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PubMed2006

A potentially insect-implantable trehalose electrooxidizing anode.

The dominant sugar in the body fluids of many insects is not glucose, the sugar of the vertebrates, but trehalose. In a step toward a cell that would operate in insects, we describe here a trehalose electrooxidizing anode. The novel component of the anode is its engineered, trehalose oxidation catalyzing, FAD-glucose-3-dehydrogenase (G3DH). Screening for gene-sources of G3DH pointed to the G3DH of Agrobacterium tumefaciens. Sequencing of the A. tumefaciens genome revealed a 1.7 kb fragment which contained the G3DH coding gene. The fragment was isolated, cloned and expressed in E. coli strain BL-21, to yield the approximately 65 kDa his-tagged flavoenzyme, with a specific activity of approximately 2.5U/mg protein. Electrical wiring of its reaction center to a carbon electrode through a high apparent electron diffusion coefficient (5.8 x 10(-6)cm(2)/s) redox hydrogel with a -0.2V versus Ag/AgCl redox potential resulted in the trehalose electrooxidizing anode. Trehalose was electrooxidized at pH 7.2 already at -0.36 V versus Ag/AgCl. At 0 V versus Ag/AgCl the trehalose electrooxidation current density was 0.1 mA/cm(2).

Animals↗
PubMed2005

Trehalose differentially inhibits aggregation and neurotoxicity of beta-amyloid 40 and 42.

A key event in Alzheimer's disease (AD) pathogenesis is the conversion of the peptide beta-amyloid (Abeta) from its soluble monomeric form into various aggregated morphologies in the brain. Preventing aggregation of Abeta is being actively pursued as a primary therapeutic strategy for treating AD. Trehalose, a simple disaccharide, has been shown to be effective in preventing the deactivation of numerous proteins and in protecting cells against stress. Here, we show that trehalose is also effective in inhibiting aggregation of Abeta and reducing its cytotoxicity, although it shows differential effects toward Abeta40 and Abeta42. When co-incubated with Abeta40, trehalose inhibits formation of both fibrillar and oligomeric morphologies as determined by fluorescence staining and atomic force microscopy (AFM). However, when co-incubated with Abeta42, trehalose inhibits formation only of the fibrillar morphology, with significant oligomeric formation still present. When aggregated mixtures were incubated with SH-SY5Y cells, trehalose was shown to reduce the toxicity of Abeta40 mixtures, but not Abeta42. These results provide additional evidence that aggregation of Abeta into soluble oligomeric forms is a pathological step in AD and that Abeta42 in particular is more susceptible to forming these toxic oligomers than Abeta40. These results also suggest that the use of trehalose, a highly soluble, low-priced sugar, as part of a potential therapeutic cocktail to control Abeta peptide aggregation and toxicity warrants further study.

Alzheimer Disease↗
PubMed2005

Heterologous expression of Escherichia coli ppsA (phosphoenolpyruvate synthetase) and galU (UDP-glucose pyrophosphorylase) genes in Corynebacterium glutamicum, and its impact on trehalose synthesis.

Trehalose is a disaccharide with a wide range of applications in the food industry. We recently proposed a strategy for trehalose production based on a Corynebacterium glutamicum strain expressing the Escherichia coli enzyme UDP-glucose pyrophosphorylase (GalU). Biochemical network analysis suggest a further bottleneck for trehalose synthesis resulting from the coupling of phosphotransferase (PTS) mediated glucose uptake, and glucose catabolism in C. glutamicum. To overcome this coupling, we propose the expression of E. coli phosphoenolpyruvate synthetase (PpsA), in addition to GalU expression, in C. glutamicum. Although GalU expression improved trehalose synthesis in C. glutamicum, the simultaneous expression of GalU and PpsA did not result in a further increase in trehalose yield, but resulted in an increased catabolic rate of glucose, which could be ascribed to the operation of a futile cycle between phosphoenolpyruvate and pyruvate. The impact of GalU and PpsA expression on polysaccharide content, side product excretion and metabolic fluxes is discussed, as well as alternative ways to decouple glucose uptake and catabolism, in order to increase trehalose yield.

Corynebacterium glutamicum↗
PubMed2001

Effect of trehalose in low concentration on the binding and transport of porphyrins in liposome-human serum albumin system.

The influence of trehalose on the interaction of liposomes with porphyrins and with human serum albumin (HSA) was studied. Small unilamellar liposomes were prepared from 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and from DMPC/1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol (DMPG) 19:1 w/w% and incorporated with mesoporphyrin IX (MP) or magnesium mesoporphyrin (MgMP). The fluorescence intensity and anisotropy of porphyrins were measured within the temperature range of 15-33 degrees C, in the presence and in the absence of 3x10(-2) M trehalose, to study the location of the porphyrins inside the liposomes and their partition between the liposomes and HSA. Based on the presented data and our earlier results (I. Bárdos-Nagy, R. Galántai, A.D. Kaposi, J. Fidy, Int. J. Pharm. 175 (1998) 255-267) we conclude that trehalose - even at this relatively low concentration - interacts with the head groups of the liposomes and that the presence of DMPG enhances the effect. This effect seems to hinder the binding of HSA to the liposome surface and influences the location of MgMP within the liposomes. In the case of MP, the porphyrin partition between the liposomes and HSA was affected by trehalose, while for MgMP, trehalose changed the structural conditions of porphyrin binding to the liposomes. The amount of trehalose used did not have a general trend to modify the association constants of porphyrin derivatives either to liposomes or to HSA.

Biological Transport↗
PubMed2002

Electron transfer kinetics in photosynthetic reaction centers embedded in trehalose glasses: trapping of conformational substates at room temperature.

We report on room temperature electron transfer in the reaction center (RC) complex purified from Rhodobacter sphaeroides. The protein was embedded in trehalose-water systems of different trehalose/water ratios. This enabled us to get new insights on the relationship between RC conformational dynamics and long-range electron transfer. In particular, we measured the kinetics of electron transfer from the primary reduced quinone acceptor (Q(A)(-)) to the primary photo oxidized donor (P(+)), by time-resolved absorption spectroscopy, as a function of the matrix composition. The composition was evaluated either by weighing (liquid samples) or by near infrared spectroscopy (highly viscous or solid glasses). Deconvolution of the observed, nonexponential kinetics required a continuous spectrum of rate constants. The average rate constant ( = 8.7 s(-1) in a 28% (w/w) trehalose solution) increases smoothly by increasing the trehalose/water ratio. In solid glasses, at trehalose/water ratios > or = 97%, an abrupt increase is observed ( = 26.6 s(-1) in the driest solid sample). A dramatic broadening of the rate distribution function parallels the above sudden increase. Both effects fully revert upon rehydration of the glass. We compared the kinetics observed at room temperature in extensively dried water-trehalose matrices with the ones measured in glycerol-water mixtures at cryogenic temperatures and conclude that, in solid trehalose-water glasses, the thermal fluctuations among conformational substates are inhibited. This was inferred from the large broadening of the rate constant distribution for electron transfer obtained in solid glasses, which was due to the free energy distribution barriers having become quasi static. Accordingly, the RC relaxation from dark-adapted to light-adapted conformation, which follows primary charge separation at room temperature, is progressively hindered over the time scale of P(+)Q(A)(-) charge recombination, upon decreasing the water content. In solid trehalose-water glasses the electron transfer process resulted much more affected than in RC dried in the absence of sugar. This indicated a larger hindering of the internal dynamics in trehalose-coated RC, notwithstanding the larger amount of residual water present in comparison with samples dried in the absence of sugar.

Algorithms↗
PubMed2003

Trehalose conserves expression of bullous pemphigoid antigen 180 during desiccation and freezing.

Bullous pemphigoid antigen 180 (BP180) is targeted by autoantibodies in a variety of subepidermal blistering skin diseases. We have recently developed a simple, highly specific and sensitive immunofluorescence (IF) assay for the detection of circulating antibodies against BP180. This novel assay involves the expression of full-length (FL) BP180 in Sf21 insect cells that are then examined under IF microscopy after staining with anti-BP180 antibodies. Application of this assay as a routine diagnostic tool requires long-term storage of FL-BP180, which can result in substantial loss of expression. Here, we show that the disaccharide trehalose, a natural cryo- and lyoprotectant, is capable of preserving the FL-BP180 antigen expressed in Sf21 insect cells under various (dry) storage conditions including 40 degrees C, room temperature (RT), 4-8, -20, and -80 degrees C. The protective effect was dose-dependent reaching a maximum at about 200 mM trehalose. Trehalose was superior to other sugars or conventional cryoprotective agents (e.g. sucrose, myo-inositol, DMSO) in preventing greatly reduced antigen expression. Trehalose conserved the expression of both extra- and intracellular epitopes of FL-BP180. Interestingly, protection of the intracellular domain was only observed when trehalose was introduced into the cytosol. Trehalose significantly prolonged the storage time of FL-BP180 expressed in Sf21 insect cells, thus permitting the routine use of the IF assay in clinics for the detection of serum antibodies. The method described here has potential applications for the preservation of other transmembrane proteins.

Animals↗
PubMed2003

Enhanced conversion of soluble starch to trehalose by a mutant of Saccharomycopsis fibuligera sdu.

In our previous studies, it was found that Saccharomycopsis fibuligera sdu cells could accumulate 18.0% (gg(-1)) trehalose from soluble starch in SSY medium. However, the yeast strain contained high activities of acid and neutral trehalases, which were reported to mobilize trehalose accumulated by the cells during fermentation. In order to enhance the yield of trehalose, it is necessary to remove the trehalase activities from the cells. By mutagenesis of ethylmethanesulfonate, one mutant that assimilated trehalose very slowly, but grew on other carbon sources as fast as its parent strain, was isolated. In Biostat B2 2-1 fermentation, trehalose accumulation of the mutant was much higher than that of the wild type when grown in YPS medium containing starch. The activities of acid and neutral trehalases of this mutant were much lower than those of the wild type, respectively. We think the reduction of acid and neutral trehalase activities is considered to be responsible for the increased yield of trehalose accumulated by the mutant.

Bioreactors↗
PubMed1997

The thermodynamic mechanism of protein stabilization by trehalose.

The stabilization of ribonuclease A by alpha-alpha-trehalose was studied by preferential interaction and thermal unfolding. The protein is stabilized by trehalose at pH 2.8 and pH 5.5. Wyman linkage analysis showed increased exclusion of trehalose from the protein domain on denaturation. Preferential interaction measurements were carried out at 52 degrees C at pH 5.5 and 2.8, where the protein is native and unfolded, respectively, and at 20 degrees C where the protein is native at both pH values. At the low temperature, the interaction was preferential exclusion. At 52 degrees C the interaction was that of preferential binding, greater to the native than the unfolded protein, the increment on denaturation being identical to that deduced from the Wyman analysis. The stabilizing effect of trehalose can be fully accounted by the change in transfer free energy on unfolding. The temperature dependence of the preferential interactions of 0.5 M trehalose with ribonuclease A showed that it is the smaller preferential binding to the unfolded protein than to the native one which gives rise to the stabilization. A thermodynamic analysis of the data led to approximate values of the transfer enthalpies and transfer entropies for the trehalose-ribonuclease A system.

Hydrogen-Ion Concentration↗
PubMed1999

Involvement of the compatible solutes trehalose and sucrose in the response to salt stress of a cyanobacterial Scytonema species isolated from desert soils.

The response to moderate salt stress of a Scytonema species isolated from a soil crust in the arid region of central Australia was studied. An increase in intracellular trehalose and sucrose concentrations was detected by NMR and HPLC analysis following salt stress, maximal amounts being produced by exposure to 150 mM NaCl after 48 h. When the organism was subsequently returned to normal growth conditions, the cellular concentrations of these solutes decreased. The biosynthesis of trehalose and sucrose was studied and found, in both cases, to involve both sugar phosphate synthase and phosphatase enzymes. The combined synthase activities and the individual phosphatase activities in cell extracts were increased by salt stress. Trehalose phosphorylase was the only catabolic enzyme detected for trehalose; neither trehalase nor phosphotrehalase activities could be detected. This is the first report of trehalose phosphorylase activity in cyanobacteria. Both trehalose and sucrose phosphorylase activities increased in salt-stressed cells, whereas the activity of invertase did not change.

Chromatography, High Pressure Liquid↗
PubMed1999

Combined effects of trehalose and cations on the thermal resistance of beta-galactosidase in freeze-dried systems.

The purpose of this study was to investigate the combined effects of trehalose and cations on the preservation of beta-galactosidase in freeze-dried systems and their relationship to physical properties. Differential scanning calorimetry was employed to measure the glass transition temperature (T(g)) and the endothermal peak area, related to the amount of crystalline trehalose dihydrate present in the samples. In systems in which the trehalose matrix was humidified to conditions which allowed a high proportion of trehalose to crystallize, the enzyme was rapidly inactivated upon heating at 70 degrees C. In these conditions the addition of CsCl, NaCl and particularly KCl or MgCl(2), improved the enzyme stability with respect to that observed in matrices containing only trehalose. For a given moisture content, addition of salts produced very little change on the glass transition temperature; therefore the protective effect could not be attributed to a higher T(g) value. The crystallization of trehalose dihydrate in the humidified samples was delayed in the trehalose/salt systems (principally in the presence of Mg(2+)) and a parallel improvement of enzyme stability was observed.

Calorimetry, Differential Scanning↗
PubMed2003

Glucogenic blood sugar formation in an insect Manduca sexta L.: asymmetric synthesis of trehalose from 13C enriched pyruvate.

Gluconeogenesis and blood sugar formation were examined in Manduca sexta, fed carbohydrate- and fat-free diets with varying levels of casein. De novo carbohydrate synthesis was examined by nuclear magnetic resonance spectroscopy of the 13C enrichment in blood trehalose and alanine derived from (2-(13)C)pyruvate and (2,3-(13)C(2))pyruvate administered to 5th instar larvae. Gluconeogenic flux and blood trehalose concentration were positively correlated with protein consumption. On all diets, the 13C distribution in trehalose was asymmetric, with C6 more highly enriched than C1. The C6/C1 13C enrichment ratio, however, decreased with increased protein consumption and gluconeogenic flux. Although the asymmetric 13C enrichment pattern in trehalose is consistent with pentose cycling via the pentose phosphate pathway following de novo synthesis, experiments employing [2,3-(13)C(2)]pyruvate demonstrated that pentose cycling is not detected in insects under these nutritional conditions. Analysis of the multiplet NMR signal structure in trehalose due to spin-spin coupling between adjacent 13C enriched carbons showed the absence of uncoupling expected by pentose phosphate pathway activity. Here we suggest that the asymmetric 13C distribution in trehalose results from a disequilibrium of the triose phosphate isomerase-catalyzed reaction.

Animals↗
PubMed1998

Multiple effects of trehalose on protein folding in vitro and in vivo.

The disaccharide trehalose is produced in large quantities by diverse organisms during a variety of stresses. Trehalose prevents proteins from denaturing at high temperatures in vitro, but its function in stress tolerance in vivo is controversial. We report that trehalose stabilizes proteins in yeast cells during heat shock. Surprisingly, trehalose also suppresses the aggregation of denatured proteins, maintaining them in a partially-folded state from which they can be activated by molecular chaperones. The continued presence of trehalose, however, interferes with refolding, suggesting why it is rapidly hydrolyzed following heat shock. These findings reconcile conflicting reports on the role of trehalose in stress tolerance, provide a novel tool for accessing protein folding intermediates, and define new parameters for modulating stress tolerance and protein aggregation.

Animals↗
PubMed2003

Trehalose favors a cutinase compact intermediate off-folding pathway.

The folding of cutinase, an enzyme displaying lipolytic activity, has been studied in the presence of trehalose. Equilibrium unfolding data show that trehalose increases the free energy change between folded and unfolded states. Unfolding kinetics reveal the presence of an intermediate which is ca. 60% folded in terms of solvent exposure. Trehalose stabilizes this intermediate relative to the folded state. In contrast, the intermediate revealed by folding kinetics is more compact than the transition state, as shown by the positive slope observed at low denaturant concentration in the chevron plot, as well as the decrease in the observable rate constant for folding with the increase in trehalose concentration. This intermediate displays more than 50% of area buried from the solvent (relative to the native state) compared to around 40% for the transition state for folding and therefore appears to be off the folding pathway. Trehalose stabilizes and guanidine hydrochloride destabilizes this compact intermediate. Both unfolding and folding kinetics show that compact conformational states are stabilized by trehalose, in agreement with current models on the effect of compatible solutes. This effect occurs even for compact states that decelerate the folding as in the case of the intermediate revealed by folding kinetics.

Carboxylic Ester Hydrolases↗
PubMed2006

Inertial suppression of protein dynamics in a binary glycerol-trehalose glass.

The traditional approach used to predict the ability of a glassy matrix to maximally preserve the activity of a protein solute is the glass transition temperature (T(g)) of the glass. Recently it has been shown that the addition of a low T(g) diluent (glycerol) can rigidify the structure of a high T(g) glassy matrix in binary glycerol-trehalose glasses. The optimal density of glycerol in trehalose minimizes the average mean square displacements of non-exchangeable protons in the glass samples. The amount of glycerol added to a trehalose glass coincides with the maximal recovery of biological activity in a separate study using similar binary glass samples. In this study, we use molecular dynamics (MD) simulations to investigate the dynamics of a hydrated protein encased in glycerol, unary trehalose and binary glycerol-trehalose glasses. We have found that we are able to reproduce the rigidification of the glycerol-trehalose glassy matrix and that there is a direct correlation between bulk glass dynamics and the extent of atomic fluctuation of protein atoms. The detailed microscopic picture that emerges is that protein dynamics are suppressed mainly by inertia of the bulk glass and to a lesser extent specific interactions at the protein-solvent interface. Thus, the inertia of the glassy matrix may be an influential factor in the determination of pharmaceutically relevant formulations.

Glass↗
PubMed1999

Trehalose accumulation by tropical yeast strains submitted to stress conditions.

Trehalose, a non-reducing disaccharide that accumulates in Saccharomyces cerevisiae, has been implicated in survival under various stress conditions by acting as membrane protectant, as a supplementary compatible solute or as a reserve carbohydrate which may be mobilized during stress. However, most of these studies have been done with strains isolated from European or Asian habitats of temperate climate. In this study, yeasts living in tropical environments, isolated from different microhabitats in Southeastern Brazil, were used to evaluate whether trehalose contributes to survival under osmotic, ethanol and heat stress. The survival under severe stress was compared to a well-characterized laboratorial wild-type strain (D273-10B). Most of the Saccharomyces cerevisiae strains isolated from Drosophila in Tropical Rain Forest were able to accumulate trehalose after a preconditioning treatment at 40 degrees C for 1 h. The amount of intracellular trehalose levels was better correlated with survival during a challenging heat shock at 50.5 degrees C for 8 min. Saccharomyces cerevisiae and Candida guilliermondii were observed to be thermotolerant as well as osmotolerant. No clear correlation between intracellular trehalose levels and survival could be derived during ethanol stress. In some cases, the amount of trehalose accumulated before the ethanol stress seemed to play an important role for the survival of these strains.

Animals↗
PubMed1997

Thermophysical properties of trehalose and its concentrated aqueous solutions.

PURPOSE: To address the lack of fundamental thermophysical data for trehalose and its aqueous systems by measuring equilibrium and non-equilibrium properties of such systems. METHODS/RESULTS: Differential scanning calorimetry (DSC) and dynamic mechanical analysis were used to measure glass transition temperatures of trehalose and its solutions. X-ray diffractometry was used to verify the structure of amorphous trehalose. Controlled-stress rheometry was used to measure viscosity of several aqueous trehalose systems at ambient and sub-ambient temperatures. Over this temperature range, the density of these solutions was also measured with a vibrating tube densimeter. The equilibrium phase diagram of aqueous trehalose was determined by measuring the solubility and freezing point depression. CONCLUSIONS: Our solubility measurements, which have allowed long times for attainment of chemical equilibrium, are substantially different from those reported earlier that used different techniques. Our measurements of the glass transition temperature of trehalose are higher than reported values. A simple model for the glass transition is presented to describe our experimental observations.

Calorimetry, Differential Scanning↗
PubMed2004

Adoption of long-term cultures to evaluate the cryoprotective potential of trehalose for freezing hematopoietic stem cells.

Dimethylsulfoxide (DMSO), which is widely used as a cryoprotectant for hematopoietic stem cells (HSC), has considerable toxicity for both the thawed cells and the patient. The aim of this study was to evaluate the cryoprotective potential of trehalose in comparison to DMSO for human HSC. Human bone marrow (BM) and peripheral blood stem cells (PBSC) of volunteer donors were cryopreserved in the presence of different concentrations of trehalose with and without insulin, as well as with DMSO 10%. After thawing to 37 degrees C colony-forming unit (CFU) assays were performed. Long-term marrow-cultures (LTMC) were established and used for the detection of long-term culture-initiating cells (LTCIC). The total amount of CFUs detected was 104+/-134 (mean+/-s.d.) in DMSO-preserved cells and 179+/-34 in trehalose-protected cells. For LTMC the best feeder layer proved to be fresh human BM and the most useful concentration of trehalose was 0.5 M. Using these culture conditions we could detect after 5 weeks LTMC a total of 172+/-28 CFUs for trehalose-protected cells and 170+/-52 for DMSO-preserved cells. In conclusion, trehalose exerts a similar cryoprotective potential for hematopoietic progenitor and stem cells like DMSO and could possibly replace DMSO at least in part as cryoprotectant in the setting of hematopoietic cell transplantation.

Bone Marrow Cells↗
PubMed2005

Trehalose metabolism and glucose sensing in plants.

Plants sense and respond to changes in carbon and nitrogen metabolites during development and growth according to the internal needs of their metabolism. Sugar-sensing allows plants to switch off photosynthesis when carbohydrates are abundant. These processes involve regulation of gene and protein activity to allow plants the efficient use of energy storage. Besides being a key element in carbon metabolism, glucose (Glc) has unravelled as a primary messenger in signal transduction. It has been proved that hexokinase (HXK) is a Glc sensor. An unusual disaccharide named trehalose is present in very low levels in most plants except for the desiccation-tolerant plants known as 'resurrection' plants where trehalose functions as an osmoprotectant. We have shown that overexpression of the Arabidopsis trehalose-6-phosphate synthase gene (AtTPS1) in Arabidopsis promotes trehalose and trehalose-6-phosphate (T6P) accumulation. Seedlings expressing AtTPS1 displayed a Glc-insensitive phenotype. Transgenic lines germinated normally on Glc, in contrast to wild-type seedlings showing growth retardation and absence of chlorophyll and root elongation. Gene-expression analysis in transgenic plants showed up-regulation of several genes involved in sugar signalling and metabolism. These data suggest that AtTPS1 and accordingly T6P and trehalose play an important role in the regulation of Glc sensing and signalling genes during plant development.

Arabidopsis↗