Eye Lens Proteins Research Articles

Functional Elements in Molecular Chaperone α-Crystallin: Identification of Binding Sites in αB-Crystallin

alpha-Crystallin, the predominant eye lens protein with sequence homology to small heat shock proteins, acts like a molecular chaperone by suppressing the aggregation of damaged crystallins and proteins. To gain an insight into the amino acid sequences in alpha-crystallin involved in chaperone-like function, we used a cleavable, fluorescent, photoactive, crosslinking agent, sulfosuccinimidyl-2 (7-azido-4-methylcoumarin-3-acetamido)-ethyl-1,3' dithiopropionate (SAED), to derivatize yeast alcohol dehydrogenase (ADH) and allowed it to complex with bovine alpha-crystallin at 48 degrees C. The complex was photolyzed and reduced with DTT and the subunits of alpha-crystallin, alpha A- and alpha B-, were separated. Fluorescence analysis showed that both alpha A- and alpha B-crystallins interacted with ADH during chaperone-like function. Tryptic digestion, amino acid sequencing, and mass spectral analysis of alpha B-crystallin revealed that APSWIDTGLSEMR (57-69) and VLGDVIEVHGKHEER (93-107) sequences were involved in binding with ADH.

Alpha-fetoprotein as a biologic response modifier: relevance to domain and subdomain structure.

In the present review, the structure of alpha-fetoprotein (AFP) is discussed in consideration of AFP membership and position in the albuminoid supergene family in relation to other gene family members. Ontogenetic AFP gene expression is then discussed in view of AFP mRNA presence in various tissues at different times during development. The multiple molecular forms of AFP is also presented in relation to published reports of AFP binding proteins and cell surface receptors. The review proceeds on to present AFP as a potential model of a modular/cassette protein based on sequence comparison with cleaved fragments of prohormones and biological response modifiers. Such cleaved fragments could potentially serve as peptide messengers for vascular, neuroendocrine, and digestive biological activities. Following a discussion on fibrin binding and serine proteases, AFP-cytoskeletal, extracellular matrix, and cellular adhesion interactions are considered. AFP as a carrier/transport protein based on structural relationships is further elucidated by examination of the various ligands bound to AFP and its hormone interaction. Since AFP binds heavy metals, the question is posed of whether AFP could function as an antioxidant. An analysis of transcription factors, tumor suppressors, and homeodomain proteins follows, which is interfaced with the concept of programmed cell death in light of amino acid sequence matches detected on the AFP molecule. Emphasis was naturally placed upon the homeodomain protein sequence stretches since AFP is a fetal, phase-specific protein found throughout embryogenesis, histogenesis, and organogenesis. In keeping with histogenesis, a discussion of AFP and eye lens protein development is presented. Finally, AFP sequence analysis presented in light of members of the immunoglobulin superfamily, autoimmune disorders, and various disease states culminates the review. A closing discussion then summarizes regions of presumptive matched protein identities on each of AFP's three domains.

Steroidal hormones and uteroplacental cytokines

Age related cataract is a major cause of visual loss worldwide that is a result of opacification of the eye lens proteins. One of the major reasons behind this deterioration is UV induced oxidative damage. The study reported here is focused on an investigation of the oxidative stress induced damage to γB-crystallin under UV exposure. Human γB-crystallin has been expressed and purified from E. coli. We have found that epicatechin gallate (ECG) has a higher affinity towards the protein compared to epigallocatechin (EGC). The in vitro study of UV irradiation under oxidative damage to the protein in the presence of increasing concentrations of GTPs is indicative of their effective role as potent inhibitors of oxidative damage. Docking analyses show that the GTPs bind to the cleft between the domains of human γB-crystallin that may be associated with the protection of the protein from oxidative damage.

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.

Mutational analysis of hydrophobic domain interactions in gamma B-crystallin from bovine eye lens.

gamma B-crystallin is a monomeric member of the beta gamma-superfamily of vertebrate eye lens proteins. It consists of two similar domains with all-beta Greek key topology associating about an approximate two-fold axis. At pH 2, with urea as the denaturant, the domains show independent equilibrium unfolding transitions, suggesting different intrinsic stabilities. Denaturation experiments using recombinant one- or two-domain proteins showed that the N-terminal domain on its own exhibits unaltered intrinsic stability but contributes significantly to the stability of its C-terminal partner. It has been suggested that docking of the domains is determined by a hydrophobic interface that includes phenylalanine at position 56 of the N-terminal domain. In order to test this hypothesis, F56 was substituted by site-directed mutagenesis in both complete gamma B-crystallin and its isolated N-terminal domain. All mutations destabilize the N-terminal domain to about the same extent but affect the C-terminal domain in a different way. Replacement by the small alanine side chain or the charged aspartic acid residue results in a significant destabilization of the C-terminal domain, whereas the more bulky tryptophan residue causes only a moderate decrease in stability. In the mutants F56A and F56D, equilibrium unfolding transitions obtained by circular dichroism and intrinsic fluorescence differ, suggesting a more complex denaturation behavior than the one observed for gamma B wild type. These results confirm how mutations in one crystallin domain can affect the stability of another when they occur at the interface. The results strongly suggest that size, hydrophobicity, and optimal packing of amino acids involved in these interactions are critical for the stability of gamma B-crystallin.

α-Crystallin Sequences Support a Galliform/Anseriform Clade

An unresolved issue in higher avian systematics is the position of the fowl-like and the duck-like birds, Galliformes and Anseriformes, respectively. Most studies place these orders at the base of the neognath radiation. While DNA hybridization data support a sister-group relationship of Galliformes and Anseriformes, macromolecular sequence analyses have not yet been able to provide a clear-cut answer. In this study, we present nucleotide sequences coding for the eye lens proteins alpha A- and alpha B-crystallin of a palaeognath, a galliform, an anseriform, and two other neognathous birds. Phylogenetic analyses of this data set clearly support a galliform/anseriform clade, to the exclusion of other neognaths.

Targeted disruption of the mouse alpha A-crystallin gene induces cataract and cytoplasmic inclusion bodies containing the small heat shock protein alpha B-crystallin.

alpha A-crystallin (alpha A) and alpha B-crystallin (alpha B) are among the predominant proteins of the vertebrate eye lens. In vitro, the alpha-crystallins, which are isolated together as a high molecular mass aggregate, exhibit a number of properties, the most interesting of which is their ability to function as molecular chaperones for other proteins. Here we begin to examine the in vivo functions of alpha-crystallin by generating mice with a targeted disruption of the alpha A gene. Mice that are homozygous for the disrupted allele produce no detectable alpha A in their lenses, based on protein gel electrophoresis and immunoblot analysis. Initially, the alpha A-deficient lenses appear structurally normal, but they are smaller than the lenses of wild-type littermates. alpha A-/- lenses develop an opacification that starts in the nucleus and progresses to a general opacification with age. Light and transmission electron microscopy reveal the presence of dense inclusion bodies in the central lens fiber cells. The inclusions react strongly with antibodies to alpha B but not significantly with antibodies to beta- or gamma-crystallins. In addition, immunoblot analyses demonstrate that a significant portion of the alpha B in alpha A-/- lenses shifts into the insoluble fraction. These studies suggest that alpha A is essential for maintaining lens transparency, possibly by ensuring that alpha B or proteins closely associated with this small heat shock protein remain soluble.

SS3D-P2: a three dimensional substructure search program for protein motifs based on secondary structure elements.

This paper discusses the implementation of a three-dimensional (3D) structure motif search of proteins. Each protein structure is represented by a set of secondary structure elements (SSEs) which involves alpha-helix segments and beta-strand segments. In describing it, every SSE is further reduced into a two-node graph that consists of the starting amino acid residue, the ending residue and a pseudo-bond between them. The searching algorithm is based on a graph theoretical clique-finding algorithm that has been used for 3D substructure searching in small organic molecules. The program SS3D-P2 was validated using proteins that have well-known 3D motifs, and it correctly found the Greek key motif within an eye lens protein, crystallin, that consists of four anti-parallel beta strands. The program was also successfully applied to searching for the more complex 3D motif, TIM-type beta-barrel motif, with a protein structure database from the Protein Data Bank.

A comparison of structural relationships among α-crystallin, human Hsp27, γ-crystallins and βB2-crystallin

The 3D structures of alpha-crystallin, a major eye lens protein, and related small heat shock proteins are unresolved. It has been assumed that alpha-crystallin is primarily a beta-sheet globular protein similar to alpha-crystallin (Siezen and Argos, Biochim. Biophys. Acta, 1983, 748, 56-67) containing sequence repeats in its two domains (Wistow, FEBS Lett. 1985, 181, 1-6). Positional flexibility of amino acid residues and far UV-circular dichroism spectroscopy were used to investigate structural relationships among these proteins. The utility of flexibility plots for predicting protein structure is demonstrated by the excellent correlation of these plots with the known 3D X-ray structures of beta/gamma-crystallins. Similar analyses of alpha-crystallin subunits, alpha A and alpha B, and human heat shock protein 27 show that the C-terminal domains and connecting segments of these proteins are very similar while the N-terminal domains have significant structural differences. Unlike beta/gamma-crystallins, both Hsp27 and alpha-crystallin subunits are asymmetrical with highly flexible C-terminal domains. Flexibility is considered essential for protein functional activity. Therefore, the C-terminal region may play an active role in alpha-crystallin and small heat shock protein function. Differences in flexibility profiles and estimated secondary structure distribution in alpha-crystallin by three recent/updated algorithms from far UV-CD spectra support our predicted 3D structure and the concept that alpha-crystallin and members of beta/gamma superfamily are structurally dissimilar.

The conformational stability of α-crystallin is rather low: Calorimetric results

The eye lens protein and chaperonin, alpha-crystallin, was studied by differential scanning microcalorimetry, spectroscopy and size exclusion chromatography. The thermal transition of alpha-crystallin proceeds at Ttrs = 59.8 +/- 0.6 degrees C with an enthalpy change of delta H = 336 +/- 9 kJ per mol subunit. Disagreement between previous delta H values could be attributed to a side reaction that leads, depending on the scan rate, to the formation of a non-productive folding form. The conformational stability of alpha-crystallin is rather low (delta G = 24 +/- 5 kJ/mol of subunit). The minimal cooperative unit of alpha-crystallin is the monomeric subunit.

Characterization and enzyme activity of argininosuccinate lyase/δ-crystallin of the embryonic duck lens

Argininosuccinate lyase (ASL)/delta-crystallin, a major soluble protein of the transparent eye lens of birds and reptiles, is a mixture of tetramers comprising all possible combinations of two similar polypeptides (delta 1 and delta 2). Only the delta 2 polypeptide has ASL activity. In the present investigation we have purified each of the 5 major isoforms (delta A to delta E, pI 5.2 to 5.8) of delta-crystallin tetramers from the embryonic duck lens by isoelectric focussing and established by peptide sequencing that the delta 1 and delta 2 polypeptides are encoded in the previously identified, linked delta 1 and delta 2 genes, respectively. The relative amounts of the different tetramers in the 14-day-old embryonic lens were consistent with equal expression of the 2 delta-crystallin genes and no preference for assembly of the 2 delta polypeptides. The relative amount of ASL activity of the tetramers was a linear function of the relative amount of their delta 2 polypeptides, with delta A (only delta 1) lacking enzymatic activity altogether. delta B (3 delta 1:1 delta 2), delta C (2 delta 1:2 delta 2), delta D (1 delta 1:3 delta 2) and delta E (4 delta 2) all gave normal Michaelis-Menten kinetics for fumarate production from argininosuccinate at 40 degrees C and had a similar Km (average Km for mixture was 0.15 mM). delta E had a Km of 0.187 mM and a Vmax of 9 mumol/min per mg protein. Unlike bovine and like human ASL, both reported previously, embryonic duck ASL/delta-crystallin showed no evidence of cooperativity or activation by GTP. Each isoform had a similar far ultraviolet circular dichroism spectrum and thermal stability between 20 degrees C and 60 degrees C, with denaturation occurring at 65 degrees C. Our data suggest that gene duplication, structural modifications leading to greater thermal stability of the delta 1 and delta 2 polypeptides, and selective loss of ASL activity in the delta 1 polypeptide all occurred during the recruitment of ASL for a refractive role in the duck lens, resulting in the generation of ASL isoenzymes.

An eye lens protein-water structure: 1.2 A resolution structure of gammaB-crystallin at 150 K.

gammabeta-crystallin is a structural protein of the eye lens with a role in the maintenance of an even distribution of protein and water over distances around the wavelength of light, preserving lens transparency. The structure of the 174-residue bovine protein has already been determined at room temperature to 1.47 A resolution. By flash freezing the protein crystals, data have now been collected to a nominal resolution limit of 1.2 A as radiation damage was essentially eliminated. The protein-water model has been refined against this data using the program RESTRAIN converging to an R factor of 18.5% with all data. Atomic positions are clearly indicated in the electron-density maps. Discrete bimodal disorder has been visualized for a few side chains. Out of a total of 498 water molecules present in the crystal asymmetric unit, 394 have been modelled and refined at unit occupancy. The solvent structure is extremely well ordered with an average B value of 23.4 A(2). Partially occupied sites have been identified where disorder in the protein induces concomitant disorder in the local solvent structure. The solvent structure covers 97% of the solvent-exposed surface of the protein in the crystal. 126 water molecules are distributed in second and higher hydration shells. There are networks of hydrogen-bonded solvent extending up to 64 molecules in a network, comprising trimers and tetramers as well as five- and six-membered water-ring structures. The hydration of the protein surface is dominated by arginine and aspartate side chains. Extensive cages of highly ordered solvent molecules are also observed around exposed non-polar groups.

Development of monoclonal antibodies against a riboflavin-tryptophan photoinduced adduct: reactivity to eye lens proteins.

We describe here the development of monoclonal antibodies to the hapten tryptophan-riboflavin, generated by irradiation of a solution of bovine serum albumin in the presence of riboflavin. The specificity of the three obtained monoclonal antibodies, named 1E6, 5H5, 5A8 all belonging to the IgG1 isotype, was assessed by a competitive enzyme-linked immunosorbent assay in the presence of an increasing concentration of the tryptophan-riboflavin adduct, obtained from an irradiated riboflavin-sensitized tryptophan solution. It was demonstrated that the tryptophan-riboflavin antibodies react with the soluble proteins of the eye lens; this reaction was more intense in the old rat lenses as compared to the young ones, and a maximum binding of the antibodies was obtained with the soluble protein fraction from the human cataractous lens. By indirect immunofluorescence, a reactivity associated with the protein matrix, localized in the lens central zone, was observed. In the peripheral zone of the lens, where the younger cells are found, a marked immunofluorescent emission was observed on structures preferentially localized in the nuclei.

Lens-Specific Expression of a Chicken βA3/A1-Crystallin Promoter Fragment in Transgenic Mice

Beta A3/A1-crystallin is one of the major refractive proteins of the chicken eye lens. Previously we showed that a fragment from -382 to +22 bp of the beta A3/A1-crystallin gene functions as a promoter in transfected lens cells. Here we show by use of the bacterial chloramphenicol acetyltransferase reporter gene that the -143/+22 fragment is sufficient for lens-specific promoter activity in transgenic mice. DNase I footprinting shows that lens nuclear proteins protect several regions within the minimal promoter fragment.

Mutations in the founder of the MIP gene family underlie cataract development in the mouse.

The major intrinsic protein (MIP) of the vertebrate eye lens is the first identified member of a sequence-related family of cell-membrane proteins that appears to have evolved by gene duplication. Several members of the MIP family transport water (aquaporins), glycerol and other small molecules in microbial, plant and animal cells. Mutations in two aquaporin homologues of MIP underlie an autosomal recessive form of nephrogenic diabetes insipidus and absence of the Colton blood group antigens in humans, whereas, mutation of a third MIP-like gene underlies 'big brain' development in Drosophila. Here we show that distinct mutations in the murine Mip gene underlie one form of autosomal dominant cataract in the mouse. The cataract Fraser mutation is a transposon-induced splicing error that substitutes a long terminal repeat sequence for the carboxy-terminus of MIP. The lens opacity mutation is an amino-acid substitution that inhibits targeting of MIP to the cell-membrane. These allelic cataract mutations provide the first direct evidence that MIP plays a crucial role in the development of a transparent eye lens.

P-H5-06 Dielectric properties of rabbit's eye lens protein following exposure to beta radiation: El Refaei FM, 1 Gamal MM, 1 Ali FM 2 1 Biophysics Unit. Research Inst. of Ophth., Giza (ARE) 2 Biophysics Department, Cairo Univ. Giza (ARE)

P-H5-06 Dielectric properties of rabbit's eye lens protein following exposure to beta radiation: El Refaei FM, 1 Gamal MM, 1 Ali FM 2 1 Biophysics Unit. Research Inst. of Ophth., Giza (ARE) 2 Biophysics Department, Cairo Univ. Giza (ARE)

Evolution, structure and function of the small heat shock proteins in plants

The a-crystallin-related, small heat shock proteins (smHSPs) are ubiquitous in nature, but are unusually abundant and diverse in higher plants as opposed to other eukaryotes. The smHSPs range in size from ∼17 to 30 kDa and share a conserved C-terminal domain common to all eukaryotic smHSPs and to the α-crystallin proteins of the vertebrate eye lens. In higher plants six nuclear gene families encoding smHSPs have been defined. Each gene family encodes proteins found in a distinct cellular compartment, including the cytosol, chloroplast, ER, and mitochondrion. Evolutionary analysis suggests that the smHSP gene families arose by gene duplication and divergence prior to the radiation of angiosperms. In general, the smHSPs are not found in normal vegetative tissues, but accumulate to high levels in response to heat stress. Specific smHSPs are also expressed during various phases of plant development as part of the endogenous developmental programme. Thus, although the smHSPs are apparently not essential for basal cell functions as are the high molecular weight HSPs such as HSP90, HSP70 and HSP60, their functions are likely to be critical for survival and recovery from heat stress as well as for specific developmental processes. Biochemical analysis indicates that smHSPs are found in high molecular weight complexes between 200-400 kDa that are most likely composed solely of multiple smHSP subunits. Purified recombinant plant smHSPs facilitate reactivation of chemically denatured enzymes in a nucleotide-independent fashion and also prevent heat-induced aggregation or reverse inactivation of protein substrates. Based on these data, it is suggested that smHSPs act in vivo as a type of molecular chaperone to bind partially denatured proteins preventing irreversible protein inactivation and aggregation, and that smHSP chaperone activity contributes to the development of thermotolerance.

Alternating charge clusters of side chains: new surface structural invariants observed in calf eye lens gamma-crystallins.

A detailed stereochemical analysis of the oppositely charged side chains of amino acid residues on the surface of calf eye lens protein gamma-crystallin B has been carried out. The refined structural data of very high quality obtained at 1.47 A resolution have been used. Charge-charge interactions were considered to be valuable for all the charged oxygen and nitrogen atoms situated at distances, d, between 2.4 and 7.0 A. This means we consider short contact ion pairs as those with intercharge distances 2.4 < d < or = 4.0 A and distant contact ion pairs as those with distances 4.0 < d < or = 7.0 A. Hydrogen bonding of the charged atomic groups with the structural water molecules also has been considered. We have not looked at the side groups of histidines which are charged only partially at neutral pH. Five clusters of charged side chains which were large enough were observed. The clusters are comprised of four to six residues which compose 54.3% of the total charged residues in the protein. The clusters contain from eight to 12 charged atoms and look like the bent chains of oppositely charged atoms. All clusters are of plane geometry and their maximal linear dimensions are from 11 to 18 A. The root mean square deviations of charged atoms from the cluster plane varied from 0.63 to 0.86 A for four clusters and was only 1.85 A for the largest cluster. All clusters include a number of water molecules situated on the cluster boundary and grouped near the cluster plane. It was shown that the amino acid sequence positions of charged residues are conservative for all the proteins of the gamma-crystallin family of vertebrates including fish, frog, mouse, rat, calf and human. The cluster properties were discussed both in their functional aspect for gamma-crystallins and in other aspects common for globular proteins. As a result, the alternating charge clusters should be considered as newly recognized surface structural invariants. The importance of the charged side chain clusters is claimed for the updated concept of the protein surface.

Phylogenetic relationships of the hoatzin, an enigmatic South American bird.

The hoatzin (Opisthocomus hoazin) lives in the humid lowlands of northern and central South America, often in riparian habitats. It is a slender bird approximately 65 cm in length, brownish with lighter streaks and buffy tips to the long tail feathers. The small head has a ragged, bristly crest of reddish-brown feathers, and the bare skin of the face is bright blue. It resembles a chachalaca (Ortalis, Cracidae) in size and shape, but its plumage and markings are similar to those of the smaller guira cuckoo (Guira guira). The hoatzin (pronounced Watson) has been a taxonomic puzzle since it was described in 1776. It usually has been viewed as related to the gallinaceous birds, but alliances to other groups have been suggested, including the cuckoos. We present DNA sequence evidence from the 12S and 16S rRNA mitochondrial genes, and from the nuclear gene that codes for the eye lens protein, alpha A-crystallin. The results indicate that the hoatzin is most closely related to the typical cuckoos and that the divergence occurred at or near the base of the cuculiform phylogenetic tree.

Rapid Refolding Studies on the Chaperone-like α-Crystallin: EFFECT OF α-CRYSTALLIN ON REFOLDING OF β- AND γ-CRYSTALLINS

alpha-Crystallin, a multimeric protein present in the eye lens, is shown to have chaperone-like activity in preventing thermally induced aggregation of enzymes and other crystallins. We have studied the rapid refolding of alpha-crystallin, and compared it with other calf eye lens proteins, namely beta- and gamma-crystallins. alpha-Crystallin forms a clear solution upon rapid refolding from 8 M urea. The refolded alpha-crystallin has native-like secondary, tertiary, and quaternary structures as revealed by circular dichroism and fluorescence characteristics as well as gel filtration and sedimentation velocity measurements. On rapid refolding, beta- and gamma-crystallins aggregate and form turbid solutions. The presence of alpha-crystallin in the refolding buffer marginally increases the recovery of beta- and gamma-crystallins in the soluble form. However, unfolding of these crystallins together with alpha-crystallin using 8 M urea and subsequent refolding significantly increases the recovery of these proteins in the soluble form. These results indicate that an intermediate of alpha-crystallin formed during refolding is more effective in preventing the aggregation of beta- and gamma-crystallins. This supports our earlier hypothesis (Raman, B., and Rao, C. M. (1994) J. Biol. Chem. 269, 27264-27268) that the chaperone-like activity of alpha-crystallin is more pronounced in its structurally perturbed state.