Interaction of newly synthesized α-crystallin with isolated lens plasma membranes

Translation of lens polyribosomes in a reticulocyte cell-free system results mainly in the synthesis of the water-soluble crystallins. After incubation of the translation products with isolated lens fiber plasma membranes, the newly synthesized α-crystallin interacts with this fraction and becomes water-insoluble. Urea extraction of the reisolated plasma membranes shows that part of the polymeric α-crystallin, in particular the αA chains, becomes urea-insoluble. When the membranes were isolated under conditions that stabilize complex formation with the cytoskeleton, only αA₂ seems to interact with this complex. In contrast, interaction with β- and γ-crystallin could not be observed.     

Thermal and acid denaturation of bovine lens α-crystallin

The chaperone-like protein α-crystallin is a ～35 subunit hetero-oligomer consisting of αA and αB subunits in a 3:1 molar ratio and has the function of maintaining eye lens transparency. We studied the thermal denaturation of α-crystallin by differential scanning calorimetry (DSC), circular dichroism (CD), and dynamic light scattering (DLS) as a function of pH. Our results show that between pH 7 and 10 the protein undergoes a reversible thermal transition. However, the thermodynamic parameters obtained by DSC are inconsistent with the complete denaturation of an oligomeric protein of the size of α-crystallin. Accordingly, the CD data suggest the presence of extensive residual secondary structure above the transition temperature. Within the pH range from 4 to 7 the increased aggregation propensity around the isoelectric point (pI ～ 6) precludes observation of a thermal transition. As pH decreases below 4 the protein undergoes a substantial unfolding. The secondary structure content of the acid-denatured state shows little sensitivity to heating. We propose that the thermal transition above pH 7 and the acid-induced transition at ambient temperature result in predominant denaturation of the αB subunit. Although the extent of denaturation of the αA subunit cannot be estimated from the current data, the existence of a native-like conformation is suggested by the preserved association of the subunits and the chaperone-like activity. A key difference between the thermal and the acid denaturation is that the latter is accompanied by dissociation of αB subunits from the remaining αA-oligomer, as supported by DLS studies.     

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.     

NMR spin–echo studies of hydration properties of the molecular chaperone α-crystallin in the bovine lens

The water-binding properties of bovine lens α-crystallin, collagen from calf skin and bovine serum albumin (BSA), were investigated with various techniques. The water absorptive capacity was obtained in high vacuum desorption experiments volumetrically, and also gravimetrically in controlled atmosphere experiments. NMR spin–echo technique was used to study the hydration of protein samples and to determine the spin–spin relaxation times (T₂) from the protons of water, absorbed on the proteins. Isolated bovine lenses were sectioned into 11–12 morphological layers (from anterior cortex through nucleus to posterior cortex). Crystallin profiles were obtained for each lens layer using thin-layer isoelectric focusing in polyacrylamide gel (IEF). The water content in relation to dry weight of proteins was measured in individual morphological lens layers. During the water vapor uptake P/P₀=0.75, α-crystallin did not absorb water, suggesting that hydrophobic regions of the protein are exposed to the aqueous solvent. At P/P₀=1.0, the absorption of water by α-crystallin was 17% with a single component decay character of spin–echo (T₂=3 ms). Addition of water to α-crystallin to about 50% of its w/w in the protein sample showed T₂=8 ms with only one single component decay of the spin–echo signal. The single component decay character of the spin–echo indicates at the tightly bound water by α-crystallin. Under a relative humidity P/P₀=1.0, collagen and BSA absorbed correspondingly 19.3% and 28% of water and showed a two-component decay curve with T₂ of about 5 and 40 ms. The findings demonstrate the presence of two water fractions in collagen and BSA which are separated in space. The IEF data suggest a tight binding of water with α-crystallin with similar distribution patterns in the lens layers. The IEF data demonstrate a possible chaperone-like function for α-crystallin in the nucleus and inner cortex of the lens, but not in the outer cortex. To conclude, it was found that α-crystallin can immobilize and bind water to a greater extent than other proteins such as collagen and BSA. These results shed new light on structural properties of α-crystallin and have important implications for understanding the mechanism of the chaperone-like action of this protein in the lens and non-ocular tissues.     

Acetylation of αA-crystallin in the human lens: Effects on structure and chaperone function

α-Crystallin is a major protein in the human lens that is perceived to help to maintain the transparency of the lens through its chaperone function. In this study, we demonstrate that many lens proteins including αA-crystallin are acetylated in vivo. We found that K70 and K99 in αA-crystallin and, K92 and K166 in αB-crystallin are acetylated in the human lens. To determine the effect of acetylation on the chaperone function and structural changes, αA-crystallin was acetylated using acetic anhydride. The resulting protein showed strong immunoreactivity against a N^ε-acetyllysine antibody, which was directly related to the degree of acetylation. When compared to the unmodified protein, the chaperone function of the in vitro acetylated αA-crystallin was higher against three of the four different client proteins tested. Because a lysine (residue 70; K70) in αA-crystallin is acetylated in vivo, we generated a protein with an acetylation mimic, replacing Lys70 with glutamine (K70Q). The K70Q mutant protein showed increased chaperone function against three client proteins compared to the Wt protein but decreased chaperone function against γ-crystallin. The acetylated protein displayed higher surface hydrophobicity and tryptophan fluorescence, had altered secondary and tertiary structures and displayed decreased thermodynamic stability. Together, our data suggest that acetylation of αA-crystallin occurs in the human lens and that it affects the chaperone function of the protein.     

Acetylation of αA-crystallin in the human lens: effects on structure and chaperone function

α-Crystallin is a major protein in the human lens that is perceived to help to maintain the transparency of the lens through its chaperone function. In this study, we demonstrate that many lens proteins including αA-crystallin are acetylated in vivo. We found that K70 and K99 in αA-crystallin and, K92 and K166 in αB-crystallin are acetylated in the human lens. To determine the effect of acetylation on the chaperone function and structural changes, αA-crystallin was acetylated using acetic anhydride. The resulting protein showed strong immunoreactivity against a N(ε)-acetyllysine antibody, which was directly related to the degree of acetylation. When compared to the unmodified protein, the chaperone function of the in vitro acetylated αA-crystallin was higher against three of the four different client proteins tested. Because a lysine (residue 70; K70) in αA-crystallin is acetylated in vivo, we generated a protein with an acetylation mimic, replacing Lys70 with glutamine (K70Q). The K70Q mutant protein showed increased chaperone function against three client proteins compared to the Wt protein but decreased chaperone function against γ-crystallin. The acetylated protein displayed higher surface hydrophobicity and tryptophan fluorescence, had altered secondary and tertiary structures and displayed decreased thermodynamic stability. Together, our data suggest that acetylation of αA-crystallin occurs in the human lens and that it affects the chaperone function of the protein.     

Effects of selenium hyperaccumulation on plant-plant interactions: evidence for elemental allelopathy?

? Few studies have investigated plant-plant interactions involving hyperaccumulator plants. Here, we investigated the effect of selenium (Se) hyperaccumulation on neighboring plants. ? Soil and litter Se concentrations were determined around the hyperaccumulators Astragalus bisulcatus and Stanleya pinnata and around the nonhyperaccumulators Medicago sativa and Helianthus pumilus. We also compared surrounding vegetative cover, species composition and Se concentration in two plant species (Artemisia ludoviciana and Symphyotrichum ericoides) growing either close to or far from Se hyperaccumulators. Then, Arabidopsis thaliana germination and growth were compared on soils collected next to the hyperaccumulators and the nonhyperaccumulators. ? Soil collected around hyperaccumulators contained more Se (up to 266 mg Se kg(-1) ) than soil collected around nonhyperaccumulators. Vegetative ground cover was 10% lower around Se hyperaccumulators compared with nonhyperaccumulators. The Se concentration was higher in neighboring species A. ludoviciana and S. ericoides when growing close to, compared with far from, Se hyperaccumulators. A. thaliana showed reduced germination and growth, and higher Se accumulation, when grown on soil collected around Se hyperaccumulators compared with soil collected around nonaccumulators. ? In conclusion, Se hyperaccumulators may increase the surrounding soil Se concentration (phytoenrichment). The enhanced soil Se contents around hyperaccumulators can impair the growth of Se-sensitive plant species, pointing to a possible role of Se hyperaccumulation in elemental allelopathy.     

Impact of N-glycosylation on Fcγ receptor / IgG interactions: unravelling differences with an enhanced surface plasmon resonance biosensor assay based on coiled-coil interactions

The N-glycosylation profile of immunoglobulin G (IgG) is considered a critical quality attribute due to its impact on IgG-Fc gamma receptor (FcγR) interactions, which subsequently affect antibody-dependent cell-based immune responses. In this study, we investigated the impact of the FcγR capture method, as well as FcγR N-glycosylation, on the kinetics of interaction with various glycoforms of trastuzumab (TZM) in a surface plasmon resonance (SPR) biosensor assay. More specifically, we developed a novel strategy based on coiled-coil interactions for the stable and oriented capture of coil-tagged FcγRs at the biosensor surface. Coil-tagged FcγR capture outperformed all other capture strategies applied to the SPR study of IgG-FcγR interactions, as the robustness and reproducibility of the assay and the shelf life of the biosensor chip were excellent (> 1,000 IgG injections with the same biosensor surface). Coil-tagged FcγRs displaying different N-glycosylation profiles were generated either by different expression systems, in vitro glycoengineering or by size-exclusion chromatography, and roughly characterized by lectin blotting. Of salient interest, the overlay of their kinetics of interaction with several TZM glycoforms revealed key differences on both association and dissociation kinetics, confirming a complex influence of the FcγR N-glycosylation and its inherent heterogeneity upon receptor interaction with mAbs. This work is thus an important step towards better understanding of the impact of glycosylation upon binding of IgGs, either natural or engineered, to their receptors.     

The structure of Get4 reveals an α-solenoid fold adapted for multiple interactions in tail-anchored protein biogenesis

Tail-anchored proteins play important roles in protein translocation, membrane fusion and apoptosis. They are targeted to the endoplasmic reticulum membrane via the guided-entry of tail-anchored proteins (Get) pathway. We present the 2 Å crystal structure of Get4 which participates in early steps of the Get pathway. The structure shows an α-solenoid fold with particular deviations from the regular pairwise arrangement of α-helices. A conserved hydrophobic groove accommodates the flexible C-terminal region in trans. The structural organization of the Get4 helical hairpin motifs provides a scaffold for protein-protein interactions in the Get pathway.     

Interaction of α-taxilin Localized on Intracellular Components with the Microtubule Cytoskeleton

Intracellular vesicle traffic plays an essential role in the establishment and maintenance of organelle identity and biosynthetic transport. We have identified α-taxilin as a binding partner of the syntaxin family, which is involved in intracellular vesicle traffic. Recently, we have found that α-taxilin is over-expressed in malignant tissues including hepatocellular carcinoma and renal cell carcinoma. However, a precise role of α-taxilin in intracellular vesicle traffic and carcinogenesis remains unclear. Then, we first investigated here the intracellular distribution of α-taxilin in Hela cells. Immunofluorescence studies showed that α-taxilin distributes throughout the cytoplasm and exhibits a tubulo-vesicular pattern. Biochemical studies showed that α-taxilin is abundantly localized on intracellular components as a peripheral membrane protein. Moreover, we found that α-taxilin distributes in microtubule-dependent and syntaxin-independent manners, that α-taxilin directly binds to polymerized tubulin in vitro, and that N-ethylmaleimide but not brefeldin A affects the intracellular distribution of α-taxilin. These results indicate that α-taxilin is localized on intracellular components in a syntaxin-independent manner and that the α-taxilin-containing intracellular components are associated with the microtubule cytoskeleton and suggest that α-taxilin functions as a linker protein between the α-taxilin-containing intracellular components and the microtubule cytoskeleton.     

The ligand-binding site of bovine beta-lactoglobulin: evidence for a function?

Ever since the fortuitous observation that beta-lactoglobulin (beta-Lg), the major whey protein in the milk of ruminants, bound retinol, the details of the binding have been controversial. beta-Lg is a lipocalin, like plasma retinol-binding protein, so that ligand association was expected to make use of the central cavity in the protein. However, an early crystallographic analysis and some of the more recent solution studies indicated binding elsewhere. We have now determined the crystal structures of the complexes of the trigonal form of beta-Lg at pH 7.5 with bound retinol (R=21.4% for 7329 reflections between 20 and 2.4 A resolution, R(free)=30.6%) and with bound retinoic acid (R=22.7% for 7813 reflections between 20 and 2.34 A resolution, R(free)=29.8%). Both ligands are found to occupy the central calyx in a manner similar to retinol binding in retinol-binding protein. We find no evidence of binding at the putative external binding site in either of these structural analyses. Further, competition between palmitic acid and retinol reveals only palmitate bound to the protein. An explanation is provided for the lack of ligand binding to the orthorhombic crystal form also obtained at pH 7.5. Finally, the possible function of beta-Lg is discussed in the light of its species distribution and similarity to other lipocalins.     

The peptide chain of tyrosyl tRNA synthetase: No evidence for a super-secondary structure of four α-helices

A detailed backbone model has been built for 274 residues of tyrosyl tRNA synthetase, based on an X-ray diffraction study. This includes eight helical sections and a six-stranded pleated sheet. The four helices near the carboxyl terminal end are not arranged like the helices of TMV disk protein and hemerythrin, and the structure gives no support to the idea that four antiparallel helices form a common structural unit in proteins.     

Effects of temperature and concentration on bovine lens α-crystallin secondary structure: a circular dichroism spectroscopic study

Elucidation of the structure of α-crystallin, the major protein in all vertebrate lenses, is important for understanding its role in maintaining transparency and its function in other tissues under both normal and pathological conditions. Progress toward a unified consensus concerning the tertiary and quaternary structures of α-crystallin depends, in part, on an accurate estimation of its secondary structure. For the first time, three algorithms, SELCON, K2D and CONTIN were used to analyze far ultra-violet circular dichroism (UV–CD) spectra of bovine lens α-crystallin to estimate the secondary structure and to determine the effects of temperature and concentration. Under all experimental conditions tested, the analyses show that α-crystallin contains 14% α-helix, 35% β-sheet and the remainder, random coil and turns. The results suggest that α-crystallin is best classified as a mixed protein. In addition, increased temperature and concentration of α-crystallin result in increased α-helices with a compensatory decrease in β-sheets. Such structural alterations in α-crystallin may be functionally important during terminal differentiation of the lens fiber cells that is accompanied by increased protein concentrations and its role as a chaperone-like protein.     

Interaction of α-thalassemia genes with each other and with HbC in an American black family

The relative rates of in vitro synthesis of hemoglobin chains have been studied in an American black family in which the mother is doubly heterozygous for alpha-thalassemia and HbC and the father is heterozygous for alpha-thalassemia. The alpha/non-alpha synthetic ratio was equally unbalanced in both the bone marrow and the peripheral blood of the mother. Although HbC comprised 35% of her hemoglobin (compared to 42.2 +/- 2.2 in individuals with HbC trait and balanced globin synthesis), synthetic data showed that the newly synthesized beta C chain was 44% of the total newly synthesized beta chains. Isolated membranes contained more newly synthesized beta C than beta A chains. Three of the offspring were within the normal range, and the remaining three had alpha-thalassemia. There were two spontaneous abortions during the second trimester of pregnancy. Hydrops fetalis did not occur, and none of the children had HbH disease or HbC trait.     

13C-NMR off-resonance rotating frame spin-lattice relaxation studies of bovine lens γ-crystallin self association: effect of ‘macromolecular crowding’

The NMR technique of ¹³C off-resonance rotating frame spin-lattice relaxation, which provides an accurate assessment of the effective rotational correlation time (τ_0,eff) for macromolecular rotational diffusion, was applied to the study of γ-crystallin association as a function of protein concentration and temperature. Values of the effective rotational correlation time for γ-crystallin rotational diffusion were obtained at moderate to high protein concentrations (80–350 mg/ml) and at temperatures above, and below, the cold cataract phase transition temperature. With increasing concentration γ-crystallin was observed to increasingly associate as reflected by larger values of τ_0,eff Decreasing temperature in the range of 35 to 22°C was found to result in no change in the temperature corrected value of τ_0,eff at a γ-crystallin concentration of 80 mg/ml, whereas at temperatures of 18°C or below, this parameter was approx. twofold larger, suggesting the occurrence of a well defined phase transition, which correlated well with the cold cataract phase transition temperature. At higher protein concentrations, by contrast, τ_0,eff (temperature corrected) was found to increase by approx. 1.6- to 2-times in the temperature interval 35°C to 22°C, a result consistent with the dependence of the cold cataract phase transition temperature on γ-crystallin concentration. Analysis of intensity ratio dispersion curves, using an assumed model of isodesmic association, permitted the estimation of the association constant characterizing the aggregation under particular conditions of concentration and temperature. The significant increase in the value of the association constant with moderate increases in protein concentration was rationalized by invoking the effect of ‘macromolecular crowding’. The results obtained in this study suggest that in the intact lens, where high protein concentrations prevail, γ-crystallin is unlikely to be found in the monomeric state, but more likely, as a significantly aggregated species, representing a broad molecular weight distribution.     

Interaction of human α-lactalbumin with fatty acids: Determination of binding parameters

The interaction of holo- and apo-forms of human alpha-lactalbumin with fatty acids was studied by a partition equilibrium method. Apo-alpha-lactalbumin, obtained by treatment with EDTA, displays one binding site for fatty acids, the association constants for oleic and palmitic acids being 1.9.10(6) and 4.2.10(5) M(-1), respectively. However, holo-alpha-lactalbumin was unable to bind fatty acids as measured by this technique. Likewise, no fatty acids bound to holo-alpha-lactalbumin, isolated using nondenaturing conditions, were detected by gas chromatography. These results demonstrate that the conformational change induced in alpha-lactalbumin by the removal of calcium enables the protein to interact with fatty acids.