B2-crystallin subunits. These are in fact the only crystallins at present known to occur outside the lens in mammals. Our findings thus indicate that anti-crystallin autoantibodies are specifically directed against those crystallins that appear to be lens-restricted, while immunological tolerance would exist for the extra-lenticularly occurring crystallins.

Role of calpain in hydrogen peroxide induced cataract.

The purpose of these experiments was to examine the relationship between oxidation cataract and proteolysis in cultured rat lens. Hydrogen peroxide cataract showed insolubilization of protein, loss of 31 kDa beta B1-crystallin polypeptide, decreases in soluble calpain, and increases in insoluble calpain. This suggested that calpain may be activated in hydrogen peroxide treated lenses, since beta B1 is a known calpain substrate, and calpain undergoes autolysis and degradation when activated. Furthermore, the cysteine protease inhibitor E64 was partially effective in preventing development of H2O2-cataract. E64 also prevented the loss of the 31 kDa beta B1-crystallin polypeptide and decreased the loss of calpain in the lens. These results suggested that development of hydrogen peroxide induced cataract in rat lenses was associated with activation of calpain.

Profile of messenger RNA decay in the emory mouse lens in cataractogenesis and in aging

The Emory mouse is presumed to be a model for studies on human senile cataracts. The cataract develops in 5-8 months after birth, and it does not appear to have an osmotic component. To date, no specific metabolic lesion has been uncovered as a probable cause to this cataract. Several studies have shown that the Emory mouse undergoes accelerated aging changes, possibly leading to development of senile-type cataracts. In this study we quantitated changes that might occur in the population of various mRNAs for proteins presumed to be essential for lens transparency, and for proteins that may contribute to development of cataracts. By Northern blot hybridization analysis we quantitated the mRNAs for: alpha A-crystallin, beta B1-crystallin, gamma-crystallin, the main lens intrinsic membrane protein, MP26, and aldose reductase; all in lenses of Emory mouse early cataract strain (EMEC), and of an age-matched cataract resistant strain (CR). These measurements were done in increments of 1 month over a 6-month period, then at both 9 and 12 months. The results show that all of these mRNAs decrease with age and with development of cataracts; although in some cases the initial concentrations at 1 month appear to be lower in the EMEC than in the CR strain. The most dramatic change occurred with the MP26 mRNA. In the CR strain, MP26 mRNA maintained its high concentration for a period of about 6 months before it began its decline to the 12 month level (about 25% of the 1 month level).(ABSTRACT TRUNCATED AT 250 WORDS)

Lens fiber cell differentiation and expression of crystallins in Co-cultures of human fetal lens epithelial cells and fibroblasts

Growth of the ocular lens is directed by the division and differentiation of a single layer of epithelial cells located at the equatorial region. It is conceivable that this region of the lens capsule presents a special microenvironment modulated by molecular cues emanating from the surrounding tissues. In an effort to investigate the source and nature of these molecular cues, we co-cultured human fetal lens epithelial cells and fibroblasts derived from the ciliary body. We observed morphological differentiation as evidenced by the appearance of differentiating lentoid structures associated with fibroblasts. Characterization of the expression of lens-specific proteins revealed that in addition to alpha B-crystallin, these lentoid structures contain the lens fiber cell-specific proteins, alpha A-crystallin, beta B2-crystallin and gamma S-crystallin. None of these crystallins could be found in the surrounding undifferentiated lens epithelial cells. Interestingly, alpha B-crystallin usually present in lens epithelial cells when cultured alone, was found to be markedly decreased, both in synthesis and content in the cells surrounding the differentiated structures, suggesting that the process of differentiation in vitro may concomitantly produce a factor(s) which modulates alpha B-crystallin expression in these cells.

Deletion mutation in an eye lens beta-crystallin. An animal model for inherited cataracts.

The most prevalent proteins in the lens of the eye are called crystallins, and it is thought that aberrant crystallins may cause opacification of lens tissue. The Philly mouse, a strain with an inherited cataract, has an abnormal beta B2-crystallin, the principal beta-crystallin in the mouse. The cDNA that codes for the beta B2-crystallin protein has been cloned and sequenced from both the normal and the cataractous Philly mouse. The normal mouse beta B2 cDNA is 756 nucleotides in length with 618 nucleotides of open reading frame. An in-frame deletion of 12 nucleotides has occurred in the Philly mouse cDNA, which results in the loss of 4 amino acids. The sequence of the mutant beta B2 was analyzed against the reported structure of the normal bovine beta B2-crystallin determined by x-ray crystallography. The region, in which the deletion of the amino acids occurs near the COOH terminus, is essential for the formation of the tertiary structure of the beta B2-crystallin. The loss of these residues could explain the alterations that are seen with the Philly beta B2 protein and may account for the instability of the Philly beta B2 protein. This abnormal beta B2-crystallin may be the cause of the cataract in this animal.

Chicken βB1-Crystallin Gene Expression: Presence of Conserved Functional Polyomavirus Enhancer-Like and Octamer Binding-Like Promoter Elements Found in Non-Lens Genes

Expression of the chicken beta B1-crystallin gene was examined. Northern (RNA) blot and primer extension analyses showed that while abundant in the lens, the beta B1 mRNA is absent from the liver, brain, heart, skeletal muscle, and fibroblasts of the chicken embryo, suggesting lens specificity. Promoter fragments ranging from 434 to 126 bp of 5'-flanking sequence (plus 30 bp of exon 1) of the beta B1 gene fused to the bacterial chloramphenicol acetyltransferase gene functioned much more efficiently in transfected embryonic chicken lens epithelial cells than in transfected primary muscle fibroblasts or HeLa cells. Transient expression of recombinant plasmids in cultured lens cells, DNase I footprinting, in vitro transcription in a HeLa cell extract, and gel mobility shift assays were used to identify putative functional promoter elements of the beta B1-crystallin gene. Sequence analysis revealed a number of potential regulatory elements between positions -126 and -53 of the beta B1 promoter, including two Sp1 sites, two octamer binding sequence-like sites (OL-1 and OL-2), and two polyomavirus enhancer-like sites (PL-1 and PL-2). Deletion and site-specific mutation experiments established the functional importance of PL-1 (-116 to -102), PL-2 (-90 to -76), and OL-2 (-75 to -68). DNase I footprinting using a lens or a HeLa cell nuclear extract and gel mobility shifts using a lens nuclear extract indicated the presence of putative lens transcription factors binding to these DNA sequences. Competition experiments provided evidence that PL-1 and PL-2 recognize the same or very similar factors, while OL-2 recognizes a different factor. Our data suggest that the same or closely related transcription factors found in many tissues are used for expression of the chicken beta B1-crystallin gene in the lens.

Chicken beta B1-crystallin gene expression: presence of conserved functional polyomavirus enhancer-like and octamer binding-like promoter elements found in non-lens genes.

Expression of the chicken beta B1-crystallin gene was examined. Northern (RNA) blot and primer extension analyses showed that while abundant in the lens, the beta B1 mRNA is absent from the liver, brain, heart, skeletal muscle, and fibroblasts of the chicken embryo, suggesting lens specificity. Promoter fragments ranging from 434 to 126 bp of 5'-flanking sequence (plus 30 bp of exon 1) of the beta B1 gene fused to the bacterial chloramphenicol acetyltransferase gene functioned much more efficiently in transfected embryonic chicken lens epithelial cells than in transfected primary muscle fibroblasts or HeLa cells. Transient expression of recombinant plasmids in cultured lens cells, DNase I footprinting, in vitro transcription in a HeLa cell extract, and gel mobility shift assays were used to identify putative functional promoter elements of the beta B1-crystallin gene. Sequence analysis revealed a number of potential regulatory elements between positions -126 and -53 of the beta B1 promoter, including two Sp1 sites, two octamer binding sequence-like sites (OL-1 and OL-2), and two polyomavirus enhancer-like sites (PL-1 and PL-2). Deletion and site-specific mutation experiments established the functional importance of PL-1 (-116 to -102), PL-2 (-90 to -76), and OL-2 (-75 to -68). DNase I footprinting using a lens or a HeLa cell nuclear extract and gel mobility shifts using a lens nuclear extract indicated the presence of putative lens transcription factors binding to these DNA sequences. Competition experiments provided evidence that PL-1 and PL-2 recognize the same or very similar factors, while OL-2 recognizes a different factor. Our data suggest that the same or closely related transcription factors found in many tissues are used for expression of the chicken beta B1-crystallin gene in the lens.