Abstract
We report the successful expression and purification of functional human muscle glycogen synthase (GYS1) in complex with human glycogenin-1 (GN1). Stoichiometric GYS1:GN1 complex was produced by co-expression of GYS1 and GN1 using a bicistronic pFastBac™-Dual expression vector, followed by affinity purification and subsequent size-exclusion chromatography. Mass spectrometry analysis identified that GYS1 is phosphorylated at several well-characterised and uncharacterised Ser/Thr residues. Biochemical analysis, including activity ratio (in the absence relative to that in the presence of glucose-6-phosphate) measurement, covalently attached phosphate estimation as well as phosphatase treatment, revealed that recombinant GYS1 is substantially more heavily phosphorylated than would be observed in intact human or rodent muscle tissues. A large quantity of highly-pure stoichiometric GYS1:GN1 complex will be useful to study its structural and biochemical properties in the future, which would reveal mechanistic insights into its functional role in glycogen biosynthesis.
Highlights
Glycogen is a branched polymer of glucose that serves as a repository of rapidly accessible energy and, plays an important role in maintaining glucose and energy homeostasis at both cellular and organism levels [1]
The GYS1:GN1 complex produced in this manner was >95% pure as assessed by SDS–PAGE and had a yield of $1 mg/L of Sf9 culture (Fig. 1B)
Regardless, the two forms assayed here exist at the extremes of phosphorylation state are not representative of physiological states in vivo, but do compare favourably with values reported for recombinant GYS1 purified from HEK293 cells which is heavily phosphorylated and essentially inactive in the absence of G6P [17]. These results demonstrate that the GYS1:GN1 complex as prepared from insect cells is functional and exhibits properties similar to protein preparations from mammalian expression systems
Summary
Glycogen is a branched polymer of glucose that serves as a repository of rapidly accessible energy and, plays an important role in maintaining glucose and energy homeostasis at both cellular and organism levels [1]. There are two glycogen synthase isoforms: muscle glycogen synthase (GYS1; encoded by GYS1), which is ubiquitously expressed, but most abundantly expressed in skeletal and cardiac muscles, and the liver-specific isoform (GYS2; encoded by GYS2). Activity of both isoforms is regulated by a complex interplay between reversible phosphorylation at multiple sites and allosteric regulators of which glucose-6-phosphate (G6P), an activator, plays the most important role [1,4,5]
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