Galactose Flux Research Articles

Hexose transporter SWEET5 confers galactose sensitivity to Arabidopsis pollen germination via a galactokinase.

Galactose is an abundant and essential sugar used for the biosynthesis of many macromolecules in different organisms, including plants. Galactose metabolism is tightly and finely controlled, since excess galactose and its derivatives are inhibitory to plant growth. In Arabidopsis (Arabidopsis thaliana), root growth and pollen germination are strongly inhibited by excess galactose. However, the mechanism of galactose-induced inhibition during pollen germination remains obscure. In this study, we characterized a plasma membrane-localized transporter, Arabidopsis Sugars Will Eventually be Exported Transporter 5, that transports glucose and galactose. SWEET5 protein levels started to accumulate at the tricellular stage of pollen development and peaked in mature pollen, before rapidly declining after pollen germinated. SWEET5 levels are responsible for the dosage-dependent sensitivity to galactose, and galactokinase is essential for these inhibitory effects during pollen germination. However, sugar measurement results indicate that galactose flux dynamics and sugar metabolism, rather than the steady-state galactose level, may explain phenotypic differences between sweet5 and Col-0 in galactose inhibition of pollen germination.

Harnessing the nutritional potential of concentrated whey for enhanced galactose flux in fermentative yeast

The primary effluent of Indian dairy industries, cheese whey, and its main component lactose is considered an environmental pollutant due to its high chemical and biological oxygen demand. The model fermentative yeast, Saccharomyces cerevisiae, does not possess the necessary enzyme to utilize lactose in whey. In the present study, metabolic engineering of wild type S. cerevisiae strain with CRISPR Cas9 system was used to construct a cell factory that can flux higher galactose from the concentrated whey medium. The improved utilization of galactose confers fermentative metabolism with improved ethanol production rate. The strain provided a high titer of the ethanol (0.32 ± 0.007 g/g) with improved galactose flux from concentrated whey at the expense of the biomass, glycerol, and acetic acid. Our present study provides evidence that galactose to ethanol fermentation is a feasible alternative for industrial waste stream treatment in developing countries.

Cross-utilization of β-galactosides and cellobiose in Geobacillus stearothermophilus

Strains of the Gram-positive, thermophilic bacterium Geobacillus stearothermophilus possess elaborate systems for the utilization of hemicellulolytic polysaccharides, including xylan, arabinan, and galactan. These systems have been studied extensively in strains T-1 and T-6, representing microbial models for the utilization of soil polysaccharides, and many of their components have been characterized both biochemically and structurally. Here, we characterized routes by which G. stearothermophilus utilizes mono- and disaccharides such as galactose, cellobiose, lactose, and galactosyl-glycerol. The G. stearothermophilus genome encodes a phosphoenolpyruvate carbohydrate phosphotransferase system (PTS) for cellobiose. We found that the cellobiose-PTS system is induced by cellobiose and characterized the corresponding GH1 6-phospho-β-glucosidase, Cel1A. The bacterium also possesses two transport systems for galactose, a galactose-PTS system and an ABC galactose transporter. The ABC galactose transport system is regulated by a three-component sensing system. We observed that both systems, the sensor and the transporter, utilize galactose-binding proteins that also bind glucose with the same affinity. We hypothesize that this allows the cell to control the flux of galactose into the cell in the presence of glucose. Unexpectedly, we discovered that G. stearothermophilus T-1 can also utilize lactose and galactosyl-glycerol via the cellobiose-PTS system together with a bifunctional 6-phospho-β-gal/glucosidase, Gan1D. Growth curves of strain T-1 growing in the presence of cellobiose, with either lactose or galactosyl-glycerol, revealed initially logarithmic growth on cellobiose and then linear growth supported by the additional sugars. We conclude that Gan1D allows the cell to utilize residual galactose-containing disaccharides, taking advantage of the promiscuity of the cellobiose-PTS system.

Unravelling evolutionary strategies of yeast for improving galactose utilization through integrated systems level analysis

Identification of the underlying molecular mechanisms for a derived phenotype by adaptive evolution is difficult. Here, we performed a systems-level inquiry into the metabolic changes occurring in the yeast Saccharomyces cerevisiae as a result of its adaptive evolution to increase its specific growth rate on galactose and related these changes to the acquired phenotypic properties. Three evolved mutants (62A, 62B, and 62C) with higher specific growth rates and faster specific galactose uptake were isolated. The evolved mutants were compared with a reference strain and two engineered strains, SO16 and PGM2, which also showed higher galactose uptake rate in previous studies. The profile of intermediates in galactose metabolism was similar in evolved and engineered mutants, whereas reserve carbohydrates metabolism was specifically elevated in the evolved mutants and one evolved strain showed changes in ergosterol biosynthesis. Mutations were identified in proteins involved in the global carbon sensing Ras/PKA pathway, which is known to regulate the reserve carbohydrates metabolism. We evaluated one of the identified mutations, RAS2(Tyr112), and this mutation resulted in an increased specific growth rate on galactose. These results show that adaptive evolution results in the utilization of unpredicted routes to accommodate increased galactose flux in contrast to rationally engineered strains. Our study demonstrates that adaptive evolution represents a valuable alternative to rational design in bioengineering of improved strains and, that through systems biology, it is possible to identify mutations in evolved strain that can serve as unforeseen metabolic engineering targets for improving microbial strains for production of biofuels and chemicals.

Pathophysiology of impaired ovarian function in galactosaemia

Classical galactosaemia is an inherited inborn error of the major galactose assimilation pathway, caused by galactose-1-phosphate uridyltransferase (GALT) deficiency. Many GALT mutations have been described, with different clinical consequences. In severe forms, newborns present with a life-threatening, acute toxic syndrome that rapidly regresses under a galactose-restricted diet. However, long-term complications, particularly cognitive and motor abnormalities, as well as hypergonadotrophic hypogonadism in female patients are still unavoidable. The pathogenesis of galactose-induced ovarian toxicity remains unclear but probably involves galactose itself and its metabolites such as galactitol and UDP-galactose. Possible mechanisms of ovarian damage include direct toxicity of galactose and metabolites, deficient galactosylation of glycoproteins and glycolipids, oxidative stress and activation of apoptosis. As there is no aetiological treatment, clinical management of ovarian failure in galactosaemic patients principally relies on hormonal replacement therapy to induce pubertal development and to prevent bone loss and other consequences of estrogen deprivation. Further investigations will be necessary to better understand the metabolic flux of galactose through its biochemical pathways and the mechanisms of these secondary complications. The aim of this article is to present an extensive review on the pathogenesis and clinical management of galactose-induced premature ovarian failure.

Galactose Biosynthesis in Arabidopsis: Genetic Evidence for Substrate Channeling from UDP-D-Galactose into Cell Wall Polymers

The biosynthesis of plant cell wall polysaccharides requires the concerted action of nucleotide sugar interconversion enzymes, nucleotide sugar transporters, and glycosyl transferases [1]. How cell wall synthesis in planta is regulated, however, remains unclear. The root epidermal bulger 1 (reb1) mutant in Arabidopsis thaliana[2] is partially deficient in cell wall arabinogalactan-protein (AGP), indicating a role for REB1 in AGP biosynthesis [3]. We show that REB1 is allelic to ROOT HAIR DEFICIENT 1 (RHD1)[4], one of five ubiquitously expressed genes that encode isoforms of UDP-D-glucose 4-epimerase (UGE), an enzyme that acts in the formation of UDP-D-galactose (UDP-D-Gal). The RHD1 isoform is specifically required for the galactosylation of xyloglucan (XG) and type II arabinogalactan (AGII) but is not involved either in D-galactose detoxification or in galactolipid biosynthesis. Epidermal cell walls in the root expansion zone lack arabinosylated (1→6)-β-D-galactan and galactosylated XG. In cortical cells of rhd1, galactosylated XG is absent, but an arabinosylated (1→6)-β-D-galactan is present. We conclude that the flux of galactose from UDP-D-Gal into different downstream products is compartmentalized at the level of cytosolic UGE isoforms. This suggests that substrate channeling plays a role in the regulation of plant cell wall biosynthesis.

Metabolic control of resistance of human epithelial cells to H2O2 and NO stresses.

The carbon flux through the oxidative branch of the pentose phosphate pathway (PPP) can be viewed as an integrator of the antioxidant mechanisms via the generation of NADPH. It could therefore be used as a control point of the cellular response to an oxidative stress. Replacement of glucose by galactose sensitized the human epithelial cell line HGT-1 to H2O2 stress. Here we demonstrate that, due to the restricted galactose flux into the PPP, the H2O2 stress led to early cellular blebbing followed by cell necrosis, these changes being associated with a fall in the NADPH/NADP+ ratio and GSH depletion. H2O2 cytotoxicity was prevented by adding 2-deoxyglucose (2dGlc). This protection was associated with an increased flow of 2-deoxyglucose 6-phosphate into the oxidative branch of the PPP together with the prevention of the NADPH/NADP+ fall and the maintenance of intracellular GSH redox homoeostasis. Inhibitors of enzyme pathways connecting the PPP to GSH recycling abolished the 2dGlc protection. In carbohydrate-free culture conditions, 2dGlc dose-dependent protective effect was paralleled by a dose-dependent influx of 2dGlc into the PPP leading to the maintenance of the intracellular redox status. By contrast, in Glc-fed cells, the PPP was not a control point of the cellular resistance to H2O2 stress as they maintained a high NADPH/NADP+ ratio. Both 2dGlc and Glc inhibited, through the maintenance of GSH redox status, NO cytotoxicity on galactose-containing Dulbecco's modified Eagle's medium (Gal-DMEM)-fed cells. 2dGlc did not prevent the fall of ATP content in NO-treated Gal-DMEM-fed cells, indicating that NO cytotoxicity was essentially due to the disruption of GSH redox homoeostasis and not to the alteration of ATP production by the mitochondrial respiratory chain. The maintenance of ATP content in NO-treated glucose-fed cells was due to their ability to derive their energy from anaerobic glycolysis. In conclusion, Gal-DMEM and 2dGlc-supplemented Gal-DMEM provide a useful system to decipher and organize into a hierarchy the targets of several stresses at the level of intact barrier epithelial cells.

Galactose transport inhibition by cytochalasin E in rat intestine in vitro

Cytochalasins are cytoskeleton disrupters, and cytochalasin E has been reported to increase intestinal paracellular permeability. In this study, the cytochalasin E effect on galactose transport has been investigated. Ussing-type chamber experiments show an inhibitory effect of 20 microM cytochalasin E on unidirectional mucosal to serosal flux of galactose. On the contrary, the opposite unidirectional flux is not modified by the inhibitor. Results using intestinal everted sacs and rings confirm that galactose uptake by the tissue is diminished by cytochalasin E. The effect appears already after 5 min incubation, depends on cytochalasin E concentration, and does not occur in the absence of Na+. The inhibition is accompanied by an increase in the apparent K(m) of the active sugar transport (11.5 vs.15.8 mM) without significant change in the VmaX (10.6 vs. 9.1 micromol x g(-1) wet weight x 5 min(-1)). Cytochalasin E does not modify either galactose uptake by brush border membrane vesicles or Na(+)-K(+) ATPase activity in the enterocytes, indicating that the inhibitory effect on the Na(+)-dependent sugar transport cannot be explained as a direct effect on SGLT1 activity or as an indirect effect through the Na(+)-K(+) ATPase. Thus, our results suggest that cytochalasin E decreases SGLTI activity indirectly through cytoskeleton disruption.

Galactose transport inhibition by cytochalasin E in rat intestine in vitro

Cytochalasins are cytoskeleton disrupters, and cytochalasin E has been reported to increase intestinal paracellular permeability. In this study, the cytochalasin E effect on galactose transport has been investigated. Ussing-type chamber experiments show an inhibitory effect of 20 microM cytochalasin E on unidirectional mucosal to serosal flux of galactose. On the contrary, the opposite unidirectional flux is not modified by the inhibitor. Results using intestinal everted sacs and rings confirm that galactose uptake by the tissue is diminished by cytochalasin E. The effect appears already after 5 min incubation, depends on cytochalasin E concentration, and does not occur in the absence of Na+. The inhibition is accompanied by an increase in the apparent K(m) of the active sugar transport (11.5 vs.15.8 mM) without significant change in the VmaX (10.6 vs. 9.1 micromol x g(-1) wet weight x 5 min(-1)). Cytochalasin E does not modify either galactose uptake by brush border membrane vesicles or Na(+)-K(+) ATPase activity in the enterocytes, indicating that the inhibitory effect on the Na(+)-dependent sugar transport cannot be explained as a direct effect on SGLT1 activity or as an indirect effect through the Na(+)-K(+) ATPase. Thus, our results suggest that cytochalasin E decreases SGLTI activity indirectly through cytoskeleton disruption.

Galactose Expulsion during Lactose Metabolism in Lactococcus lactis subsp. cremoris FD1 Due to Dephosphorylation of Intracellular Galactose 6-Phosphate

In Lactococcus lactis subsp. cremoris FD1, galactose and lactose are both transported and phosphorylated by phosphotransferase systems. Lactose 6-phosphate (lactose-6P) is hydrolyzed intracellularly to galactose-6P and glucose. Glucose enters glycolysis as glucose-6P, whereas galactose-6P is metabolized via the tagatose-6P pathway and enters glycolysis at the tagatose diphosphate and fructose diphosphate pool. Galactose would therefore be a gluconeogenic sugar in L. lactis subsp. cremoris FD1, but since fructose 1,6-diphosphatase is not present in this strain, galactose cannot serve as an essential biomass precursor (glucose-6P or fructose-6P) but only as an energy (ATP) source. Analysis of the growth energetics shows that transition from N limitation to limitation by glucose-6P or fructose-6P gives rise to a very high growth-related ATP consumption (152 mmol of ATP per g of biomass) compared with the value in cultures which are not limited by glucose-6P or fructose-6P (15 to 50 mmol of ATP per g of biomass). During lactose metabolism, the galactose flux through the tagatose-6P pathway (r(max) = 1.2 h) is lower than the glucose flux through glycolysis (r(max) = 1.5 h) and intracellular galactose-6P is dephosphorylated; this is followed by expulsion of galactose. Expulsion of a metabolizable sugar has not been reported previously, and the specific rate of galactose expulsion is up to 0.61 g of galactose g of biomass h depending on the lactose flux and the metabolic state of the bacteria. Galactose excreted during batch fermentation on lactose is reabsorbed and metabolized when lactose is depleted from the medium. In vitro incubation of galactose-6P (50 mM) and permeabilized cells (8 g/liter) gives a supernatant containing free galactose (50 mM) but no P(i) (less than 0.5 mM). No organic compound except the liberated galactose is present in sufficient concentration to bind the phosphate. Phosphate is quantitatively recovered in the supernatant as P(i) by hydrolysis with alkaline phosphatase (EC 3.1.3.1), whereas inorganic pyrophosphatase (EC 3.6.1.1) cannot hydrolyze the compound. The results indicate that the unknown phosphate-containing compound might be polyphosphate.

Galactose fermentation by Streptococcus lactis and Streptococcus cremoris: pathways, products, and regulation.

All of the lactic streptococci examined except Streptococcus lactis ML8 fermented galactose to lactate, formate, acetate, and ethanol. The levels of pyruvate-formate lyase and lactate dehydrogenase were elevated and reduced, respectively, in galactose-grown cells compared with glucose- or lactose-grown cells. Reduced intracellular levels of both the lactate dehydrogenase activator (fructose, 1,6-diphosphate) and pyruvate-formate lyase inhibitors (triose phosphates) appeared to be the main factors involved in the diversion of lactate to the other products. S. lactis ML8 produced only lactate from galactose, apparently due to the maintenance of high intracellular levels of fructose 1,6-diphosphate and triose phosphates. The growth rates of all 10 Streptococcus cremoris strains examined decreased markedly with galactose concentrations below about 30 mM. This effect appeared to be correlated with uptake predominantly by the low-affinity galactose phosphotransferase system and initial metabolism via the D-tagatose 6-phosphate pathway. In contrast, with four of the five S. lactis strains examined, galactose uptake and initial metabolism involved more extensive use of the high-affinity galactose permease and Leloir pathway. With these strains the relative flux of galactose through the alternate pathways would depend on the exogenous galactose concentration.

D-Galactose accumulation in rabbit ileum effects of theophylline on serosal permeability

The effects of theophylline and dibutyryl cyclic AMP, on in vitro unidirectional galactose fluxes across the mucosal and serosal borders of rabbit ileum have been studied. 1. 1.|When Ringer [galactose] = 2 mM , theophylline and dibutyryl cyclic AMP reduce both mucosal-serosal and serosal-mucosal galactose flux by approx. 50%. The K I for theophylline inhibition of flux in both directions is 2 mM. 1 mM dibutyryl cyclic AMP elicits a maximal inhibitory response. Concurrent with the inhibition in transmural galactose fluxes, theophylline and dibutyryl cyclic AMP increase the tissue accumulation of [galactose] and the specific-activity ratio R of 3H: 14C-labelled galactose coming from the mucosal and serosal solutions respectively. It is deduced that theophylline and dibutyryl cyclic AMP are without effect on the mucosal unidirectional permeability to galactose but cause a symmetrical reduction in serosal entry and exit permeability. 2. 2.|Reduction in the asymmetry of the mucosal border to galactose by reducing Ringer [Na], raising Ringer [galactose] or adding ouabain reduces the theophylline-dependent increase in galactose accumulation. 3. 3.|Hypertonicity in the serosal solution increases the permeability of the serosal border to galactose and reduces tissue galactose accumulation. Serosal hypertonicity partially reverses the theophylline-dependent effects on galactose transport. Replacing Ringer chloride by sulphate abolishes the theophylline-dependent effects on galactose transport. 4. 4.|It is considered that the theophylline-dependent increase in galactose accumulation results from the reduction in serosal permeability. This is shown to be a quantitatively consistent inference. 5. 5.|Further support for the view that the asymmetric transport of galactose in rabbit ileum results from convective-diffusion is presented.

Galactose transport across the serosal border of rabbit ileum and its role in intracellular accumulation

1. 1. Unidirectional fluxes of d-galactose across the brush and serosal border of rabbit ileum were determined using the method described previously (Naftalin, R. J. and Curran, P. F. (1974) J. Membrane Biol. 16, 257–278). With Ringer [Na] + 75 mequiv., the K m for galactose influx across the brush-border is 5 mM, with 0.1 mM ouabain present K m = 50 mM, the V (2.0 μmol · cm −2 · h −1) remains unaltered. The Michaelis parameters for galactose influx across the serosal border are K m = 59±9 mM and V = 4.7±0.24 μmol · cm −2 · h −1 and for efflux K m = 85±10 mM and V = 6.8±0.7 μmol · cm −2 · h −1. 2. 2. 2-Deoxy- d-glucose and methyl β- d-glucopyranoside inhibit galactose entry exclusively at the serosal and mucosal borders respectively, whilst 3-o- methyl- d-glucose inhibits galactose influx at both borders. 0.1 mM ouabain increases the K 1 of 3- O-methylglucose for the mucosal galactose transport system from 50 to 150 mM, whilst the K 1 of 3- O-methylglucose for the serosal transport system (100 mM) is unaffected by ouabain. Inhibition of mucosal galactose transport by ouabain or by competition with other sugars results in a reciprocal increase in exit permeability and decrease in entry permeability. Inhibition of serosal galactose transport results in inhibition of both the entry and exit permeability, entry is more affected. 3. 3. There is a small degree of permeability asymmetry at the serosal border to galactose which is reduced by ouabain or removal of Na + from the Ringer. Uptake of 14C-labelled galactose from the serosal solution into the tissue is also inhibited by addition of ouabain or Na + removal. It is therefore considered that there is a weak active transport system for galactose at the serosal border. 4. 4. Net transepithelial galactose flux is sufficiently high and serosal permeability to galactose sufficiently low to be consistent with the view that galactoseiis concentrated within the tissue fluid, after convection (Nattalin, R. J. and Holman, G. D. (1974) Biochim. Biophys. Acta., 373, 453–470) across across the mucosal border because it is reflected at the serosal boundary.

The role of Na as a determinant of the asymmetric permeability of rabbit ileal brush-border to d-galactose

1. 1. Raising Ringer [Na] from 0–140 mequiv causes a significant rise in [Na] within rabbit ileal tissue fluid, with 0.1 mM ouabain present the tissue [Na] is raised still further. 2. 2. There is a Na-dependent increase in the calculated entry permeability of the brush-border to galactose and the calculated exit permeability of the brush-border to galactose falls as the tissue [Na] is increased. The maximal permeability ratio of the brush-border to galactose exceeds the inverse Na distribution ratio between tissue water and Ringer by 20-fold. 3. 3. Ouabain both reduces the Na-dependent increase in entry permeability and reduces the Na-dependent decrease in exit permeability of the brush-border to galactose. A model explaining the observed asymmetric permeability of the brush-border to galactose in terms of convective-diffusion flow is proposed. By resolving galactose flux across the brush-border into separate convective and diffusive velocity components (see Appendix), it is shown that both velocity components are increased by increasing Ringer [Na]. Addition of ouabain to the Ringer reduces the convective component to zero without affecting the diffusive component, hence it is deduced that the Na pump activity provides the force required to produce mass flow across the brush-border. As brush-border [galactose] is increased, the convective velocity decreases more than the diffusive velocity. This result is consistent with the view that the permeability asymmetry of the brush-border to galactose is the result of mass flow via narrow channels.

Effects of erythrocyte lipid and of glucose and galactose concentration on transport of the sugars across a water-butanol interface

A property of sugar transport into the human erythrocyte is that a sugar with a high affinity for the hypothetical "carrier" will enter the cell at low concentration more rapidly than a sugar with lower affinity for carrier. At high concentration the sequence will be reversed. This behavior is exemplified by glucose, which enters erythrocytes faster than galactose at 0.015 m and slower than galactose at 1.3 m. A physicochemical model with the same properties has been found: layers of butanol and water with erythrocyte lipid at the interface. With total lipid from the human erythrocyte incorporated into the model, glucose at low concentration enters the oil phase faster than galactose and at high concentration galactose enters more rapidly. In the absence of lipid, glucose flux exceeds galactose flux at all concentrations. The hypothetical carrier molecule has not been identified.