Genetically modified L3,7 and L2 lipooligosaccharides from Neisseria meningitidis serogroup B confer a broad cross-bactericidal response.

Currently available Neisseria meningitidis serogroup B (MenB) vaccines are based on outer membrane vesicles (OMVs) that are obtained from wild-type strains. They are purified with the aim of decreasing the lipooligosaccharide (LOS) content and hence reduce the reactogenicity of the vaccine even though LOS is a potential protective antigen. In <2-year-old children, these MenB vaccines confer protection only against strains expressing homologous PorA, a major and variable outer membrane protein. Our objective was to develop a safe LOS-based vaccine against MenB. To this end, we used modified porA knockout strains expressing genetically detoxified (msbB gene-deleted) L2 and L3,7 LOSs, allowing the production of LOS-enriched OMVs. The vaccine-induced antibodies were found to be bactericidal against nearly all invasive strains, irrespective of capsular serogroup. In addition, we have also demonstrated that LOS lacking the terminal galactose (with a lgtB mutation; truncated L3 LOS), but not LOS produced without the galE gene, induced a bactericidal antibody response in mice similar to that seen for LOS containing the full lacto-N-neotetraose (L3,7 LOS). In conclusion, a bivalent detoxified LOS OMV-based vaccine demonstrated the potential to afford a broad cross-protection against meningococcal disease.

Species and tissue distribution of the regulatory protein of glucokinase.

Rat liver is known to contain a regulatory protein that inhibits glucokinase (hexokinase IV or D) competitively versus glucose. This inhibition is greatly reinforced by the presence of fructose 6-phosphate and antagonized by fructose 1-phosphate and by KCl. This protein was now measured in various rat tissues and in the livers of various species by the inhibition it exerts on rat liver glucokinase. Rat, mouse, rabbit, guinea-pig and pig liver, all of which contain glucokinase, also contained between 60 and 200 units/g of tissue of a regulatory protein displaying the properties mentioned above. By contrast, this protein could not be detected in cat, goat, chicken or trout liver, or in rat brain, heart, skeletal muscle, kidney and spleen, all tissues from which glucokinase is missing. Fructose 1-phosphate stimulated glucokinase in extracts of human liver, indicating the presence of regulatory protein. In addition, antibodies raised against rat regulatory protein allowed the detection of an approximately 60 kDa polypeptide in rat, guinea pig, rabbit and human liver. The livers of the toad Bufo marinus, of Xenopus laevis and of the turtle Pseudemys scripta elegans contained a regulatory protein similar to that of the rat, with, however, the major difference that it was not sensitive to fructose 6-phosphate or fructose 1-phosphate. In rat liver, the regulatory protein was detectable 4 days before birth. Its concentration increased afterwards to reach the adult level at day 30 of extrauterine life, whereas glucokinase only appeared after day 15. In the liver of the adult rat, starvation and streptozotocin-diabetes caused a 50-60% decrease in the concentration of regulatory protein after 7 days, whereas glucokinase activity fell to about 20% of its initial level. When 4-day-starved rats were refed, or when diabetic rats were treated with insulin, the concentration of regulatory protein slowly increased to reach about 85% of the control level after 3 days, whereas the glucokinase activity was normalized after the same delay. The fact that there appears to be no situation in which glucokinase is expressed without regulatory protein is in agreement with the notion that the regulatory protein forms a functional entity with this enzyme.

Heterogeneity in glucose sensitivity among pancreatic beta-cells is correlated to differences in glucose phosphorylation rather than glucose transport.

Rat beta-cells differ in their individual rates of glucose-induced insulin biosynthesis and release. This functional heterogeneity has been correlated with intercellular differences in metabolic redox responsiveness to glucose. The present study compares glucose metabolism in two beta-cell subpopulations that have been separated on the basis of the presence (high responsive) or absence (low responsive) of a metabolic redox shift at 7.5 mM glucose. Mean rates of glucose utilization and glucose oxidation in high responsive beta-cells were 2- to 4-fold higher than in low responsive beta-cells, whereas their leucine and glutamine oxidation was only 10-50% higher. This heterogeneity in glucose metabolism cannot be attributed to differences in GLUT2 mRNA levels or in glucose transport. In both cell subpopulations, the rates of glucose transport (13-19 pmol/min/10(3) beta-cells) were at least 50-fold higher than corresponding rates of glucose utilization. On the other hand, rates of glucose phosphorylation (0.3-0.7 pmol/min/10(3) beta-cells) ranged within those of total glucose utilization (0.2-0.4 pmol/min/10(3) beta-cells). High responsive beta-cells exhibited a 60% higher glucokinase activity than low responsive beta-cells and their glucokinase mRNA level was 100% higher. Furthermore, glucose phosphorylation via low Km hexokinase was detected only in the high responsive beta-cell subpopulation. Heterogeneity in glucose sensitivity among pancreatic beta-cells can therefore be explained by intercellular differences in glucose phosphorylation rather than in glucose transport.

Binding of sorbitol 6-phosphate and of fructose 1-phosphate to the regulatory protein of liver glucokinase.

Using a binding assay in which the ligand-protein complex is separated from free ligand by precipitation with poly(ethylene glycol) 6000, we found that the regulatory protein of rat liver glucokinase bound close to 1 mol of radiolabelled sorbitol 6-phosphate, a negative effector, or of fructose 1-phosphate, a positive effector, per mol of regulatory protein. Scatchard plots were linear, the dissociation constant being 0.3 microM for both phosphate esters. Sorbitol 6-phosphate and fructose 1-phosphate competed with each other for the binding. Competition was also observed with psicose 1-phosphate, ribitol 5-phosphate, arabitol 5-phosphate and 3-phosphoglycerate, all of which are known to affect the inhibition exerted by the regulatory protein. At a concentration of 10%, poly(ethylene glycol) 6000 decreased the concentration of regulatory protein causing 50% inhibition to a larger extent in the absence (12-fold) than in the presence (3-fold) of a saturating concentration of fructose 6-phosphate, another negative effector. Furthermore, it increased by about 3-fold the apparent affinity for inhibitory phosphate esters, indicating that it induced conformational changes of the regulatory protein.

Mechanism of the stimulatory effect of a potassium-rich medium on the phosphorylation of glucose in isolated rat hepatocytes.

We have investigated the mechanism by which the replacement of a Na(+)-rich medium by a K(+)-rich medium causes an increase in the apparent affinity of glucokinase (hexokinase IV or D) for glucose in isolated hepatocytes [Bontemps, F., Hue, L. & Hers, H. G. (1978) Biochem. J. 174, 603-611]. The stimulatory effect of a K(+)-rich medium on the rate of glucose phosphorylation, as assessed by the release of tritium from [2-3H]glucose, was only partially additive with the effect of fructose, suggesting that it was also due to a decrease in the inhibition exerted on glucokinase by its regulatory protein. Measurements of metabolites indicated that the effect of the K(+)-rich medium was neither due to the formation of fructose 1-phosphate, nor to changes in the concentrations of fructose 6-phosphate or Pi, two other effectors of the regulatory protein. Replacement of Na+ by K+ in the medium resulted in a time-dependent and dose-dependent increase in cell volume that paralleled the changes in the rate of detritiation observed at 5 mM glucose. The water and chloride contents, estimated using radiolabelled compounds, were threefold and tenfold higher, respectively, in K+ cells than in Na+ cells, and the intracellular Cl- concentration about threefold higher (94 versus 29 meq/l). The effects of the K(+)-rich medium on cell volume, Cl- concentration and rate of detritiation were greatly reduced by including 80 mM trehalose or sucrose in the medium at the start of the incubation. Addition of trehalose to cells incubated for 45-50 min in the K(+)-rich medium caused an immediate decrease in cell volume whereas the rate of detritiation and the Cl- concentration underwent a transient increase followed by a decrease. Replacement of KCl by KBr, potassium acetate or potassium trichloroacetate in the K(+)-rich medium resulted in different relationships between cell volume and the rate of detritiation, in agreement with the differential effect of these salts on the activity of purified glucokinase assayed in the presence of regulatory protein. From these results we conclude that the increase in the activity of glucokinase induced by a KCl-rich medium is at least partly due to an increase in the concentration of Cl-, which relieves the inhibition exerted by the regulatory protein on purified glucokinase.

The regulatory protein of liver glucokinase.

Fructose, sorbitol and D-glyceraldehyde stimulate the rate of glucose phosphorylation in isolated hepatocytes. This effect is mediated by fructose 1-phosphate, which releases the inhibition exerted by a regulatory protein on liver glucokinase. In the presence of fructose 6-phosphate, the regulatory protein binds to, and inhibits, liver glucokinase. Fructose 1-phosphate antagonizes this inhibition by causing dissociation of the glucokinase-regulatory protein complex. Both phosphate esters act by binding to the regulatory protein, and by presumably causing changes in its conformation. The regulatory protein behaves as a fully competitive inhibitor. It inhibits liver glucokinase from various species, and rat islet glucokinase, but has no effect on hexokinases from mammalian tissues or from yeast, or on glucokinase from microorganisms. Kinetic studies indicate that the regulatory protein binds to glucokinase at a site distinct from the catalytic site. Several phosphate esters, mainly polyol-phosphates, were found to mimick the effect of fructose 6-phosphate. The most potent is sorbitol 6-phosphate, suggesting that fructose 6-phosphate is recognized by the regulatory protein in its open-chain configuration. Other phosphate esters and Pi have a fructose 1-phosphate-like effect. The stimulatory effect of fructose on glucose phosphorylation is observed not only in isolated hepatocytes but also in the livers of anesthetized rats. This suggests that fructose could be a nutritional signal causing an increase in the hepatic glucose uptake.

Competitive inhibition of liver glucokinase by its regulatory protein.

The regulatory protein of rat liver glucokinase (hexokinase IV or D) behaved as a fully competitive inhibitor of this enzyme when glucose was the variable substrate, i.e. it increased the half-saturating concentration of glucose as a linear function of its concentration without affecting V (velocity at infinite concentration of substrate). The inhibition by the regulatory protein and that by palmitoyl-CoA were synergistic with that by N-acetyl-glucosamine, indicating that the two former inhibitors bind to a site distinct from the catalytic site. In contrast, the effects of the regulatory protein and palmitoyl-CoA were competitive with each other, indicating that these two inhibitors bind to the same site. The regulatory protein exerted a non-competitive inhibition with respect to Mg-ATP at concentrations of this nucleotide less than 0.5 mM. At higher concentrations, the latter antagonized the inhibition by the regulatory protein partly by decreasing the apparent affinity for fructose 6-phosphate. The following anions inhibited glucokinase non-competitively with respect to glucose: Pi, sulfate, I-, Br-, No3-, Cl-, F- and acetate. Pi and sulfate, at concentrations in the millimolar range, decreased the inhibition by the regulatory protein by competing with fructose 6-phosphate. Monovalent anions also antagonized the inhibition by the regulatory protein with the following order of potency: I- greater than Br- greater than NO3- greater than Cl- greater than F- greater than acetate and their effect was non-competitive with respect to fructose 6-phosphate. Glucokinase from Buffo marinus and pig liver were, like the rat liver enzyme, inhibited by the regulatory protein, as well as by palmitoyl-CoA at micromolar concentrations. In contrast, neither compound inhibited hexokinases from rat brain, beef heart or yeast, or the low-Km specific glucokinase from Bacillus stearothermophilus.

Effectors of the regulatory protein acting on liver glucokinase: a kinetic investigation.

In the absence of fructose 6-phosphate, the regulatory protein of rat liver glucokinase (hexokinase IV or D) inhibited this enzyme, though with a much (15-fold) lower potency than in the presence of a saturating concentration of fructose 6-phosphate. Evidence is provided that this inhibition is not due to contaminating fructose 6-phosphate. In the presence of regulatory protein, sorbitol 6-phosphate, a potent analog of fructose 6-phosphate, exerted a hyperbolic, partial inhibition on glucokinase, the degree of which increased with the concentration of regulatory protein. Plots of the reciprocal of the difference between the rates in the absence and in the presence of sorbitol 6-phosphate versus 1/[sorbitol 6-phosphate] at various concentrations of regulatory protein were linear, and demonstrated that the apparent affinity for sorbitol 6-phosphate increased with the concentration of regulatory protein. Plots of the reciprocal of the difference between 1/v in the presence and in the absence of sorbitol 6-phosphate versus 1/[sorbitol 6-phosphate] were also linear and crossed the axis at a value independent of the concentration of regulatory protein. Fructose 1-phosphate released the inhibition exerted by the regulatory protein in a hyperbolic fashion. The concentration of this effector required for a half-maximal effect increased linearly with the concentrations of sorbitol 6-phosphate and of regulatory protein. These results are consistent with a model in which the regulatory protein exists under two conformations, one form which binds inhibitors and glucokinase, and the other which binds activators, although not glucokinase. Sorbitol 6-phosphate, 2-deoxysorbitol 6-phosphate and mannitol 1-phosphate, all analogs of the open-chain configuration of fructose 6-phosphate, inhibited glucokinase in the presence of regulatory protein at lower concentrations than fructose 6-phosphate, whereas fixed analogs of the furanose form of fructose 6-phosphate were inactive or behaved as activators. This indicated that fructose 6-phosphate in its open-chain configuration is recognized by the regulatory protein. A series of compounds exerted an activating effect. These included, in order of decreasing potency: fructose 1-phosphate, psicose 1-phosphate, ribitol 5-phosphate, analogs of fructose 1-phosphate and of ribitol 5-phosphate and, at much higher concentrations, inorganic phosphate.

The mechanism by which rat liver glucokinase is inhibited by the regulatory protein.

The fructose-6-phosphate-sensitive and fructose-1-phosphate-sensitive protein that inhibits rat liver glucokinase [Van Schaftingen, E. (1989) Eur. J. Biochem. 179, 179-184] was purified close to homogeneity by a procedure involving poly(ethyleneglycol) precipitation, chromatography on anion-exchangers and hydroxylapatite, gel filtration and chromatography on Mono S, a cation exchanger. In the last chromatographic step, the regulatory protein coeluted with a 62 kDa peptide. From the elution volume of the gel-filtration column a molecular mass of 60 kDa was determined, allowing the conclusion that the regulator is a monomer. The decrease in the apparent affinity of glucokinase for glucose, which the regulator induced, disappeared upon separation of the two proteins. Furthermore, the regulator did not appear to catalyse the formation of a heat-stable or a trypsin-resistant inhibitor of glucokinase. Finally, the inhibition exerted by the regulatory protein reached a steady value 1-2 min after the addition of the regulator. These results indicate that the regulator does not act by causing a covalent modification of glucokinase nor by catalysing the formation of a low-molecular-mass inhibitor. Raising the concentration of glucokinase in the assay from 6 mU/ml to 120 mU/ml caused a 2.5-fold increase in the concentration of regulator required to half-maximally inhibit the enzyme. The apparent mass of glucokinase, as determined by centrifugation in isokinetic sucrose gradient, was 55 kDa, and this value was unaffected by the separate presence of fructose 6-phosphate or of the regulatory protein. However, the apparent mass of the enzyme increased to 105 kDa when glucokinase was centrifuged together with both fructose 6-phosphate and the regulatory protein, although not when fructose 1-phosphate was also present. Conversely, the presence of glucokinase increased the apparent molecular mass of the regulator in the presence of fructose 6-phosphate. From these results, it is concluded that the regulatory protein inhibits glucokinase by forming a complex with this enzyme in the presence of fructose 6-phosphate, and that fructose 1-phosphate antagonises this inhibition by preventing the formation of the complex.

Regulation of glucokinase by a fructose-1-phosphate-sensitive protein in pancreatic islets.

In the post-microsomal supernatant of pancreatic islets, prepared from fasted or fed rats, D-fructose 1-phosphate increased the activity of glucokinase by 20-30% as measured in the presence of D-glucose 6-phosphate and D-fructose 6-phosphate. Such an activation was less marked than that found in liver extracts. The islet cytosol was also found to inhibit purified liver glucokinase, and this effect was antagonized by D-fructose 1-phosphate. In the presence of hexose 6-phosphates, partially purified islet glucokinase was inhibited by the hepatic glucokinase regulatory protein in a D-fructose-1-phosphate-sensitive manner. In intact islets, D-glyceraldehyde stimulated the generation of 14C-labelled D-fructose 1-phosphate from D-[U-14C]glucose and increased the production of 3H2O from D-[5-3H]glucose. These findings suggest that the activity of glucokinase in islet cells may be regulated by a protein mediating the antagonistic effects of D-fructose 6-phosphate and D-fructose 1-phosphate in a manner qualitatively similar to that operating in hepatocytes, but with lower efficiency.

Fructose 2,6-bisphosphate and carbohydrate metabolism during the life cycle of the aquatic fungus Blastocladiella emersonii.

Removal of the growth medium and resuspension of Blastocladiella emersonii vegetative cells in a sporulation medium resulted in an abrupt fall of fructose 2,6-bisphosphate concentration to about 2% of its initial value within 10 min. The concentrations of hexose 6-phosphate and of fructose 1,6-bisphosphate also decreased by, respectively, three and tenfold over the same period. All these values remained at their low level throughout the sporulation phase and during the subsequent germination of zoospores when performed in the absence of glucose. In contrast, the concentration of cyclic AMP was low during the sporulation period and exhibited a transient increase a few minutes after the initiation of germination. Other biochemical events occurring during sporulation were a 70% reduction in glycogen content and the complete disappearance of trehalose. The remaining glycogen was degraded upon subsequent germination of the zoospores. B. emersonii phosphofructo 2-kinase (PFK-2) and fructose-2,6-bisphosphatase (FBPase-2) could not be separated from each other by various chromatographic procedures, suggesting that they were part of a single bifunctional protein. On anion-exchange chromatography, two peaks of PFK-2 and FBPase-2 were resolved. Upon incubation of fractions from the two peaks or of a crude extract in the presence of [2-32P]fructose 2,6-bisphosphate, two radiolabelled subunits with molecular masses close to 90 and 54 kDa were obtained. The labelling of the subunit of higher molecular mass was greater than that of the lower one in extracts prepared in the presence of protease inhibitors and in the first peak of the Mono Q column. PFK-2 and FBPase-2 displayed kinetic properties comparable to those of mammalian enzymes, but no indication of a cyclic AMP-dependent regulation could be obtained. Phosphofructo 1-kinase and fructose-1,6-bisphosphatase from B. emersonii were, respectively, stimulated and inhibited by micromolar concentrations of fructose 2,6-bisphosphate. The physiological significance of these properties is discussed. A simple method for the determination of trehalose is also reported.

Characterization of trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase of Saccharomyces cerevisiae.

The properties of yeast trehalose-6-phosphate synthase were reinvestigated in relation with the recent claim made by Panek et al. [Panek, A. C., de Araujo, P. S., Moura-Neto, V. and Panek, A. D. (1987) Curr. Genet. II, 459-465] that the enzyme would be stimulated by ATP and partially inactivated by cAMP-dependent protein kinase. Trehalose-6-phosphate synthase activity was measured by the sum of [14C]trehalose 6-phosphate and [14C]trehalose formed from UDP-[14C]glucose and glucose 6-phosphate. The activity measured in an extract of Saccharomyces cerevisiae was not affected by any treatment of the cells, such as incubation in the presence of glucose or of dinitrophenol, which are known to greatly increase the intracellular concentration of cAMP, nor by preincubation of the extract in the presence of ATP-Mg, cAMP and bovine heart cAMP-dependent protein kinase. The activity was also not significantly different in several mutants affected in the cAMP system. The kinetic properties of the partially purified enzyme were investigated; no effect of ATP could be detected but Pi acted as a potent noncompetitive inhibitor (Ki = 2 mM). The activity of trehalose-6-phosphate phosphatase was measured by the amount of [14C]trehalose formed from [14C]trehalose 6-phosphate. The enzyme could be separated from other phosphatases and appeared to be highly specific for trehalose 6-phosphate. It was Mg dependent and its kinetics for trehalose 6-phosphate was hyperbolic. Studies performed with intact cells, crude extracts or the purified enzyme did not reveal any cAMP-dependent change in its activity. Remarkably, trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase copurified in the course of different chromatographic procedures, suggesting that they are part of a single bifunctional protein. A 50-fold purification of the two enzymes could be achieved with a yield of only 2% by chromatography on Mono S followed by gel filtration on Superose 6B.

Stimulation of glucose phosphorylation by fructose in isolated rat hepatocytes.

The phosphorylation of glucose was measured by the formation of [3H]H2O from [2-3H]glucose in suspensions of freshly isolated rat hepatocytes. Fructose (0.2 mM) stimulated 2-4-fold the rate of phosphorylation of 5 mM glucose although not of 40 mM glucose, thus increasing the apparent affinity of the glucose phosphorylating system. A half-maximal stimulatory effect was observed at about 50 microM fructose. Stimulation was maximal 5 min after addition of the ketose and was stable for at least 40 min, during which period 60% of the fructose was consumed. The effect of fructose was reversible upon removal of the ketose. Sorbitol and tagatose were as potent as fructose in stimulating the phosphorylation of 5 mM glucose. D-Glyceraldehyde also had a stimulatory effect but at tenfold higher concentrations. In contrast, dihydroxyacetone had no significant effect and glycerol inhibited the detritiation of glucose. Oleate did not affect the phosphorylation of glucose, even in the presence of fructose, although it stimulated the formation of ketone bodies severalfold, indicating that it was converted to its acyl-CoA derivative. These results allow the conclusion that fructose stimulates glucokinase in the intact hepatocyte. They also suggest that this effect is mediated through the formation of fructose 1-phosphate, which presumably interacts with a competitive inhibitor of glucokinase other than long-chain acyl-CoAs.