Mechanistic Insights into Glucan Phosphatase Activity against Polyglucan Substrates*

Background: Glucan phosphatases are essential for glycogen and starch metabolism. Results: Comparative enzymology of glucan phosphatases defines the mechanism for specific activity versus physiological glucan substrates. Conclusion: Glucan phosphatases possess a common active site motif but unique specific activities determined by phosphatase and carbohydrate binding domains. Significance: Defining glucan dephosphorylation is essential for understanding normal plant and animal physiology and human disease. Glucan phosphatases are central to the regulation of starch and glycogen metabolism. Plants contain two known glucan phosphatases, Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2), which dephosphorylate starch. Starch is water-insoluble and reversible phosphorylation solubilizes its outer surface allowing processive degradation. Vertebrates contain a single known glucan phosphatase, laforin, that dephosphorylates glycogen. In the absence of laforin, water-soluble glycogen becomes insoluble, leading to the neurodegenerative disorder Lafora Disease. Because of their essential role in starch and glycogen metabolism glucan phosphatases are of significant interest, yet a comparative analysis of their activities against diverse glucan substrates has not been established. We identify active site residues required for specific glucan dephosphorylation, defining a glucan phosphatase signature motif (CζAGΨGR) in the active site loop. We further explore the basis for phosphate position-specific activity of these enzymes and determine that their diverse phosphate position-specific activity is governed by the phosphatase domain. In addition, we find key differences in glucan phosphatase activity toward soluble and insoluble polyglucan substrates, resulting from the participation of ancillary glucan-binding domains. Together, these data provide fundamental insights into the specific activity of glucan phosphatases against diverse polyglucan substrates.

[1]  C. V. Vander Kooi,et al.  Structural Mechanism of Laforin Function in Glycogen Dephosphorylation and Lafora Disease , 2015, Molecular cell.

[2]  M. Ishihara,et al.  Glycogen Phosphomonoester Distribution in Mouse Models of the Progressive Myoclonic Epilepsy, Lafora Disease* , 2014, The Journal of Biological Chemistry.

[3]  O. Kötting,et al.  Phosphoglucan phosphatase function sheds light on starch degradation. , 2014, Trends in plant science.

[4]  A. Garz,et al.  Phosphorylation of transitory starch by α-glucan, water dikinase during starch turnover affects the surface properties and morphology of starch granules. , 2014, The New phytologist.

[5]  C. V. Vander Kooi,et al.  Phosphoglucan-bound structure of starch phosphatase Starch Excess4 reveals the mechanism for C6 specificity , 2014, Proceedings of the National Academy of Sciences.

[6]  J. Fettke,et al.  The glucan phosphorylation mediated by α-glucan, water dikinase (GWD) is also essential in the light phase for a functional transitory starch turn-over , 2014, Plant signaling & behavior.

[7]  W. Gruissem,et al.  Glucan, Water Dikinase Exerts Little Control over Starch Degradation in Arabidopsis Leaves at Night1[W][OPEN] , 2014, Plant Physiology.

[8]  C. V. Vander Kooi,et al.  Structure of the Arabidopsis Glucan Phosphatase LIKE SEX FOUR2 Reveals a Unique Mechanism for Starch Dephosphorylation[W] , 2013, Plant Cell.

[9]  P. Schmieder,et al.  Hyperphosphorylation of glucosyl C6 carbons and altered structure of glycogen in the neurodegenerative epilepsy Lafora disease. , 2013, Cell metabolism.

[10]  C. Worby,et al.  A malachite green-based assay to assess glucan phosphatase activity. , 2013, Analytical biochemistry.

[11]  N. Tonks.  Protein Tyrosine Phosphatases: From Housekeeping Enzymes to Master-Regulators of Signal Transduction , 2013 .

[12]  Matthew S. Gentry,et al.  Laforin, a protein with many faces: glucan phosphatase, adapter protein, et alii , 2013, The FEBS journal.

[13]  S. Zeeman,et al.  Starch Metabolism in Arabidopsis , 2012, The arabidopsis book.

[14]  A. Depaoli-Roach,et al.  Glycogen and its metabolism: some new developments and old themes. , 2012, The Biochemical journal.

[15]  N. Patron,et al.  The Phosphoglucan Phosphatase Like Sex Four2 Dephosphorylates Starch at the C3-Position in Arabidopsis[W][OA] , 2011, Plant Cell.

[16]  A. Blennow,et al.  Hyperphosphorylation of cereal starch , 2011 .

[17]  M. Ishihara,et al.  Phosphate incorporation during glycogen synthesis and Lafora disease. , 2011, Cell metabolism.

[18]  C. Ackerley,et al.  Glycogen hyperphosphorylation underlies lafora body formation , 2010, Annals of neurology.

[19]  C. V. Vander Kooi,et al.  Structural basis for the glucan phosphatase activity of Starch Excess4 , 2010, Proceedings of the National Academy of Sciences.

[20]  S. Zeeman,et al.  Regulation of starch metabolism: the age of enlightenment? , 2010, Current opinion in plant biology.

[21]  S. Engelsen,et al.  Helix-breaking news: fighting crystalline starch energy deposits in the cell. , 2010, Trends in plant science.

[22]  D. MacLean,et al.  A Putative Phosphatase, LSF1, Is Required for Normal Starch Turnover in Arabidopsis Leaves1[W][OA] , 2009, Plant Physiology.

[23]  S. Zeeman,et al.  The Laforin-Like Dual-Specificity Phosphatase SEX4 from Arabidopsis Hydrolyzes Both C6- and C3-Phosphate Esters Introduced by Starch-Related Dikinases and Thereby Affects Phase Transition of α-Glucans1[W] , 2009, Plant Physiology.

[24]  C. Worby,et al.  Lafora disease: insights into neurodegeneration from plant metabolism. , 2009, Trends in biochemical sciences.

[25]  Matthew S. Gentry,et al.  Conservation of the glucan phosphatase laforin is linked to rates of molecular evolution and the glucan metabolism of the organism , 2009, BMC Evolutionary Biology.

[26]  David Kerk,et al.  Evolution of protein phosphatases in plants and animals. , 2009, The Biochemical journal.

[27]  Alison M. Smith,et al.  STARCH-EXCESS4 Is a Laforin-Like Phosphoglucan Phosphatase Required for Starch Degradation in Arabidopsis thaliana[W][OA] , 2009, The Plant Cell Online.

[28]  M. Cascante,et al.  How did glycogen structure evolve to satisfy the requirement for rapid mobilization of glucose? A problem of physical constraints in structure building , 1997, Journal of Molecular Evolution.

[29]  A. Depaoli-Roach,et al.  Abnormal Metabolism of Glycogen Phosphate as a Cause for Lafora Disease* , 2008, Journal of Biological Chemistry.

[30]  C. Frohberg,et al.  Glucan, water dikinase phosphorylates crystalline maltodextrins and thereby initiates solubilization. , 2008, The Plant journal : for cell and molecular biology.

[31]  A. Delgado-Escueta,et al.  Laforin is a glycogen phosphatase, deficiency of which leads to elevated phosphorylation of glycogen in vivo , 2007, Proceedings of the National Academy of Sciences.

[32]  J. Ecker,et al.  The phosphatase laforin crosses evolutionary boundaries and links carbohydrate metabolism to neuronal disease , 2007, The Journal of cell biology.

[33]  M. Steup,et al.  Glucan, Water Dikinase Activity Stimulates Breakdown of Starch Granules by Plastidial β-Amylases1[W][OA] , 2007, Plant Physiology.

[34]  M. Steup,et al.  Glucan, Water Dikinase Activity Stimulates Breakdown of Starch Granules by Plastidial b-Amylases , 2007 .

[35]  Alison M. Smith,et al.  The diurnal metabolism of leaf starch. , 2007, The Biochemical journal.

[36]  Yang Liu,et al.  Dimerization of Laforin Is Required for Its Optimal Phosphatase Activity, Regulation of GSK3β Phosphorylation, and Wnt Signaling* , 2006, Journal of Biological Chemistry.

[37]  N. Tonks,et al.  Protein tyrosine phosphatases: from genes, to function, to disease , 2006, Nature Reviews Molecular Cell Biology.

[38]  C. Worby,et al.  Laforin, a Dual Specificity Phosphatase That Dephosphorylates Complex Carbohydrates* , 2006, Journal of Biological Chemistry.

[39]  M. Steup,et al.  Phosphorylation of C6‐ and C3‐positions of glucosyl residues in starch is catalysed by distinct dikinases , 2006, FEBS letters.

[40]  G. Moorhead,et al.  A chloroplast-localized dual-specificity protein phosphatase in Arabidopsis contains a phylogenetically dispersed and ancient carbohydrate-binding domain, which binds the polysaccharide starch. , 2006, The Plant journal : for cell and molecular biology.

[41]  Alison M. Smith,et al.  Similar Protein Phosphatases Control Starch Metabolism in Plants and Glycogen Metabolism in Mammals* , 2006, Journal of Biological Chemistry.

[42]  Partho Ghosh,et al.  Molecular basis for substrate recognition by MTMR2, a myotubularin family phosphoinositide phosphatase. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[43]  P. Geigenberger,et al.  Identification of a Novel Enzyme Required for Starch Metabolism in Arabidopsis Leaves. The Phosphoglucan, Water Dikinase1[w] , 2005, Plant Physiology.

[44]  Dr. Gonzalo R. Lafora.  Über das Vorkommen amyloider Körperchen im Innern der Ganglienzellen , 1911, Virchows Archiv für pathologische Anatomie und Physiologie und für klinische Medizin.

[45]  P. Roach,et al.  Glycogen and related polysaccharides inhibit the laforin dual-specificity protein phosphatase. , 2004, Biochemical and biophysical research communications.

[46]  S. Scherer,et al.  Laforin preferentially binds the neurotoxic starch-like polyglucosans, which form in its absence in progressive myoclonus epilepsy. , 2004, Human molecular genetics.

[47]  J. Karkalas,et al.  Starch-composition, fine structure and architecture , 2004 .

[48]  S. Scherer,et al.  Loss of function of the cytoplasmic isoform of the protein laforin (EPM2A) causes Lafora progressive myoclonus epilepsy , 2004, Human mutation.

[49]  K. Yamakawa,et al.  The carbohydrate-binding domain of Lafora disease protein targets Lafora polyglucosan bodies. , 2004, Biochemical and biophysical research communications.

[50]  A. Godzik,et al.  The dual-specific protein tyrosine phosphatase family , 2004 .

[51]  D. Barrett,et al.  Determination of reducing sugars with 3-methyl-2-benzothiazolinonehydrazone. , 2002, Analytical biochemistry.

[52]  M. T. Medina,et al.  Genotype-phenotype correlations for EPM2A mutations in Lafora's progressive myoclonus epilepsy: exon 1 mutations associate with an early-onset cognitive deficit subphenotype. , 2002, Human molecular genetics.

[53]  M. Steup,et al.  The starch-related R1 protein is an α-glucan, water dikinase , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[54]  T. Fenton,et al.  A novel higher plant protein tyrosine phosphatase interacts with SNF1-related protein kinases via a KIS (kinase interaction sequence) domain. , 2002, The Plant journal : for cell and molecular biology.

[55]  J. Dixon,et al.  A Unique Carbohydrate Binding Domain Targets the Lafora Disease Phosphatase to Glycogen* , 2002, The Journal of Biological Chemistry.

[56]  M. Steup,et al.  The starch-related R1 protein is an alpha -glucan, water dikinase. , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[57]  B. Minassian.  Lafora's disease: towards a clinical, pathologic, and molecular synthesis. , 2001, Pediatric neurology.

[58]  A. Weber,et al.  The Arabidopsis sex1 mutant is defective in the R1 protein, a general regulator of starch degradation in plants, and not in the chloroplast hexose transporter. , 2001, The Plant cell.

[59]  S. Scherer,et al.  Mutation spectrum and predicted function of laforin in Lafora’s progressive myoclonus epilepsy , 2000, Neurology.

[60]  J. Cavanagh.  Corpora-amylacea and the family of polyglucosan diseases , 1999, Brain Research Reviews.

[61]  R. Michelucci,et al.  A novel protein tyrosine phosphatase gene is mutated in progressive myoclonus epilepsy of the Lafora type (EPM2). , 1999, Human molecular genetics.

[62]  S. Scherer,et al.  Mutations in a gene encoding a novel protein tyrosine phosphatase cause progressive myoclonus epilepsy , 1998, Nature Genetics.

[63]  Alison M. Smith,et al.  A Mutant of Arabidopsis Lacking a Chloroplastic Isoamylase Accumulates Both Starch and Phytoglycogen , 1998, Plant Cell.

[64]  P Colonna,et al.  Starch granules: structure and biosynthesis. , 1998, International journal of biological macromolecules.

[65]  S. Zeeman,et al.  A starch-accumulating mutant of Arabidopsis thaliana deficient in a chloroplastic starch-hydrolysing enzyme. , 1998, The Plant journal : for cell and molecular biology.

[66]  D. Gallant,et al.  Microscopy of starch : evidence of a new level of granule organization , 1997 .

[67]  Jack E. Dixon,et al.  Crystal Structure of the Dual Specificity Protein Phosphatase VHR , 1996, Science.

[68]  D. Barford,et al.  Structural basis for phosphotyrosine peptide recognition by protein tyrosine phosphatase 1B. , 1995, Science.

[69]  G. Zhou,et al.  The catalytic role of Cys124 in the dual specificity phosphatase VHR. , 1994, The Journal of biological chemistry.

[70]  E. Meléndez-Hevia,et al.  Optimization of molecular design in the evolution of metabolism: the glycogen molecule. , 1993, The Biochemical journal.

[71]  A. Eliasson,et al.  Influence of the naturally occurring phosphate esters on the crystallinity of potato starch , 1991 .

[72]  C. Schuerch,et al.  Solid-phase synthesis of oligosaccharides. V. Preparation of an inorganic support. , 1975, Carbohydrate research.

[73]  M. Sakai,et al.  Studies in myoclonus epilepsy (Lafora body form) , 1970, Neurology.

[74]  S. Hizukuri.  Studies on Starch Phosphate , 1970 .

[75]  Collins Gh,et al.  Myoclonus epilepsy with Lafora bodies. An ultrastruc- tural and cytochemical study. , 1968 .

[76]  M. Sakai,et al.  Studies in myoclonus epilepsy (Lafora body form). I. Isolation and preliminary characterization of Lafora bodies in two cases. , 1968, Archives of neurology.

[77]  R. R. Cowden,et al.  Myoclonus epilepsy with Lafora bodies. An ultrastruc- tural and cytochemical study. , 1968, Archives of pathology.

[78]  J. Austin,et al.  Isolation and characterization of Lafora bodies in two cases of myoclonus epilepsy. , 1967, Journal of neuropathology and experimental neurology.