Cardiomyocyte protein O-GlcNAcylation is regulated by GFAT1 not GFAT2

Adam A Nabeebaccus,Sharwari Verma,Anna Zoccarato,Giulia Emanuelli,Celio Xc Santos,Katrin Streckfuss-Bömeke,Ajay M Shah

doi:10.1016/j.bbrc.2021.10.056

Abstract

In response to cardiac injury, increased activity of the hexosamine biosynthesis pathway (HBP) is linked with cytoprotective as well as adverse effects depending on the type and duration of injury. Glutamine-fructose amidotransferase (GFAT; gene name gfpt) is the rate-limiting enzyme that controls flux through HBP. Two protein isoforms exist in the heart called GFAT1 and GFAT2. There are conflicting data on the relative importance of GFAT1 and GFAT2 during stress-induced HBP responses in the heart.Using neonatal rat cardiac cell preparations, targeted knockdown of GFPT1 and GFPT2 were performed and HBP activity measured. Immunostaining with specific GFAT1 and GFAT2 antibodies was undertaken in neonatal rat cardiac preparations and murine cardiac tissues to characterise cell-specific expression. Publicly available human heart single cell sequencing data was interrogated to determine cell-type expression. Western blots for GFAT isoform protein expression were performed in human cardiomyocytes derived from induced pluripotent stem cells (iPSCs).GFPT1 but not GFPT2 knockdown resulted in a loss of stress-induced protein O-GlcNAcylation in neonatal cardiac cell preparations indicating reduced HBP activity. In rodent cells and tissue, immunostaining for GFAT1 identified expression in both cardiac myocytes and fibroblasts whereas immunostaining for GFAT2 was only identified in fibroblasts. Further corroboration of findings in human heart cells identified an enrichment of GFPT2 gene expression in cardiac fibroblasts but not ventricular myocytes whereas GFPT1 was expressed in both myocytes and fibroblasts. In human iPSC-derived cardiomyocytes, only GFAT1 protein was expressed with an absence of GFAT2.In conclusion, these results indicate that GFAT1 is the primary cardiomyocyte isoform and GFAT2 is only present in cardiac fibroblasts. Cell-specific isoform expression may have differing effects on cell function and should be considered when studying HBP and GFAT functions in the heart.

Highlights

The hexosamine biosynthesis pathway (HBP) converts fructose 6-phosphate from glycolysis into the end-product UDP-N-acetylglucosamine (UDP-GlcNAc) which is required for a range of important macromolecule glycosylation such as the O-GlcNAcylation of many proteins
Using western blotting with specific monoclonal antibodies against GFAT1 and GFAT2, we demonstrate that we could knockdown Gfpt[1] and Gfpt[2] using siRNA (Fig. 1 A and B)
We observed both expression of GFAT1 and GFAT2 at basal conditions with both isoforms showing an increase in expression with PE-stimulation

Summary

Introduction

The hexosamine biosynthesis pathway (HBP) converts fructose 6-phosphate from glycolysis into the end-product UDP-N-acetylglucosamine (UDP-GlcNAc) which is required for a range of important macromolecule glycosylation such as the O-GlcNAcylation of many proteins. The key enzyme responsible for making fructose 6-phosphate into glucosamine 6-phosphate and committing it to the HBP is glutamine-fructose amidotransferase (GFAT; gene name gfpt) It is the first and rate-limiting step of this pathway, transferring the amide group from glutamine to fructose and forming glucosamine 6-phosphate. Genetic mutations affecting GFAT1 and leading to loss of function are primarily characterised by a muscular disorder called congenital myasthenic syndrome [10e12] Another interesting difference between the isoforms is that GFAT1 is present as a splice variant in striated muscles termed long and short variants [13e15]. Gfpt-specific SNPs have been associated with risk of diabetes and their complications but the results are inconsistent [17e20] Increased activity of both isoforms has been noted in driving carcinogenesis in a variety of tissues [21e25]

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Conclusion