Expression and purification of functional human glycogen synthase-1 (hGYS1) in insect cells

Khanna, May; Imasaki, Tsuyoshi; Chikwana, Vimbai M.; Perez‐Miller, Samantha; Hunter, Gerald O.; Mosley, Amber L.; Takagi, Yuichiro; Hurley, Thomas D.

doi:10.1016/j.pep.2013.05.007

Cited by 12 publications

(25 citation statements)

References 27 publications

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“…In addition, a number of non-conventional sites were identified (T54, T372, S412, S486, S652 and S727), albeit at relatively low abundance. Several of these sites have been identified by a recent study where human GYS1 was overexpressed in insect cells [9] and also in global MS screens [20–22] . It might be of interest to perform mutagenesis analysis and examine the impact of these phosphorylation sites on the regulation of GYS1 activity and possibly cellular localisation, as the physiological significance of these non-conventional sites is unknown.…”

Section: Resultsmentioning

confidence: 97%

“…Subsequent studies have demonstrated that glycogenin interacts with glycogen synthase and enhances soluble overexpression of glycogen synthase (when ectopically co-expressed with glycogen synthase in cultured cell lines) [4,8] . This has led Khanna et al [9] to make an attempt to co-express GYS1 and glycogenin using MultiBac system in insect cells, which resulted in a successful soluble expression and purification of large quantities of human GYS1 proteins. Direct interaction of the C-terminus of GN with glycogen synthase was first reported by Skurat et al [10] based on a yeast two-hybrid study followed by cellular overexpression experiments and more recently by an X-ray crystal structure of glycogen synthase in complex with a C-terminal 34-residue fragment of GN [11] .…”

Section: Introductionmentioning

confidence: 99%

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Expression and purification of functional human glycogen synthase-1:glycogenin-1 complex in insect cells

Hunter¹,

Zeqiraj²,

Morrice³

et al. 2015

Protein Expression and Purification

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Section: Resultsmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

Expression and purification of functional human glycogen synthase-1:glycogenin-1 complex in insect cells

Hunter¹,

Zeqiraj²,

Morrice³

et al. 2015

Protein Expression and Purification

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“…The low activity ratio seen in the WT equine GS enzyme (0.02) is similar to that detectable in baculovirus-generated human GS [48]. Despite both the equine WT and mutant insect-derived enzymes’ phosphorylation (at site 3a) (Figure 5A), the mutant equine enzyme had a high activity ratio, similar to that of the S7A mutant human GS, (which cannot be phosphorylated) (Figure 5B).…”

Section: Resultsmentioning

confidence: 60%

“…Further, the R309H enzyme had significantly lower K m for UDPG even in the absence of G6P and the V max /K m value for the mutant enzyme in the absence of G6P was increased over 60 fold, when compared to the WT enzyme. In contrast, the activity ratio of the WT equine enzyme was similar to that of human GS purified in a similar manner [48]. Further, there was no detectable difference in the extent of 3a phosphorylation between the WT and mutant recombinant enzymes, but despite this, the equine recombinant enzyme behaved similarly to that of a mutant human enzyme that cannot be phosphorylated, both with and without pre-treatment with protein phosphatase 1.…”

Section: Discussionmentioning

confidence: 93%

A highly prevalent equine glycogen storage disease is explained by constitutive activation of a mutant glycogen synthase

Maile

Hingst

Mahalingan

et al. 2017

Biochimica et Biophysica Acta (BBA) - General Subjects

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Summary BACKGROUND Equine type 1 polysaccharide storage myopathy (PSSM1) is associated with a missense mutation (R309H) in the glycogen synthase (GYS1) gene, enhanced glycogen synthase (GS) activity and excessive glycogen and amylopectate inclusions in muscle. METHODS Equine muscle biochemical and recombinant enzyme kinetic assays in vitro and homology modelling in silico, were used to investigate the hypothesis that higher GS activity in affected horse muscle is caused by higher GS expression, dysregulation, or constitutive activation via a conformational change. RESULTS PSSM1-affected horse muscle had significantly higher glycogen content than control horse muscle despite no difference in GS expression. GS activity was significantly higher in muscle from homozygous mutants than from heterozygote and control horses, in the absence and presence of the allosteric regulator, glucose 6 phosphate (G6P). Muscle from homozygous mutant horses also had significantly increased GS phosphorylation at sites 2+2a and significantly higher AMPKα1 (an upstream kinase) expression than controls, likely reflecting a physiological attempt to reduce GS enzyme activity. Recombinant mutant GS was highly active with a considerably lower Km for UDP-glucose, in the presence and absence of G6P, when compared to wild type GS, and despite its phosphorylation. CONCLUSIONS Elevated activity of the mutant enzyme is associated with ineffective regulation via phosphorylation rendering it constitutively active. Modelling suggested that the mutation disrupts a salt bridge that normally stabilises the basal state, shifting the equilibrium to the enzyme's active state. GENERAL SIGNIFICANCE This study explains the gain of function pathogenesis in this highly prevalent polyglucosan myopathy.

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“…Recombinant human GYS1 glycogen synthase was produced in insect cells and purified as described by Khanna et al [32]. Hexokinase (#1012765501) and pyrophosphatase (#91078329) were from Roche.…”

Section: Methodsmentioning

confidence: 99%

Incorporation of phosphate into glycogen by glycogen synthase

Contreras

Segvich

Mahalingan

et al. 2016

Archives of Biochemistry and Biophysics

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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, perhaps C3, phosphorylation.

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Expression and purification of functional human glycogen synthase-1 (hGYS1) in insect cells

Cited by 12 publications

References 27 publications

Expression and purification of functional human glycogen synthase-1:glycogenin-1 complex in insect cells

Expression and purification of functional human glycogen synthase-1:glycogenin-1 complex in insect cells

A highly prevalent equine glycogen storage disease is explained by constitutive activation of a mutant glycogen synthase

Incorporation of phosphate into glycogen by glycogen synthase

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