Glycogen phosphorylation and Lafora disease

Covalent phosphorylation of glycogen, first described 35 years ago, was put on firm ground through the work of the Whelan laboratory in the 1990s. But glycogen phosphorylation lay fallow until interest was rekindled in the mid 2000s by the finding that it could be removed by a glycogen-binding phosphatase, laforin, and that mutations in laforin cause a fatal teenage-onset epilepsy, called Lafora disease. Glycogen phosphorylation is due to phosphomonoesters at C2, C3 and C6 of glucose residues. Phosphate is rare, ranging from 1:500 to 1:5000 phosphates/glucose depending on the glycogen source. The mechanisms of glycogen phosphorylation remain under investigation but one hypothesis to explain C2 and perhaps C3 phosphate is that it results from a rare side reaction of the normal synthetic enzyme glycogen synthase. Lafora disease is likely caused by over-accumulation of abnormal glycogen in insoluble deposits termed Lafora bodies in neurons. The abnormality in the glycogen correlates with elevated phosphorylation (at C2, C3 and C6), reduced branching, insolubility and an enhanced tendency to aggregate and become insoluble. Hyperphosphorylation of glycogen is emerging as an important feature of this deadly childhood disease

Are there errors in glycogen biosynthesis and is laforin a repair enzyme?

Glycogen, a branched polymer of glucose, is well known as a cellular reserve of metabolic energy and/or biosynthetic precursors. Besides glucose, however, glycogen contains small amounts of covalent phosphate, present as C2 and C3 phosphomonoesters. Current evidence suggests that the phosphate is introduced by the biosynthetic enzyme glycogen synthase as a rare alternative to its normal catalytic addition of glucose units. The phosphate can be removed by the laforin phosphatase, whose mutation causes a fatal myoclonus epilepsy called Lafora disease. The hypothesis is that glycogen phosphorylation can be considered a catalytic error and laforin a repair enzyme

Lafora disease offers a unique window into neuronal glycogen metabolism

Lafora disease (LD) is a fatal, autosomal recessive, glycogen-storage disorder that manifests as severe epilepsy. LD results from mutations in the gene encoding either the glycogen phosphatase laforin or the E3 ubiquitin ligase malin. Individuals with LD develop cytoplasmic, aberrant glycogen inclusions in nearly all tissues that more closely resemble plant starch than human glycogen. This Minireview discusses the unique window into glycogen metabolism that LD research offers. It also highlights recent discoveries, including that glycogen contains covalently bound phosphate and that neurons synthesize glycogen and express both glycogen synthase and glycogen phosphorylase

Glycogen and its metabolism: some new developments and old themes

Glycogen is a branched polymer of glucose that acts as a store of energy in times of nutritional sufficiency for utilization in times of need. Its metabolism has been the subject of extensive investigation and much is known about its regulation by hormones such as insulin, glucagon and adrenaline (epinephrine). There has been debate over the relative importance of allosteric compared with covalent control of the key biosynthetic enzyme, glycogen synthase, as well as the relative importance of glucose entry into cells compared with glycogen synthase regulation in determining glycogen accumulation. Significant new developments in eukaryotic glycogen metabolism over the last decade or so include: (i) three-dimensional structures of the biosynthetic enzymes glycogenin and glycogen synthase, with associated implications for mechanism and control; (ii) analyses of several genetically engineered mice with altered glycogen metabolism that shed light on the mechanism of control; (iii) greater appreciation of the spatial aspects of glycogen metabolism, including more focus on the lysosomal degradation of glycogen; and (iv) glycogen phosphorylation and advances in the study of Lafora disease, which is emerging as a glycogen storage disease

Novel method for detection of glycogen in cells

Glycogen, a branched polymer of glucose, functions as an energy reserve in many living organisms. Abnormalities in glycogen metabolism, usually excessive accumulation, can be caused genetically, most often through mutation of the enzymes directly involved in synthesis and degradation of the polymer leading to a variety of glycogen storage diseases (GSDs). Microscopic visualization of glycogen deposits in cells and tissues is important for the study of normal glycogen metabolism as well as diagnosis of GSDs. Here, we describe a method for the detection of glycogen using a renewable, recombinant protein which contains the carbohydrate-binding module (CBM) from starch-binding domain containing protein 1 (Stbd1). We generated a fusion protein containing g lutathione S-transferase, a cM c eptitope and the tbd1 BM (GYSC) for use as a glycogen-binding probe, which can be detected with secondary antibodies against glutathione S-transferase or cMyc. By enzyme-linked immunosorbent assay, we demonstrate that GYSC binds glycogen and two other polymers of glucose, amylopectin and amylose. Immunofluorescence staining of cultured cells indicate a GYSC-specific signal that is co-localized with signals obtained with anti-glycogen or anti-glycogen synthase antibodies. GYSC-positive staining inside of lysosomes is observed in individual muscle fibers isolated from mice deficient in lysosomal enzyme acid alpha-glucosidase, a well-characterized model of GSD II (Pompe disease). Co-localized GYSC and glycogen signals are also found in muscle fibers isolated from mice deficient in malin, a model for Lafora disease. These data indicate that GYSC is a novel probe that can be used to study glycogen metabolism under normal and pathological conditions

Electrophoretic deposition of primary coat onto investment casting wax patterns

The objective of the work reported in this thesis was to tailor a colloidal processing technique called electrophoretic deposition (EPD) for use within the investment casting shell formation process, where the EPD coating procedure would be used to form the primary ceramic coating on the melt-out substrate. EPD takes place due to the presence of an electric field within the suspension medium, which attracts charged particles in the suspension towards an electrode of opposite charge, onto which they are deposited. For the complex structures created using investment casting, the die cast patterns used as the substrate for the ceramic have to be easily removed, and so substrates materials that can either be melted or dissolved out the material to leave the hollow ceramic shell used. To implement EPD into the investment casting process, this substrate needed to be conducting, and so conducting particle-filled investment casting waxes were created and analysed. Carbon black and graphite filler were incorporated into waxes, and the conductivity and rheology of the resultant composites were studied, to gauge their suitability as an investment casting pattern material. On the basis of both cost and for environmental reasons, the use of aqueous suspension media for EPD was preferred over the more commonly used organic systems. EPD was carried out using zircon in aqueous suspension, and the low particle concentration suspensions were stabilised through pH modification and anionic dispersant addition. The effect of suspension parameters and EPD set-up parameters on the coatings formed on compressed graphite electrodes and conductive wax electrodes were studied, through yield measurements and cross sectional analysis using scanning electron microscopy

Mental Health Malingering and the Fraudulent Motor Insurance Claimant

Malingering is the intentional production of false or grossly exaggerated symptoms in order to obtain an advantage. Although it has been estimated that over 800,000 claims for personal injury in Road Traffic Accidents (RTA) were filed in the UK in 2012, no approximation exists forhow many involved malingering. This study attempts to understand what influences a psychiatrist to conclude that a claimant’s symptoms are not caused by an RTA and thus suggests the claimant is malingering. This article describes a study of Personality Assessment Inventory scores alongside collateral forms of evidence for 100 RTA claimants; all individuals seeking compensation for damages to their mental health. The results suggest that up to 40% of these claims could be cate-gorised as not being the result of the RTA. Significant differences emerged between those claimants diagnosed as having a mental disorder as a result of the RTA and those claimants who were classified as not having a mental disorder as a result of the RTA in regards to: employment status, level of injuries and scores on the paranoia scales of the PAI.The study emphasises how the assessment process is idiosyncratic and in need of further researc

Redox Switch for the Inhibited State of Yeast Glycogen Synthase Mimics Regulation by Phosphorylation

Glycogen synthase (GS) is the rate limiting enzyme in the synthesis of glycogen. Eukaryotic GS is negatively regulated by covalent phosphorylation and allosterically activated by glucose-6-phosphate (G6P). To gain structural insights into the inhibited state of the enzyme, we solved the crystal structure of yGsy2-R589A/R592A to a resolution of 3.3 Å. The double mutant has an activity ratio similar to the phosphorylated enzyme and also retains the ability to be activated by G6P. When compared to the 2.88 Å structure of the wild-type G-6-P activated enzyme, the crystal structure of the low-activity mutant showed that the N-terminal domain of the inhibited state is tightly held against the dimer-related interface thereby hindering acceptor access to the catalytic cleft. Based on these two structural observations, we developed a reversible redox regulatory feature in yeast GS by substituting cysteine residues for two highly conserved arginine residues. When oxidized, the cysteine mutant enzyme exhibits activity levels similar to the phosphorylated enzyme, but cannot be activated by G-6-P. Upon reduction, the cysteine mutant enzyme regains normal activity levels and regulatory response to G-6-P activation

Glycogen Phosphomonoester Distribution in Mouse Models of the Progressive Myoclonic Epilepsy, Lafora Disease

Glycogen is a branched polymer of glucose that acts as an energy reserve in many cell types. Glycogen contains trace amounts of covalent phosphate, in the range of 1 phosphate per 500–2000 glucose residues depending on the source. The function, if any, is unknown, but in at least one genetic disease, the progressive myoclonic epilepsy Lafora disease, excessive phosphorylation of glycogen has been implicated in the pathology by disturbing glycogen structure. Some 90% of Lafora cases are attributed to mutations of the EPM2A or EPM2B genes, and mice with either gene disrupted accumulate hyperphosphorylated glycogen. It is, therefore, of importance to understand the chemistry of glycogen phosphorylation. Rabbit skeletal muscle glycogen contained covalent phosphate as monoesters of C2, C3, and C6 carbons of glucose residues based on analyses of phospho-oligosaccharides by NMR. Furthermore, using a sensitive assay for glucose 6-P in hydrolysates of glycogen coupled with measurement of total phosphate, we determined the proportion of C6 phosphorylation in rabbit muscle glycogen to be ∼20%. C6 phosphorylation also accounted for ∼20% of the covalent phosphate in wild type mouse muscle glycogen. Glycogen phosphorylation in Epm2a−/− and Epm2b−/− mice was increased 8- and 4-fold compared with wild type mice, but the proportion of C6 phosphorylation remained unchanged at ∼20%. Therefore, our results suggest that C2, C3, and/or C6 phosphate could all contribute to abnormal glycogen structure or to Lafora disease

Observation of a Free-Shercliff-Layer Instability in Cylindrical Geometry

We report on observations of a free-Shercliff-layer instability in a Taylor-Couette experiment using a liquid metal over a wide range of Reynolds numbers, $Re\sim 10^3-10^6$. The free Shercliff layer is formed by imposing a sufficiently strong axial magnetic field across a pair of differentially rotating axial endcap rings. This layer is destabilized by a hydrodynamic Kelvin-Helmholtz-type instability, characterized by velocity fluctuations in the $r-\theta$ plane. The instability appears with an Elsasser number above unity, and saturates with an azimuthal mode number $m$ which increases with the Elsasser number. Measurements of the structure agree well with 2D global linear mode analyses and 3D global nonlinear simulations. These observations have implications for a range of rotating MHD systems in which similar shear layers may be produced.Comment: 5 pages, 4 figure