Characterization of Human Glycogenin-2, a Self-glucosylating Initiator of Liver Glycogen Metabolism

James Mu,Peter J Roach

doi:10.1074/jbc.273.52.34850

Abstract

Glycogenin-2 is a recently described self-glucosylating protein potentially involved in the initiation of glycogen biosynthesis (Mu, J., Skurat, A. V., and Roach, P. J. (1997) J. Biol. Chem. 272, 27589-27597). In human liver extracts, most of the glycogenin-2 was only detectable after treatment with alpha-amylase. Similarly, purifed high Mr glycogen was only detected after release by alpha-amylase treatment. Based on analysis by polymerase chain reaction, the predominant isoform in liver was glycogenin-2beta. Glycogenin-2 was found in Ewing's sarcoma RD-ES cells where, however, it was not associated with high Mr carbohydrate. Both human liver and human RD-ES cell extracts also contained glycogenin-1. Glycogenin-1 and glycogenin-2 interact with one another, based on in vitro interactions and co-immunoprecipitation from liver and cell extracts. Mutation of Tyr-196 in glycogenin-2 to a Phe residue abolished the ability of glycogenin-2 to self-glucosylate but not to interact with glycogenin-1. Stable overexpression of glycogenin-2alpha in Rat-1 fibroblast cells resulted in a 5-fold increase in the level of glycogen present in the low speed pellet but little change in the low speed supernatant. This result is important since it indicates that the level of glycogenin-2 can determine glycogen accumulation and hence has the potential to control glycogen synthesis.

Highlights

Glycogen, a polymeric storage form of glucose, is present in many cell types, but, in absolute amount, most is stored in skeletal muscle and liver where, for the most part, a different set of enzymes mediates its metabolism [1,2,3]
Glycogenin-2 Is Covalently Associated with Human Liver Glycogen—In our initial characterization of glycogenin-2 [15], we detected glycogenin-2 in a rat liver fraction and in H4IIEC3 cells
Analysis of 12,000 ϫ g supernatants from human liver extracts by Western analysis clearly indicated the presence of species of ϳ66 kDa, the expected size of glycogenin-2 based on analysis of recombinant proteins (Fig. 1A)

Summary

Introduction

A polymeric storage form of glucose, is present in many cell types, but, in absolute amount, most is stored in skeletal muscle and liver where, for the most part, a different set of enzymes mediates its metabolism [1,2,3]. Analysis of 12,000 ϫ g supernatants from human liver extracts by Western analysis clearly indicated the presence of species of ϳ66 kDa, the expected size of glycogenin-2 based on analysis of recombinant proteins (Fig. 1A). By using recombinant glycogenin-2 as a standard in the Western analysis, we estimated that the glycogenin-2 content in human liver is about 0.15 mg/g of tissue, distributed between the supernatant and pellet fractions as defined above.

Results

Conclusion