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Past contributions of the group
The group has a long-standing interest in the regulation of carbohydrate metabolism and in its inborn errors of metabolism. Previous important achievements have been the discovery of fructose-2,6-bisphosphate (in 1980—the laboratory was still headed by Prof. Henri-Géry Hers, who retired in 1988) and of glucokinase regulatory protein. In the field of inborn errors of metabolism, the group has also contributed the cloning of a glucose-6-phosphate translocase, mutated in glycogen storage disease type Ib and has —in collaboration with Prof J. Jaeken, University of Leuven— identified phosphomannomutase deficiency as the most frequent cause of CDG (congenital disorders of glycosylation). Defects of serine synthesis have also been identified in collaboration with J. Jaeken.
Fructose-2,6-bisphosphate
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Role of fructose 2,6-bisphosphate in the control of glycolysis and gluconeogenesis. Fructose-2,6-bisphosphate is a powerful stimulator of PFK1 in animals and fungi, of plant PPi-PFK and of trypanosomatid pyruvate kinase |
 The Structure of Fructose-2,6-bisphosphate |
The discovery of fructose 2,6-bisphosphate stems from studies on the mechanism by which glucagon stimulates gluconeogenesis and inhibits glycolysis in liver. Flux measurements and assays of the concentration of intracellular metabolites had shown that glucagon decreases the flux through 6-phosphofructo-1-kinase (PFK1). Further work showed that this is due to the fact that glucagon causes the disappearance of a low-molecular weight stimulator of PFK1 [1]. This stimulator behaved as a non-nucleotidic, non-reducing bisphosphate ester, which was rapidly destroyed in dilute acid at room temperature, and gave then rise to stoichiometric amounts of fructose 6-phosphate and Pi [2]. These properties indicated that the stimulator was fructose 2,6-bisphosphate, which was soon confirmed by chemical synthesis of this “new” phosphate ester.
Fructose-2,6-bisphosphate is not only a potent stimulator of phosphofructokinase but also an inhibitor of fructose-1,6-bisphosphatase. In liver, its synthesis by 6-phosphofructo 2-kinase (PFK2) and hydrolysis by fructose 2,6-bisphosphatase (FBPase2) are controlled by phosphorylation of the bifunctional enzyme PFK2-FBPase 2 by cyclic AMP-dependent protein kinase. In trypanosomatids, fructose-2,6-bisphosphate stimulates pyruvate kinase rather than PFK1.
Key publications of the lab in relation with fructose 2,6-bisphosphate
Glucokinase regulatory protein
The control of glucokinase activity by its regulatory protein and by metabolites.
Mechanism of action of the regulatory protein of glucokinase
The regulatory protein exists under two different conformations, one (R) able to bind fructose 6-phosphate and glucokinase, and the other (R’), able to bind fructose 1-phosphate but not glucokinase. The glucokinase-regulatory protein complex is inactive. In the absence of metabolite, the R’ form predominates. Fructose 6-phosphate shifts the equilibrium towards R, hence reinforcing inhibition. Fructose 1-phosphate sequesters the R’ form and has therefore the opposite effect.
Glucokinase is an enzyme that displays a low affinity for glucose, and can therefore play the role of “glucose sensor” in liver and in ß-cells of pancreatic islets. In liver, the activity of this enzyme is controlled not only by the glucose concentration, but also by a fructose 6-phosphate- and fructose-1-phosphate-sensitive protein, termed ‘regulatory protein of glucokinase’ (GKRP). The latter was identified while trying to understand the mechanism by which fructose stimulates the phosphorylation of glucose in liver [1,2]. Glucokinase regulatory protein behaves as an inhibitor of glucokinase in the presence of fructose-6-phosphate, and this inhibition is suppressed by fructose-1-phosphate, accounting for the effect of fructose to stimulate glucose phosphorylation. Both proteins form a one-to-one complex in the presence of fructose 6-phosphate, but dissociate in the presence of fructose 1-phosphate [3]. GKRP is distantly homologous to sugar phosphate-isomerases and binds both fructose 6-phosphate and fructose 1-phosphate to a single site [9]. Work by other groups has shown that GKRP controls the subcellular localization of glucokinase.
Selected publications of the lab
Glucose 6-phosphate translocase
Glucose-6-phosphatase is the enzyme that catalyzes the last step of glycogenolysis and gluconeogenesis in liver and kidneys, i.e. the hydrolysis of glucose 6-phosphate to inorganic phosphate and free glucose. Its genetic deficiency causes glycogen storage disease type I, a disease in which glycogen accumulation in liver and kidneys, hypoglycemia and accumulation of lactic acid in blood are observed. Glucose 6-phosphatase, which is known to be associated with the endoplasmic reticulum, was postulated by Arion et al. (1975) to consist of a hydrolase whose catalytic site faces the lumen of the organelle and of translocases required for the transport of glucose 6-phosphate, Pi and glucose. The cDNA of the hydrolase was cloned by Shelly et al., 1993 and shown to be mutated in the patients with glycogen storage disease type Ia (deficiency in the catalytic component). Our group cloned the glucose 6-phosphate translocase in 1997 and showed that it is mutated in patients with glycogen storage disease type Ib [1,2], a disease in which the classical metabolic symptoms of glucose 6-phosphatase deficiency are associated with neutropenia and neutrophil dysfunction. Other work provided additional evidence for the fact that glucose 6-phosphate must enter the endoplasmic reticulum before being hydrolysed by glucose 6-phosphatase or oxidized by hexose-6-phosphate dehydrogenase.
Arion’s substrate-transport model
Glucose 6-phosphate is transported into the lumen of the endoplasmic reticulum by a specific transporter before being hydrolysed by glucose-6-phosphatase, a transmembrane protein with its catalytic site oriented towards the lumen of the endoplasmic reticulum. Glycogen storage disease type Ia (GSD Ia) is due to a defect in glucose-6-phosphatase and glycogen storage disease type Ib, to a defect in the glucose 6-phosphate transporter.
Selected publications of the lab
[6] Van Schaftingen E, Gerin I. The glucose-6-phosphatase system. Biochem J. 2002, 362, 513-32. Review.
Inborn errors of metabolism
Diseases of L-serine metabolism
The amino acid L-serine is produced from the glycolytic intermediate, 3-phosphoglycerate in a three-step pathway involving 3-phosphoglycerate dehydrogenase, phosphoserine transaminase and phosphoserine phosphatase. Two different types of defects have been identified in this pathway : one, at the level of 3-phosphoglycerate dehydrogenase and the other, at the level of phosphoserine phosphatase. The fact that these defects are accompanied by marked neurological symptoms (mental retardation, seizures) indicates that endogenous L-serine production is important for brain function, possibly because the blood-brain barrier is relatively impermeable to this amino acid.
L-serine is degraded either through its direct conversion to pyruvate by L-serine dehydratase or by a series of reactions involving its conversion to 3-hydroxypyruvate, D-glycerate and 2-phosphoglycerate (Fig). The second pathway was thought to be of little importance in the degradation of L-serine in man, because human liver had been reported to be nearly devoid of D-glycerate kinase activity. D-glycerate kinase was found to be present in human liver, but to be extremely unstable in tissue extracts unless stabilizing ligands were added. D-glycerate kinase could then be shown to be deficient in D-glyceric aciduria, indicating that the main pathway of L-serine degradation proceeds via this intermediate.
Selected publications of the lab
[1] Van Schaftingen E. D-glycerate kinase deficiency as a cause of D-glyceric aciduria. FEBS Lett. 1989, 243, 127-31.
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