Incorporation of phosphate into glycogen by glycogen synthase

Christopher J. Contreras, Dyann M. Segvich, Krishna Mahalingan, Vimbai M. Chikwana, Terence L. Kirley, Thomas Hurley, Anna De Paoli-Roach, Peter Roach

Research output: Contribution to journalArticle

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Abstract

The storage polymer glycogen normally contains small amounts of covalently attached phosphate as phosphomonoesters at C2, C3 and C6 atoms of glucose residues. In the absence of the laforin phosphatase, as in the rare childhood epilepsy Lafora disease, the phosphorylation level is elevated and is associated with abnormal glycogen structure that contributes to the pathology. Laforin therefore likely functions in vivo as a glycogen phosphatase. The mechanism of glycogen phosphorylation is less well-understood. We have reported that glycogen synthase incorporates phosphate into glycogen via a rare side reaction in which glucose-phosphate rather than glucose is transferred to a growing polyglucose chain (Tagliabracci et al. (2011) Cell Metab 13, 274-282). We proposed a mechanism to account for phosphorylation at C2 and possibly at C3. Our results have since been challenged (Nitschke et al. (2013) Cell Metab 17, 756-767). Here we extend the evidence supporting our conclusion, validating the assay used for the detection of glycogen phosphorylation, measurement of the transfer of 32P from [β-32P]UDP-glucose to glycogen by glycogen synthase. The 32P associated with the glycogen fraction was stable to ethanol precipitation, SDS-PAGE and gel filtration on Sephadex G50. The 32P-signal was not affected by inclusion of excess unlabeled UDP before analysis or by treatment with a UDPase, arguing against the signal being due to contaminating [β-32P]UDP generated in the reaction. Furthermore, [32P]UDP did not bind non-covalently to glycogen. The 32P associated with glycogen was released by laforin treatment, suggesting that it was present as a phosphomonoester. The conclusion is that glycogen synthase can mediate the introduction of phosphate into glycogen, thereby providing a possible mechanism for C2, and perhaps C3, phosphorylation.

Original languageEnglish (US)
Pages (from-to)21-29
Number of pages9
JournalArchives of Biochemistry and Biophysics
Volume597
DOIs
StatePublished - May 1 2016

Fingerprint

Glycogen Synthase
Glycogen
Phosphates
Phosphorylation
Uridine Diphosphate
Phosphoric Monoester Hydrolases
Glucose
Lafora Disease
Uridine Diphosphate Glucose
Glucans
Pathology
Gel Chromatography
Polyacrylamide Gel Electrophoresis
Epilepsy
Assays
Polymers
Ethanol
Gels

Keywords

  • Glycogen
  • Glycogen synthase
  • Lafora
  • Laforin
  • Phosphorylation

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Incorporation of phosphate into glycogen by glycogen synthase. / Contreras, Christopher J.; Segvich, Dyann M.; Mahalingan, Krishna; Chikwana, Vimbai M.; Kirley, Terence L.; Hurley, Thomas; De Paoli-Roach, Anna; Roach, Peter.

In: Archives of Biochemistry and Biophysics, Vol. 597, 01.05.2016, p. 21-29.

Research output: Contribution to journalArticle

Contreras, Christopher J. ; Segvich, Dyann M. ; Mahalingan, Krishna ; Chikwana, Vimbai M. ; Kirley, Terence L. ; Hurley, Thomas ; De Paoli-Roach, Anna ; Roach, Peter. / Incorporation of phosphate into glycogen by glycogen synthase. In: Archives of Biochemistry and Biophysics. 2016 ; Vol. 597. pp. 21-29.
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AU - Contreras, Christopher J.

AU - Segvich, Dyann M.

AU - Mahalingan, Krishna

AU - Chikwana, Vimbai M.

AU - Kirley, Terence L.

AU - Hurley, Thomas

AU - De Paoli-Roach, Anna

AU - Roach, Peter

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N2 - The storage polymer glycogen normally contains small amounts of covalently attached phosphate as phosphomonoesters at C2, C3 and C6 atoms of glucose residues. In the absence of the laforin phosphatase, as in the rare childhood epilepsy Lafora disease, the phosphorylation level is elevated and is associated with abnormal glycogen structure that contributes to the pathology. Laforin therefore likely functions in vivo as a glycogen phosphatase. The mechanism of glycogen phosphorylation is less well-understood. We have reported that glycogen synthase incorporates phosphate into glycogen via a rare side reaction in which glucose-phosphate rather than glucose is transferred to a growing polyglucose chain (Tagliabracci et al. (2011) Cell Metab 13, 274-282). We proposed a mechanism to account for phosphorylation at C2 and possibly at C3. Our results have since been challenged (Nitschke et al. (2013) Cell Metab 17, 756-767). Here we extend the evidence supporting our conclusion, validating the assay used for the detection of glycogen phosphorylation, measurement of the transfer of 32P from [β-32P]UDP-glucose to glycogen by glycogen synthase. The 32P associated with the glycogen fraction was stable to ethanol precipitation, SDS-PAGE and gel filtration on Sephadex G50. The 32P-signal was not affected by inclusion of excess unlabeled UDP before analysis or by treatment with a UDPase, arguing against the signal being due to contaminating [β-32P]UDP generated in the reaction. Furthermore, [32P]UDP did not bind non-covalently to glycogen. The 32P associated with glycogen was released by laforin treatment, suggesting that it was present as a phosphomonoester. The conclusion is that glycogen synthase can mediate the introduction of phosphate into glycogen, thereby providing a possible mechanism for C2, and perhaps C3, phosphorylation.

AB - The storage polymer glycogen normally contains small amounts of covalently attached phosphate as phosphomonoesters at C2, C3 and C6 atoms of glucose residues. In the absence of the laforin phosphatase, as in the rare childhood epilepsy Lafora disease, the phosphorylation level is elevated and is associated with abnormal glycogen structure that contributes to the pathology. Laforin therefore likely functions in vivo as a glycogen phosphatase. The mechanism of glycogen phosphorylation is less well-understood. We have reported that glycogen synthase incorporates phosphate into glycogen via a rare side reaction in which glucose-phosphate rather than glucose is transferred to a growing polyglucose chain (Tagliabracci et al. (2011) Cell Metab 13, 274-282). We proposed a mechanism to account for phosphorylation at C2 and possibly at C3. Our results have since been challenged (Nitschke et al. (2013) Cell Metab 17, 756-767). Here we extend the evidence supporting our conclusion, validating the assay used for the detection of glycogen phosphorylation, measurement of the transfer of 32P from [β-32P]UDP-glucose to glycogen by glycogen synthase. The 32P associated with the glycogen fraction was stable to ethanol precipitation, SDS-PAGE and gel filtration on Sephadex G50. The 32P-signal was not affected by inclusion of excess unlabeled UDP before analysis or by treatment with a UDPase, arguing against the signal being due to contaminating [β-32P]UDP generated in the reaction. Furthermore, [32P]UDP did not bind non-covalently to glycogen. The 32P associated with glycogen was released by laforin treatment, suggesting that it was present as a phosphomonoester. The conclusion is that glycogen synthase can mediate the introduction of phosphate into glycogen, thereby providing a possible mechanism for C2, and perhaps C3, phosphorylation.

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