Mutations in human αA-crystallin/sHSP affect subunit exchange interaction with αB-crystallin

Background

Mutation in αA-crystallin contributes to the development of congenital cataract in humans. Heterooligomerization of αA-crystallin and αB-crystallin is essential for maintaining transparency in the eye lens. The effect of congenital cataract causing mutants of αA-crystallin on subunit exchange and interaction with αB-crystallin is unknown. In the present study, interaction of the mutants of αA-crystallin with αB-crystallin was studied both in vitro and in situ by the fluorescence resonance energy transfer (FRET) technique.

Methodology/Principal Findings

In vitro FRET technique was used to demonstrate the rates of subunit exchange of αB-wt with the following αA-crystallin mutants: R12C, R21L, R21W, R49C, R54C, and R116C. The subunit exchange rates (k values) of R21W and R116C with αB-wt decreased drastically as compared to αA-wt interacting with αB-wt. Moderately decreased k values were seen with R12C, R49C and R54C while R21L showed nearly normal k value. The interaction of αA- mutants with αB-wt was also assessed by in situ FRET. YFP-tagged αA mutants were co-expressed with CFP-tagged αB-wt in HeLa cells and the spectral signals were captured with a confocal microscope before and after acceptor laser photobleaching. The interaction of R21W and R116C with αB-wt was decreased nearly 50% as compared to αA-wt while the rest of the mutants showed slightly decreased interaction. Thus, there is good agreement between the in vitro and in situ FRET data.

Conclusions/Significance

Structural changes occurring in these mutants, as reported earlier, could be the underlying cause for the decreased interaction with αB may contribute to development of congenital cataract.     

Identification and characterization of a copper-binding site in αA-crystallin

Previous studies have shown that both αA- and αB-crystallins bind Cu2+, suppress the formation of Cu2+-mediated active oxygen species, and protect ascorbic acid from oxidation by Cu2+. αA- and αB-crystallins are small heat shock proteins with molecular chaperone activity. In this study we show that the mini-αA-crystallin, a peptide consisting of residues 71-88 of αA-crystallin, prevents copper-induced oxidation of ascorbic acid. Evaluation of binding of copper to mini-αA-crystallin showed that each molecule of mini-αA-crystallin binds one copper molecule. Isothermal titration calorimetry and nanospray mass spectrometry revealed dissociation constants of 10.72 and 9.9 μM, respectively. 1,1'-Bis(4-anilino)naphthalene-5,5'-disulfonic acid interaction with mini-αA-crystallin was reduced after binding of Cu2+, suggesting that the same amino acids interact with these two ligands. Circular dichroism spectrometry showed that copper binding to mini-αA-crystallin peptide affects its secondary structure. Substitution of the His residue in mini-αA-crystallin with Ala abolished the redox-suppression activity of the peptide. During the Cu2+-induced ascorbic acid oxidation assay, a deletion mutant, αAΔ70-77, showed about 75% loss of ascorbic acid protection compared to the wild-type αA-crystallin. This difference indicates that the 70-77 region is the primary Cu2+-binding site(s) in human native full-size αA-crystallin. The role of the chaperone site in Cu2+ binding in native αA-crystallin was confirmed by the significant loss of chaperone activity by the peptide after Cu2+ binding.     

The benefits of being β-crystallin heteromers: βB1-crystallin protects βA3-crystallin against aggregation during co-refolding

β-Crystallins are the major structural proteins in mammalian lens, and their stability is critical in maintaining the transparency and refraction index of the lens. Among the seven β-crystallins, βA3-crystallin and βB1-crystallin, an acidic and a basic β-crystallin, respectively, can form heteromers in vivo. However, the physiological roles of the heteromer have not been fully elucidated. In this research, we studied whether the basic β-crystallin facilitates the folding of acidic β-crystallin. Equilibrium folding studies revealed that the βA3-crystallin and βB1-crystallin homomers and the βA3/βB1-crystallin heteromer all undergo similar five-state folding pathways which include one dimeric and two monomeric intermediates. βA3-Crystallin was found to be the most unstable among the three proteins, and the transition curve of βA3/βB1-crystallin was close to that of βB1-crystallin. The dimeric intermediate may be a critical determinant in the aggregation process and thus is crucial to the lifelong stability of the β-crystallins. A comparison of the Gibbs free energy of the equilibrium folding suggested that the formation of heteromer contributed to the stabilization of the dimer interface. On the other hand, βA3-crystallin, the only protein whose refolding is challenged by serious aggregation, can be protected by βB1-crystallin in a dose-dependent manner during the kinetic co-refolding. However, the protection is not observed in the presence of the pre-existed well-folded βB1-crystallin. These findings suggested that the formation of β-crystallin heteromers not only stabilizes the unstable acidic β-crystallin but also protects them against aggregation during refolding from the stress-denatured states.     

Specific dissociation of αB subunits from α-crystallin

Exposure of bovine α-crystallin to 0.1 M glycine at pH 7 decreases the average molar mass of the protein from 700 to 420 kDa. When the pH is lowered to 2.5, in the same buffer, the αB chains specifically dissociate from the aggregates, leaving a particle of 290 kDa containing only αA chains. The decrease in the molar mass corresponds to the mass of the αB chains in the original aggregate. The pH-dependent dissociation is fully reversible. Similar changes were observed with rat and kangaroo α-crystallins but the dogfish protein was not affected. Sedimentation velocity analyses and fluorescence spectroscopy yielded a pK, for the dissociation, of 3.7 for α-crystallin and 4.0 for a homopolymer constructed from purified αB₂ polypeptides. An αA₂ homopolymer was virtually unaffected by the lowering of pH. The products from the dissociation were isolated and their properties studied by sedimentation analysis and acrylamide quenching of tryptophan fluorescence. The αB chains were found to be completely denatured, whereas the structure of the αA chains, in the 290 kDa, particle, were only slightly altered. Comparisons of the sequences of the various proteins examined suggested that decreased ionization of aspartic acid 127 in the αB chain was responsible for the specific dissociation of this polypeptide.     

The molecular weight of the basic polypetide chain αB2 of α-crystallin

The molecular weights calculated from the amino acid sequences of the A and B chains of the lens protein -crystallin differ only slightly (19830 and 20070, respectively). SDS gel electrophoresis of these chains and comparison with marker proteins yield apparent molecular weights of 19500 for A and 22500 for B. The discrepancy between the value of 22500 and the real molecular weight of 20070 for B vanishes by the combined use of SDS and 6 M urea in the polyacrylamide gels.     

Characterization of the mutant α-mannosidase in bovine mannosidosis

Residual acidic α-mannosidase, varying in amount up to approx. 15% of normal values, can be measured in various organs of a calf with mannosidosis. The highest specific activity and relative proportion of residual activity were found in the liver. Chromatography on DEAE-cellulose showed that the residual activity was associated with two components, which were eluted at comparable positions with those found in normal tissues. The residual activity had a lower thermal stability and a higher K_m value for a synthetic substrate than did the normal enzyme. No differences in molecular weight or electrophoretic mobility between normal acidic α-mannosidase and the residual activity were observed by gel filtration and electrophoresis on cellulose acetate respectively. The isoelectric focusing profiles for the α-mannosidase in the normal and pathological livers were very similar. It is suggested that a mutant enzyme, resulting from a mutation in a structural gene, accounts for the residual acidic α-mannosidase in mannosidosis. The mutant enzyme, which cross-reacts with antiserum raised against normal bovine acidic α-mannosidase, is present at a decreased concentration compared with the normal enzyme. There is a correlation between the concentrations of residual activity and cross-reacting material in mannosidosis. α-Mannosidase with a pH optimum of 5.75 and which is activated by Zn²⁺ was also detected in the liver of the calf with mannosidosis. However, it is probably not a product of the defective gene because addition of Zn²⁺ indicated that it was also present in normal tissues.     

Accumulation of the β-subunit of polygalacturonase 1 in normal and mutant tomato fruit

Polyclonal antiserum raised against the native PG1 isoform of tomato fruit (Lycopersicon esculentum Mill.) polygalacturonase [poly(1,4--d-galacturonide) glycanohydrolase, EC 3.2.1.15] bound to each of the subunits of the protein and also to a range of other fruit proteins. Affinity purification was used to remove antibody molecules that bound to the native form of the PG2 isoform. The resulting serum bound to native PG1, denatured PG2 and -subunits of PG1 but not to native PG2 or other fruit proteins. This anti-PG1 serum was used to monitor the occurrence of the PG1 -subunit and PG2 in detergent extracts of tomato tissues. The -subunit polypeptide was detected in pericarp but not locule tissue of fruit, including fruit of the rin and nor mutants. It increased in amount in the pericarp tissues from an early stage to the mature green stage, clearly prior to any appreciable accumulation of the PG2 subunit. The -subunit polypeptide was not detected in stem or leaf tissues. A PG2-specific antiserum was used to study the interaction of PG2 with the isolated -subunit. The PG2 isoform was bound to the -subunit over a wide range of salt concentrations and pH; the interaction was independent of the presence of reducing agents. It is concluded that strong non-covalent forces are involved in the interaction. The results are consistent with a model in which the -subunit is positioned in the cell wall structure and provides a specific binding site for the active PG2 subunit when this is synthesised during ripening.Abbreviations B breaker - MG mature green - M_r relative molecular mass - nor non-ripening mutant - PAGE polyacrylamide gel electrophoresis - PG polygalacturonase - rin ripening inhibitor mutant - SDS sodium dodecyl sulphate     

Increased hydrophobicity and decreased backbone flexibility explain the lower solubility of a cataract-linked mutant of γD-crystallin

A number of point mutations in γD-crystallin are associated with human cataract. The Pro23-to-Thr (P23T) mutation is perhaps the most common, is geographically widespread, and presents itself in a variety of phenotypes. It is therefore important to understand the molecular basis of lens opacity due to this mutation. In our earlier studies, we noted that P23T shows retrograde and sharply lowered solubility, most likely due to the emergence of hydrophobic patches involved in protein aggregation. Binding of 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonate (Bis-ANS) dye (a probe commonly used for detecting surface hydrophobicity) competed with aggregation, suggesting that the residues involved in Bis-ANS binding are also involved in protein aggregation. Here, using NMR spectroscopy in conjunction with Bis-ANS binding, we identify three residues (Y16, D21, and Y50) in P23T that are involved in binding the dye. Furthermore, using ¹⁵N NMR relaxation experiments, we show that, in the mutant protein, backbone fluctuations are restricted to the picosecond-to-nanosecond and microsecond timescales relative to the wild type. Our present studies specify the residues involved in these two pivotal characteristics of the mutant protein, namely increased surface hydrophobicity and restricted mobility of the protein backbone, which can explain the nucleation and further propagation of protein aggregates. Thus, we have now identified the residues in the P23T mutant that give rise to novel hydrophobic surfaces, as well as those regions of the protein backbone where fluctuations in different timescales are restricted, providing a comprehensive understanding of how lens opacity could result from this mutation.     

The importance of the last strand at the C-terminus in βB2-crystallin stability and assembly

Congenital cataract is the leading cause of childhood blindness worldwide. Investigations of the effects of inherited mutations on protein structure and function not only help us to understand the molecular mechanisms underlying congenital hereditary cataract, but also facilitate the study of complicated cataract and non-lens abnormities caused by lens-specific genes. In this research, we studied the effects of the V187M, V187E and R188H mutations on βB2-crystallin structure and stability using a combination of biophysical, cellular and molecular dynamic simulation analysis. Both V187 and R188 are located at the last strand of βB2-crystallin Greek-key motif 4. All of the three mutations promoted βB2-crystallin aggregation in vitro and at the cellular level. These three mutations affected βB2-crystallin quite differentially: V187M influenced the hydrophobic core of the C-terminal domain, V187E was a Greek-key motif breaker with the disruption of the backbone H-bonding network, while R188H perturbed the dynamic oligomeric equilibrium by dissociating the dimer and stabilizing the tetramer. Our results highlighted the importance of the last strand in the structural integrity, folding, assembly and stability of β-crystallins. More importantly, we proposed that the perturbation of the dynamic equilibrium between β-crystallin oligomers was an important mechanism of congenital hereditary cataract. The selective stabilization of one specific high-order oligomer by mutations might also be deleterious to the stability and folding of the β-crystalllin homomers and heteromers. The long-term structural stability and functional maintenance of β-crystallins are achieved by the precisely regulated oligomeric equilibrium.     

C-Terminal truncation affects subunit exchange of human αA-crystallin with αB-crystallin

In human lenses, C-terminal cleavage of αA-crystallin at residues 172,168, and 162 have been reported. The effect of C-terminal truncation of αA-crystallin on subunit exchange and heterooligomer formation with αB-crystallin and homooligomer formation with native αA-crystallin is not known. We have conducted fluorescence resonance energy transfer studies which have shown that the rates of subunit exchange of αA_1–172 and αA_1–168 with αB-wt were two-fold lower than for αA-wt interacting with αB-wt. The subunit exchange rate between αA_1–162 and αB-wt was six-fold lower. These data suggest that cleavage of the C-terminal residues could significantly affect heterooligomerization. On the other hand, the subunit exchange rates between αA-wt and the truncated αA-crystallins were either unchanged or only slightly decreased, which suggest that homooligomerization may not be significantly influenced by C-terminal truncation. The main conclusion from this study is that cleavage of C-terminal residues of αA-crystallin including the nine residues of the flexible tail is expected to significantly affect the formation of heteroaggregates. Reconstitution experiments showed that the presence of an intact C-terminus is essential for the formation of fully integrated heteroaggregates with equal proportion of αA and αB subunits.     

How α-crystallin prevents the aggregation of insulin

Using steady-state, polarized, and phase-modulation fluorometry, the dithiothreitol-induced denaturation of insulin and formation of its complex with alpha-crystallin in solution were studied. Prevention of the aggregation of insulin by alpha-crystallin is due to formation of chaperone complexes, i.e. interaction of chains of the denatured insulin with alpha-crystallin. The conformational changes in alpha-crystallin that occur during complex formation are rather small. It is unlikely that N-termini are directly involved in the complex formation. The 8-anilino-1-naphthalenesulfonate (ANS) is not sensitive to the complex formation. ANS emits mainly from alpha-crystallin monomers, dimers, and tetramers, but not from oligomers or aggregates. The possibility of highly sensitive detection of aggregates by light scattering using a spectrofluorometer with crossed monochromators is demonstrated.     

Distinct interactions of αA-crystallin with homologous substrate proteins, δ-crystallin and argininosuccinate lyase, under thermal stress

δ-Crystallin is a taxon-specific eye lens protein that was recruited from argininosuccinate lyase (ASL) through gene sharing. ASL is a metabolic enzyme that catalyzes the reversible conversion of argininosuccinate into arginine and fumarate and shares about 70% sequence identity and similar overall topology with δ-crystallin. ASL has a lower thermal stability than δ-crystallin. In this study, we show that the small heat shock protein, αA-crystallin, functions as a molecular chaperone, and enhanced thermal stability of both δ-crystallin and ASL. The stoichiometry for efficient protection of the two substrate proteins by αA-crystallin was determined by slowly increasing the temperature. N- or C-terminal truncated mutants of δ-crystallin co-incubated with αA-crystallin showed higher thermal stability than wild-type enzyme, and the stoichiometry for efficient protection was the same. Thermal unfolding of δ-crystallin or ASL in the presence of αA-crystallin followed a similar three-state model, as determined by circular dichroism analyses. A stable intermediate which retained about 30% α-helical structure was observed. Protection from thermal denaturation by αA-crystallin was by interaction with partly unfolded ASL or δ-crystallin to form high molecular weight heteroligomers, as judged by size-exclusive chromatography and SDS-PAGE analyses. Aggregate formation of ASL was significantly reduced in the presence of αA-crystallin. The extent of protection of ASL and δ-crystallin at different ratios of αA-crystallin were described by hyperbolic and sigmoidal curves, respectively. These results suggest the preferential recognition of partly unfolded ASL by αA-crystallin. In contrast, unstable δ-crystallin might trigger a cooperative interaction by higher stoichiometries of αA-crystallin leading to fuller protection. The different interactions of αA-crystallin with the two homologous but functionally different substrate proteins show its behavior as a chaperone is variable.     

Further studies on the sub-units of α-crystallin

1. A new procedure is described for the purification of alpha-crystallin, including: preparative zone electrophoresis, density-gradient centrifugation and gel filtration. The total amino acid composition of highly purified samples prepared according to this procedure has been determined. 2. Evidence is presented for the presence of intermediates in the urea-induced splitting of alpha-crystallin into sub-units. A possible mechanism for this splitting is proposed. 3. The recombination of sub-units has been studied by polyacrylamide-gel electrophoresis and ultracentrifugal analysis. As judged from these criteria, only a partial recovery of starting material was obtained. 4. The origin of the minor bands in the electrophoretic pattern of alpha-crystallin on 7m-urea-polyacrylamide gel has been investigated. No evidence was found that their presence is due to carbamoylation or sulphide-disulphide interchange. They probably arise from isomerization. 5. The mean molecular weight of the sub-units was calculated to be 24000 (Archibald's method). Determination of the sedimentation-diffusion equilibrium revealed a value of 21000 at the meniscus. Assuming that all sub-units contain one cysteine residue/molecule, 23000 can be derived for the mean molecular weight.     

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

The 3D structures of α-crystallin, a major eye lens protein, and related small heat shock proteins are unresolved. It has been assumed that α-crystallin is primarily a β-sheet globular protein similar to γ-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 β/γ-crystallins. Similar analyses of α-crystallin subunits, αA and αB, 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 β/γ-crystallins, both Hsp27 and α-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 α-crystallin and small heat shock protein function. Differences in flexibility profiles and estimated secondary structure distribution in α-crystallin by three recent/updated algorithms from far UV-CD spectra support our predicted 3D structure and the concept that α-crystallin and members of β/γ-superfamily are structurally dissimilar.     

Association of α-subunits with nucleotide-modified β-subunits induces asymmetry in the catalytic sites of the F1-ATPase α3β3

The photoaffinity spin-labeled ATP analog, 2-N₃-SL-adenosine triphosphate (ATP), was used to covalently modify isolated β-subunits from F₁-ATPase of the thermophilic bacterium PS3. Approximately 1.2 mol of the nucleotide analog bound to the isolated subunit in the dark. Irradiation leads to covalent incorporation of the nucleotide into the binding site. ESR spectra of the complex show a signal that is typical for protein-immobilized radicals. Addition of isolated α-subunits to the modified β-subunits results in ESR spectra with two new signals indicative of two distinctly different environments of the spin-label, e.g., two distinctly different conformations of the catalytic sites. The relative ratio of the signals is approx 2∶1 in favor of the more closed conformation. The data show for the first time that when nucleotides are bound to isolated β-subunits, binding of α-subunits induces asymmetry in the catalytic sites even in the absence of the γ-subunit. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to PDV.     

Incorporation of a β-turn mimic in the N-terminal fragment of the B1 domain of streptococcal protein G

Summary A dibenzofuran-based β-turn mimic has been incorporated in the B1_2–29 fragment of the B1 domain of streptococcal protein G. This amino acid sequence adopts a β-hairpin structure in the complete B1 domain (B1_2–56). The modified peptide was studied by CD and NMR spectroscopy and its solution behavior was compared with the conformation adopted by the same sequence in the modified B1 domain.     

Interaction of chaperone α-crystallin with unfolded state of α-amylase: Implications for reconstitution of the active enzyme

α-Crystallin is reported to act like molecular chaperone by suppressing the aggregation of damaged crystallins in eye lens. In this work, it is shown that α-crystallin increases the reactivation of guanidine hydrochloride (GdnHCl)-denatured α-amylase from porcine pancreas. 8-Anilinonaphthalene-sulphonate (ANS) binding studies reveal the involvement of hydrophobic interactions in the formation of the complex of α-crystallin and α-amylase. On the basis of our fluorescence spectroscopic and gel-filtration results, we propose that α-crystallin blocks the unfavorable pathways that lead to irreversible denaturation of α-amylase and keep it in folding-competent intermediate state.