Key amino acid residues of the AGT1 permease required for maltotriose consumption and fermentation by Saccharomyces cerevisiae.

AIMS: The AGT1 gene encodes for a general α-glucoside-H+ symporter required for efficient maltotriose fermentation by Saccharomyces cerevisiae. In the present study, we analysed the involvement of four charged amino acid residues present in this transporter that are required for maltotriose consumption and fermentation by yeast cells. METHODS AND RESULTS: By using a knowledge-driven approach based on charge, conservation, location, three-dimensional (3D) structural modelling and molecular docking analysis, we identified four amino acid residues (Glu-120, Asp-123, Glu-167 and Arg-504) in the AGT1 permease that could mediate substrate binding and translocation. Mutant permeases were generated by site-directed mutagenesis of these charged residues, and expressed in a yeast strain lacking this permease (agt1∆). While mutating the Arg-504 or Glu-120 residues into alanine totally abolished (R504A mutant) or greatly reduced (E120A mutant) maltotriose consumption by yeast cells, as well as impaired the active transport of several other α-glucosides, in the case of the Asp-123 and Glu-167 amino acids, it was necessary to mutate both residues (D123G/E167A mutant) in order to impair maltotriose consumption and fermentation. CONCLUSIONS: Based on the results obtained with mutant proteins, molecular docking and the localization of amino acid residues, we propose a transport mechanism for the AGT1 permease. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results present new insights into the structural basis for active α-glucoside-H+ symport activity by yeast transporters, providing the molecular bases for improving the catalytic properties of this type of sugar transporters.

Shared control of maltose and trehalose utilization in Candida utilis.

Trehalose biosynthesis and its hydrolysis have been extensively studied in yeast, but few reports have addressed the catabolism of exogenously supplied trehalose. Here we report the catabolism of exogenous trehalose by Candida utilis. In contrast to the biphasic growth in glucose, the growth of C. utilis in a mineral medium with trehalose as the sole carbon and energy source is aerobic and exhibits the Kluyver effect. Trehalose is transported into the cell by an inducible trehalose transporter (K M of 8 mM and V MAX of 1.8 mol trehalose min-1 mg cell (dry weight)-1. The activity of the trehalose transporter is high in cells growing in media containing trehalose or maltose and very low or absent during the growth in glucose or glycerol. Similarly, total trehalase activity was increased from about 1.0 mU/mg protein in cells growing in glucose to 39.0 and 56.2 mU/mg protein in cells growing in maltose and trehalose, respectively. Acidic and neutral trehalase activities increased during the growth in trehalose, with neutral trehalase contributing to about 70% of the total activity. In addition to the increased activities of the trehalose transporter and trehalases, growth in trehalose promoted the increase in the activity of alpha-glucosidase and the maltose transporter. These results clearly indicate that maltose and trehalose promote the increase of the enzymatic activities necessary to their catabolism but are also able to stimulate each other's catabolism, as reported to occur in Escherichia coli. We show here for the first time that trehalose induces the catabolism of maltose in yeast.

The use of protein structure/activity relationships in the rational design of stable particulate delivery systems.

The recombinant heat shock protein (18 kDa-hsp) from Mycobacterium leprae was studied as a T-epitope model for vaccine development. We present a structural analysis of the stability of recombinant 18 kDa-hsp during different processing steps. Circular dichroism and ELISA were used to monitor protein structure after thermal stress, lyophilization and chemical modification. We observed that the 18 kDa-hsp is extremely resistant to a wide range of temperatures (60% of activity is retained at 80 degrees C for 20 min). N-Acylation increased its ordered structure by 4% and decreased its beta-T1 structure by 2%. ELISA demonstrated that the native conformation of the 18 kDa-hsp was preserved after hydrophobic modification by acylation. The recombinant 18 kDa-hsp resists to a wide range of temperatures and chemical modifications without loss of its main characteristic, which is to be a source of T epitopes. This resistance is probably directly related to its lack of organization at the level of tertiary and secondary structures.

Maltotriose fermentation by Saccharomyces cerevisiae.

Maltotriose, the second most abundant sugar of brewer's wort, is not fermented but is respired by several industrial yeast strains. We have isolated a strain capable of growing on a medium containing maltotriose and the respiratory inhibitor, antimycin A. This strain produced equivalent amounts of ethanol from 20 g l(-1) glucose, maltose, or maltotriose. We performed a detailed analysis of the rates of active transport and intracellular hydrolysis of maltotriose by this strain, and by a strain that does not ferment this sugar. The kinetics of sugar hydrolysis by both strains was similar, and our results also indicated that yeast cells do not synthesize a maltotriose-specific alpha-glucosidase. However, when considering active sugar transport, a different pattern was observed. The maltotriose-fermenting strain showed the same rate of active maltose or maltotriose transport, while the strain that could not ferment maltotriose showed a lower rate of maltotriose transport when compared with the rates of active maltose transport. Thus, our results revealed that transport across the plasma membrane, and not intracellular hydrolysis, is the rate-limiting step for the fermentation of maltotriose by these Saccharomyces cerevisiae cells.

Kinetics of active alpha-glucoside transport in Saccharomyces cerevisiae.

alpha-Glucosides are the most abundant fermentable sugars in the industrial applications of Saccharomyces cerevisiae, and the active transport across the plasma membrane is the rate-limiting step for their metabolism. In this report we performed a detailed kinetic analysis of the active alpha-glucoside transport system(s) present in a wild-type strain, and in strains with defined alpha-glucoside permeases. Our results indicate that the wild-type strain harbors active transporters with high and low affinity for maltose and trehalose, and low-affinity transport systems for maltotriose and alpha-methylglucoside. The maltose permease encoded by the MAL21 gene showed a high affinity (K(m) approximately 5 mM) for maltose, and a low affinity (K(m) approximately 90 mM) for trehalose. On the other hand, the alpha-glucoside permease encoded by the AGT1 gene had a high affinity (K(m) approximately 7 mM) for trehalose, a low affinity (K(m) approximately 18 mM) for maltose and maltotriose, and a very low affinity (K(m) approximately 35 mM) for alpha-methylglucoside.

Large unilamellar vesicles as trehalose-stabilised vehicles for vaccines: storage time and in vivo studies.

Liposomes, as a pharmaceutical formulation must display a long shelf life. The recombinant heat-shock protein from Mycobacterium leprae (18-kDa hsp) or its N-acylated derivative, when entrapped within or externally associated with large unilamellar vesicles, acts as a T-epitope source. Freeze-fracture electron microscopy shows unequivocally that trehalose avoids aggregation and fusion of these vesicles. Formulations containing trehalose retained up to 98% of the entrapped protein. The highest antibody level is obtained with formulations containing trehalose. The adjuvant effect depends on the liposomal membrane integrity.

Inactivation of maltose permease and maltase in sporulating Saccharomyces cerevisiae.

Maltose transport and maltase activities were inactivated during sporulation of a MAL constitutive yeast strain harboring different MAL loci. Both activities were reduced to almost zero after 5 h of incubation in sporulation medium. The inactivation of maltase and maltose permease seems to be related to optimal sporulation conditions such as a suitable supply of oxygen and cell concentration in the sporulating cultures, and occurs in the fully derepressed conditions of incubation in the sporulation acetate medium. The inactivation of maltase and maltose permease under sporulation conditions in MAL constitutive strains suggests an alternative mechanism for the regulation of the MAL gene expression during the sporulation process.

Kinetics of active sucrose transport in Saccharomyces cerevisiae.

The kinetic analysis of active sucrose-H+ uptake by Saccharomyces cerevisiae revealed the presence of two transport systems with high and low affinity for sucrose. The MAL2T permease has a low affinity (K(m) = 120 +/-20 mM) for sucrose, while the alpha-glucoside transporter encoded by the AGT1 gene is a high affinity sucrose-H+ symporter (K(m) = 7.9+/-0.8 mM) that increases the specific growth rate of cells growing on sucrose.

Preservation of frozen yeast cells by trehalose.

Two different methods commonly used to preserve intact yeast cells-freezing and freeze-drying-were compared. Different yeast cells submitted to these treatments were stored for 28 days and cell viability assessed during this period. Intact yeast cells showed to be less tolerant to freeze-drying than to freezing. The rate of survival for both treatments could be enhanced by exogenous trehalose (10%) added during freezing and freeze-drying treatments or by a combination of two procedures: a pre-exposure of cells to 40 degrees C for 60 min and addition of trehalose. A maximum survival level of 71.5 +/- 6.3% after freezing could be achieved at the end of a storage period of 28 days, whereas only 25.0 +/- 1.4% showed the ability to tolerate freeze-drying treatment, if both low-temperature treatments were preceded by a heat exposure and addition of trehalose to yeast cells. Increased survival ability was also obtained when the pre-exposure treatment of yeast cells was performed at 10 degrees C for 3 h and trehalose was added: these treatments enhanced cell survival following freezing from 20.5 +/- 7. 7% to 60.0 +/- 3.5%. Although both mild cold and heat shock treatments could enhance cell tolerance to low temperature, only the heat treatment was able to increase the accumulation of intracellular trehalose whereas, during cold shock exposure, the intracellular amount of trehalose remained unaltered. Intracellular trehalose levels seemed not to be the only factor contributing to cell tolerance against freezing and freeze-drying treatments; however, the protection that this sugar confers to cells can be exerted only if it is to be found on both sides of the plasma membrane.

Active alpha-glucoside transport in Saccharomyces cerevisiae.

The AGT1 permease is a alpha-glucoside-H+ symporter responsible for the active transport of maltose, trehalose, maltotriose, alpha-methylglucoside, melezitose and sucrose. In wild-type as well as in MAL constitutive strains, alpha-methylglucoside seemed to be the best inducer of transport activity, while trehalose had no inducing effect. Based on the initial rates of transport it seems that the sugar preferentially transported by this permease is trehalose, followed by sucrose.

Sphingomyelinases in the venom of the spider Loxosceles intermedia are responsible for both dermonecrosis and complement-dependent hemolysis.

The bite of spiders of the genus Loxosceles can induce a variety of biological effects, including dermonecrosis and complement (C) dependent haemolysis. The aim of this study was to characterise the toxins in the venom responsible for the different biological effects. We have previously shown that a 35 kDa protein, named F35, purified from Loxosceles intermedia venom, incorporates into the membranes of human erythrocytes and renders them susceptible to the alternative pathway of autologous C. Here we have further purified the F35 protein which was resolved by reversed phase chromatography into three tightly contiguous peaks termed P1, P2, and P3. P1 and P2 were shown to be homogeneous by SDS-PAGE and N-terminal aminoacid analysis, while P3 consisted of two highly homologous proteins. N-terminal sequencing of all four proteins showed a high degree of homology, which was confirmed by cross-reactivity of antisera raised against the individual purified proteins. Functional characterisation of P1 and P2 indicated the presence of sphingomyelinase activity and either protein in isolation was capable of inducing all the in vivo effects seen with whole spider venom, including C-dependent haemolysis and dermonecrosis. In all assays, P2 was more active than P1, while P3 was completely inactive. These data show that different biological effects of L. intermedia venom can be assigned to the sphingomyelinase activity of two highly homologous proteins, P1 and P2. Identification of these proteins as inducers of the principal pathological effects induced by whole venom will aid studies of the mechanism of action of the venom and the development of a effective therapy.

Conformational stability and antibody response to the 18kDa heat-shock protein formulated into different vehicles.

Protein stability is one of the most important obstacles for successful formulation in the development of new-generation vaccines. Here, the 18kDa heat-shock protein (18kDa-hsp) was chemically modified though conjugation with bovine serum albumin or by esterification with N-hydroxysuccinimide ester of palmitic acid. The biologically active conformation of the protein was preserved after chemical modification. The immune responses to the recombinant 18kDa-hsp from Mycobacterium leprae were studied in different presentations: free, copolymerized with bovine serum albumin in aggregates (18kDa-hsp-BSA), and either surface linked to liposomes or entrapped into liposomes. Measuring the antibody production of immunized genetically selected mice has compared the adjuvant effects of liposomes and proteic copolymer. Among the two liposome preparations, the strongest response was obtained with the surface-exposed antigen-liposomes. The copolymer 18kDa-hsp-BSA conferred a high titer of antibody in injected mice, and persisted 70 d after immunization. This approach should prove very useful for designing more effective vaccines by using 18kDa-hsp as carrier protein.

Expression of high-affinity trehalose-H+ symport in Saccharomyces cerevisiae.

The expression of the high-affinity trehalose-H+ symport was investigated in various Saccharomyces cerevisiae strains and culture conditions. Previous kinetic studies of trehalose transport in yeast have revealed the existence of at least two different uptake mechanisms: a high-affinity trehalose-H+ symport activity repressed by glucose, and a constitutive low-affinity transport activity, a putative facilitated diffusion process. Exogenously added trehalose was not an inducer of the high-affinity transport activity, and a correlation between trehalose and maltose uptake by yeast cells was found. Our results indicate that the maltose-H+ symporters encoded by MAL11, MAL21, and MAL41 are not responsible for the trehalose transport activity. The analysis of both trehalose and maltose transport activities in wild-type and in laboratory strains with defined MAL genes showed that the trehalose-H+ symporter was under control of MAL regulatory genes. Our results also suggest that the recently characterized AGT1 gene of S. cerevisiae may encode the high-affinity trehalose-H+ symporter. During diauxic growth on glucose the transport activity was low during the first exponential phase of growth, increased as glucose was exhausted from the medium, and decreased again as the cells reached the late stationary phase. This pattern was coincident with that of the intracellular levels of trehalose. The strong correlation between these two parameters may be of physiological significance during adaptation of yeast cells to stress conditions.

Arbutin inhibits PLA2 in partially hydrated model systems.

Arbutin is a glycosylated hydroquinone found at high concentrations in certain plants capable of surviving extreme and sustained dehydration. In this paper, we examine a potential role of this molecule in anhydrobiosis. We have studied its effects on the physical properties of phospholipids and on preservation of liposomes during drying. Arbutin depresses the gel to liquid crystalline phase transition temperature of dry phospholipids, as measured by differential scanning calorimetry, with a pattern similar to that seen in phospholipids dried with the disaccharide trehalose. Unlike trehalose, however, arbutin does not protect dry liposomes from leaking their contents. Also, using Fourier transform infrared spectroscopy, we found an increase in the vibrational frequency of the phosphate asymmetric stretch in partially hydrated phospholipids in the presence of arbutin. Trehalose, by contrast, depresses the frequency of the phosphate in dry phospholipids, indicating that the modes of interaction of trehalose and arbutin with the bilayer are different. Previously, we have shown that phospholipases can be active in liposomes with surprisingly low water contents. Based on the structural similarity of arbutin to a known inhibitor of phospholipase A2 (PLA2), it appeared possible that arbutin might serve as an inhibitor of phospholipases. Liposomes of varying composition were lyophilized in the presence and absence of phospholipases. When the liposomes were partially rehydrated at 76% relative humidity, arbutin inhibited PLA2, but did not inhibit phospholipases B or C. Accumulation of enzyme product in the liposome membranes was measured by analytical thin layer chromatography, and was taken as a measure of enzyme activity. Arbutin did not inhibit any of the enzymes in the presence of excess water. Based on these data, hypotheses are presented concerning the mechanism of PLA2 inhibition by arbutin in the mostly dehydrated state.

The role of trehalose in cell stress.

Water is usually thought to be required for the living state, but many organisms can withstand anhydrobiosis when essentially all of their body water has been removed. The mechanisms for survival to this kind of stress could be similar in microbes, plants and animals. One common feature is the accumulation of sugars by anhydrobiotic organisms. Trehalose, which is one of the most effective saccharides in preventing phase transition events in the lipid bilayer, is accumulated by anhydrobiotic organisms in large amounts. It lowers membrane phase transitions in dry yeast cells, thus preventing imbibitional damages when cells are rehydrated. Yeast cells have a trehalose carrier in the plasma membrane which endows them with the ability to protect both sides of the membrane. Kinetic analysis of the trehalose transport activity in Saccharomyces cerevisiae cells revealed the existence of a multicomponent system with a constitutive low-affinity uptake component and a high-affinity H(+)-trehalose symporter regulated by glucose repression.

Kinetics and energetics of trehalose transport in Saccharomyces cerevisiae.

Cells of Saccharomyces cerevisiae are able to transport trehalose against a concentration gradient, without efflux or counterflow of the labeled substrate. Uptake was inhibited by uncouplers, acetic acid, and organic mercury compounds. The addition of trehalose resulted in alkalinization of the medium. The ratio of H+ depletion to trehalose uptake by yeast cells was approximately 1:1, which indicates the existence of a trehalose-H+ symporter in these cells. The optimum pH for this active H+-trehalose symport was 5.0, and both the Km and the Vmax were negatively affected by increasing or decreasing the extracellular pH from its optimum value. Kinetic studies showed the existence of at least two different trehalose transport activities in yeast cells: a high-affinity H+-trehalose symporter (Km = 4 mM), and a low-affinity transport activity (Km > 100 mM) that could be a facilitated diffusion process. The high-affinity H+-trehalose symporter was repressed by glucose, whereas the low-affinity uptake was constitutively expressed in S. cerevisiae.

Phospholipase A2 activity in dehydrated systems: effect of the physical state of the substrate.

In the presence of excess water, enzymatic activity of phospholipase A2 (PLA2) depends on the physical state of the lipid substrate. In order to determine if this also holds true in dehydrated systems, the physical parameters of charge, hydration state, and head group spacing of liposome membranes and their effects on PLA2 lipid hydrolysis were studied. Liposomes of varying composition were freeze-dried in the presence of PLA2 and partially rehydrated at controlled relative humidities. Accumulation of free fatty acids in the liposomal membranes was used as a measure of PLA2 activity. We found that PLA2, which was not activated during lyophilization, was most active during partial rehydration of the liposomes. The hydration state, charge and headgroup spacing of the membrane were all important in determining PLA2 activity in the dehydrated system.

A dependable method for the synthesis of [14C]trehalose.

A new method for the preparation of [14C]trehalose was developed, based on the ability of yeast cells to accumulate trehalose under stress. The method is simple and reliable. It utilizes a yeast strain in which the gene that encodes for phosphoglucoisomerase has been deleted. Thus, exogenously supplied glucose is not metabolized, but is instead converted to trehalose. The [14C]-trehalose obtained is pure, it is hydrolyzed by trehalase, and it is not susceptible to the action of alpha-glucosidase. The yield of this method is in the order of 35% of the [14C]glucose supplied.

The interaction of Saccharomyces cerevisiae trehalase with membranes.

Plasma membranes isolated from cells of Saccharomyces cerevisiae previously submitted to a heat-shock showed a 10-fold increase in membrane-bound trehalase activity. Trehalase was purified to a high specific activity and was shown to be inhibited by glucose 6-phosphate and by the addition of a neutral phospholipid-like surfactant. Purified trehalase binds spontaneously to egg phosphatidylcholine small unilamellar vesicles, when in its active, phosphorylated form. When the enzyme was treated with alkaline phosphatase no binding was observed. The significance of this reversible binding for the control of trehalose metabolism in yeast cells is still unknown.

Correlation between free bilirubin and indirect bilirubin in normal newborn infants with non-hemolytic jaundice and effect of hemolysis on free bilirubin measurement by the peroxidase method.

The present study was undertaken to determine the correlation between free bilirubin and indirect bilirubin in normal newborn infants with non-hemolytic jaundice, and the possible effect of hemolysis on free bilirubin measurement by the peroxidase method. A prospective study protocol was applied at the Neonatal Unit of the Department of Pediatrics, Escola Paulista de Medicina. Forty-three newborn infants were submitted to measurement of free bilirubin and bilirubin fractions and the extent of hemolysis of the sample was determined. Data were analyzed statistically by the Student t-test. A positive and moderate correlation (r = 0.668; p < 0.01) was detected between free bilirubin and indirect bilirubin. The linear regression equation calculated by the least squares method was as follows: f(x) = 4.562 + 0.382x. The concentration of free bilirubin was inversely proportional to sample hemolysis, the difference being greater at 50 mg/dl hemolysis. Despite these results, however, the use of this correlation is delicate due to the impossibility of establishing it in individual cases. Also, since the samples may show some degree of hemolysis, this factor should be minimized by appropriate sample collection before free bilirubin measurement.