Processivity and subcellular localization of glycogen synthase depend on a non-catalytic high affinity glycogen-binding site.

Glycogen synthase, a central enzyme in glucose metabolism, catalyzes the successive addition of α-1,4-linked glucose residues to the non-reducing end of a growing glycogen molecule. A non-catalytic glycogen-binding site, identified by x-ray crystallography on the surface of the glycogen synthase from the archaeon Pyrococcus abyssi, has been found to be functionally conserved in the eukaryotic enzymes. The disruption of this binding site in both the archaeal and the human muscle glycogen synthases has a large impact when glycogen is the acceptor substrate. Instead, the catalytic efficiency remains essentially unchanged when small oligosaccharides are used as substrates. Mutants of the human muscle enzyme with reduced affinity for glycogen also show an altered intracellular distribution and a marked decrease in their capacity to drive glycogen accumulation in vivo. The presence of a high affinity glycogen-binding site away from the active center explains not only the long-recognized strong binding of glycogen synthase to glycogen but also the processivity and the intracellular localization of the enzyme. These observations demonstrate that the glycogen-binding site is a critical regulatory element responsible for the in vivo catalytic efficiency of GS.

Control of liver glycogen synthase activity and intracellular distribution by phosphorylation.

Eukaryotic glycogen synthase activity is regulated by reversible phosphorylation at multiple sites. Of the two GS isoforms found in mammals, the muscle enzyme (muscle glycogen synthase) has received more attention and the relative importance of every known phosphorylation site in the control of its activity and intracellular distribution has been previously addressed. We have analyzed the impact of the dephosphorylation at the homologous sites of the glycogen synthase liver (LGS) isoform. Serine residues at these sites were replaced by non-phosphorylatable alanine residues, singly or in pairs, and the resultant LGS variants were expressed in cultured cells using adenoviral vectors. The sole mutation at site 2 (Ser7) yielded an enzyme that was almost fully active and able to induce glycogen deposition in primary hepatocytes incubated in the absence of glucose and in FTO2B cells, a cell line that does not normally synthesize glycogen. Mutation at site 2 was also sufficient to trigger the aggregation and translocation of LGS from the cytoplasm to the hepatocyte cell cortex in the absence of glucose. However, this redistribution was not observed in hepatocytes incubated without glucose when an additional mutation (E509A), which renders the enzyme inactive, was introduced. This result suggests that LGS translocation is strictly dependent on glycogen synthesis.

Crystal structure of an archaeal glycogen synthase: insights into oligomerization and substrate binding of eukaryotic glycogen synthases.

Glycogen and starch synthases are retaining glycosyltransferases that catalyze the transfer of glucosyl residues to the non-reducing end of a growing alpha-1,4-glucan chain, a central process of the carbon/energy metabolism present in almost all living organisms. The crystal structure of the glycogen synthase from Pyrococcus abyssi, the smallest known member of this family of enzymes, revealed that its subunits possess a fold common to other glycosyltransferases, a pair of beta/alpha/beta Rossmann fold-type domains with the catalytic site at their interface. Nevertheless, the archaeal enzyme presents an unprecedented homotrimeric molecular arrangement both in solution, as determined by analytical ultracentrifugation, and in the crystal. The C-domains are not involved in intersubunit interactions of the trimeric molecule, thus allowing for movements, likely required for catalysis, across the narrow hinge that connects the N- and C-domains. The radial disposition of the subunits confers on the molecule a distinct triangular shape, clearly visible with negative staining electron microscopy, in which the upper and lower faces present a sharp asymmetry. Comparison of bacterial and eukaryotic glycogen synthases, which use, respectively, ADP or UDP glucose as donor substrates, with the archaeal enzyme, which can utilize both molecules, allowed us to propose the residues that determine glucosyl donor specificity.

Determinants of the nucleocytoplasmic shuttling of muscle glycogen synthase.

Muscle glycogen synthase (MGS) presents a nuclear speckled pattern in primary cultured human muscle and in 3T3-L1 cells deprived of glucose and with depleted glycogen reserves. Nuclear accumulation of the enzyme correlates inversely with cellular glycogen content. Although the glucose-induced export of MGS from the nucleus to the cytoplasm is blocked by leptomycin B, and therefore mediated by CRM1, no nuclear export signal was identified in the sequence of the protein. Deletion analysis shows that the region comprising amino acids 555-633 of human MGS, which encompasses an Arg-rich cluster involved in the allosteric activation of the enzyme by Glc6P, is crucial for its nuclear concentration and aggregation. Mutation of these Arg residues, which desensitizes the enzyme towards Glc6P, interferes with its nuclear accumulation. In contrast, the known phosphorylation sites of MGS that regulate its activity are not involved in the control of its subcellular distribution. Nuclear human MGS co-localizes with the promyelocytic leukaemia oncoprotein and p80-coilin, a marker of Cajal bodies. The subnuclear distribution of MGS is altered by incubation with transcription inhibitors. These observations suggest that, in addition to its metabolic function, MGS may participate in nuclear processes.

Crystallization and preliminary X-ray analysis of the glycogen synthase from Pyrococcus abyssi.

Glycogen synthase catalyzes the transfer of glucosyl residues from ADP- or UDP-glucose to the non-reducing end of a growing alpha-1,4-glucan chain. To date, no crystallographic structure of an animal/fungal glycogen synthase (family 3 of the glycosyl transferases) or a bacterial/plant glycogen/starch synthase (family 5) has been reported. This paper describes the recombinant expression, crystallization and preliminary X-ray analysis of the glycogen synthase from the hyperthermophilic archaeon Pyrococcus abyssi, the smallest enzyme of the members of families 3 and 5 of the glycosyl transferases. Crystals from this protein and from its selenomethionyl variant were grown in 100 mM sodium citrate pH 5.6 containing 20% PEG and 20% dioxane by the hanging-drop vapour-diffusion method at 293 K. The crystals, which grew as thin needles, diffracted to 3.5 A resolution and belong to space group C2, with unit-cell parameters a = 202, b = 73, c = 149 A, beta = 131 degrees. The crystallographic and biochemical data are consistent with either a dimer or a tetramer in the crystal asymmetric unit and a volume solvent content of 70 or 39%, respectively.

Alternative splicing of the human proto-oncogene c-H-ras renders a new Ras family protein that trafficks to cytoplasm and nucleus.

We characterized a novel protein of the Ras family, p19 (H-RasIDX). The c-H-ras proto-oncogene undergoes alternative splicing of the exon termed IDX. We show that the alternative p19 mRNA is stable and as abundant as p21 (p21 H-Ras4A) mRNA in all of the human tissues and cell lines tested. IDX is spliced into stable mRNA in different mammalian species, which present a high degree of nucleotide conservation. Both the endogenous and the transiently expressed p19 protein are detected in COS-1 and HeLa cells and show nuclear diffuse and speckled patterns as well as cytoplasmic localization. In yeast two-hybrid assays, p19 did not interact with two known p21 effectors, Raf1 and Rin1, but was shown to interact with RACK1, a scaffolding protein that promotes multiprotein complexes in different signaling pathways. This observation suggests that p19 and p21 play differential and complementary roles in the cell.

Control of glycogen deposition.

Traditionally, glycogen synthase (GS) has been considered to catalyze the key step of glycogen synthesis and to exercise most of the control over this metabolic pathway. However, recent advances have shown that other factors must be considered. Moreover, the control of glycogen deposition does not follow identical mechanisms in muscle and liver. Glucose must be phosphorylated to promote activation of GS. Glucose-6-phosphate (Glc-6-P) binds to GS, causing the allosteric activation of the enzyme probably through a conformational rearrangement that simultaneously converts it into a better substrate for protein phosphatases, which can then lead to the covalent activation of GS. The potency of Glc-6-P for activation of liver GS is determined by its source, since Glc-6-P arising from the catalytic action of glucokinase (GK) is much more effective in mediating the activation of the enzyme than the same metabolite produced by hexokinase I (HK I). As a result, hepatic glycogen deposition from glucose is subject to a system of control in which the 'controller', GS, is in turn controlled by GK. In contrast, in skeletal muscle, the control of glycogen synthesis is shared between glucose transport and GS. The characteristics of the two pairs of isoenzymes, liver GS/GK and muscle GS/HK I, and the relationships that they establish are tailored to suit specific metabolic roles of the tissues in which they are expressed. The key enzymes in glycogen metabolism change their intracellular localization in response to glucose. The changes in the intracellular distribution of liver GS and GK triggered by glucose correlate with stimulation of glycogen synthesis. The translocation of GS, which constitutes an additional mechanism of control, causes the orderly deposition of hepatic glycogen and probably represents a functional advantage in the metabolism of the polysaccharide.

Glucose 6-phosphate produced by gluconeogenesis and by glucokinase is equally effective in activating hepatic glycogen synthase.

Glucose 6-phosphate (Glc-6-P) produced in cultured hepatocytes by direct phosphorylation of glucose or by gluconeogenesis from dihydroxyacetone (DHA) was equally effective in activating glycogen synthase (GS). However, glycogen accumulation was higher in hepatocytes incubated with glucose than in those treated with DHA. This difference was attributed to decreased futile cycling through GS and glycogen phosphorylase (GP) in the glucose-treated hepatocytes, owing to the partial inactivation of GP induced by glucose. Our results indicate that the gluconeogenic pathway and the glucokinase-mediated phosphorylation of glucose deliver their common product to the same Glc-6-P pool, which is accessible to liver GS. As observed in the treatment with glucose, incubation of cultured hepatocytes with DHA caused the translocation of GS from a uniform cytoplasmic distribution to the hepatocyte periphery and a similar pattern of glycogen deposition. We hypothesize that Glc-6-P has a major role in glycogen metabolism not only by determining the activation state of GS but also by controlling its subcellular distribution in the hepatocyte.

Zonal distribution of glycogen synthesis in isolated rat hepatocytes.

Incubation of hepatocytes isolated from fasted rats with [14C]glucose for short periods of time showed that the initial stages of glycogen synthesis occur near the plasma membrane. Incubation with [14C]glucose followed by cold glucose demonstrated that glycogen synthesis is always active at the hepatocyte periphery and that previously synthesised glycogen moves towards the centre of the cell, while its place is filled by newly synthesised molecules. However, the reverse experiment, incubation with cold glucose before addition of [14C]glucose, showed that, as glycogen synthesis progresses, it also becomes gradually active in more internal sites of the hepatocyte. These results indicate a spatial order in the synthesis of hepatic glycogen.

Glycogen synthase: towards a minimum catalytic unit?

Classically, alpha-1,4-glucan synthases have been divided into two families, animal/fungal glycogen synthases (GS) and bacterial/plant starch synthases (G(S)S), according to differences in sequence, sugar donor specificity and regulatory mechanisms. Detailed sequence analysis, predicted secondary structure comparison and threading analysis show that these two families are structurally related and that some domains of GSs were acquired to meet regulatory requirements. Archaeal G(S)S present structural and functional features that are conserved in one, the other or both families. Therefore, they are the link between GS and G(S)S and harbor the minimal sequence and structural features that constitute the minimum catalytic unit of the alpha-1,4-glucan synthase superfamily.

Crystallization and preliminary X-ray analysis of the hydroperoxidase I C-terminal domain from Escherichia coli.

Hydroperoxidases (HP) are normally large haem-containing bifunctional enzymes capable of acting as both catalases and peroxidases. The C-terminal domain of HPI from Escherichia coli (KatG), extending from residue Tyr422 to Leu726, was found to be resistant to trypsin proteolysis. The segment of katG encoding this domain was cloned and overexpressed to produce a haemless protein that is soluble even at concentrations above 30 mg ml(-1). This protein shows a 25% sequence identity with cytochrome c peroxidase (CCP) from Saccharomyces cerevisae, despite lacking the characteristic catalytic and iron-binding residues. Crystals from this protein were grown in 0.6 M sodium citrate buffered to pH 7.5 with HEPES by the hanging-drop vapour-diffusion method at 293 K. These crystals diffracted beyond 2.0 A resolution and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 84.2, b = 98.7, c = 302.8 A. Three pseudo-origin peaks in the Patterson maps indicate an unusual packing compatible with the presence of three molecules in the crystal asymmetric unit and a solvent content of about 80% by volume.

Liver glycogen synthase but not the muscle isoform differentiates between glucose 6-phosphate produced by glucokinase or hexokinase.

Using adenovirus-mediated gene transfer into FTO-2B cells, a rat hepatoma cell line, we have overexpressed hexokinase I (HK I), glucokinase (GK), liver glycogen synthase (LGS), muscle glycogen synthase (MGS), and combinations of each of the two glucose-phosphorylating enzymes with each one of the GS isoforms. FTO-2B cells do not synthesize glycogen even when incubated with high doses of glucose. Adenovirus-induced overexpression of HK I and/or LGS, two enzymes endogenously expressed by these cells, did not produce a significant increase in the levels of active GS and the total glycogen content. In contrast, GK overexpression led to the glucose-dependent activation of endogenous or overexpressed LGS and to the accumulation of glycogen. Similarly overexpressed MGS was efficiently activated by the glucose-6-phosphate (Glc-6-P) produced by either endogenous or overexpressed HK I and by overexpressed GK. These results indicate the existence of at least two pools of Glc-6-P in the cell, one of them is accessible to both isoforms of GS and is replenished by the action of GK, whereas LGS is excluded from the cellular compartment where the Glc-6-P produced by HK I is directed. These findings are interpreted in terms of the metabolic role that the two pairs of enzymes, HK I-MGS in the muscle and GK-LGS in the hepatocyte, perform in their respective tissues.