Abstract
Our previous studies on a β1,6-N-acetylglucosaminyltransferase, GnT-IX (GnT-Vb), a homolog of GnT-V, indicated that the enzyme has a broad GlcNAc transfer activity toward N-linked and O-mannosyl glycan core structures and that its brain-specific gene expression is regulated by epigenetic histone modifications. In this study, we demonstrate the existence of an endogenous inhibitory factor for GnT-IX that functions as a key regulator for GnT-IX enzymatic activity in Neuro2a (N2a) cells. We purified this factor from N2a cells and found that it is identical to ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3), as evidenced by mass spectrometry and by the knockdown and overexpression of ENPP3 in cultured cells. Kinetic analyses revealed that the mechanism responsible for the inhibition of GnT-IX caused by ENPP3 is the ENPP3-mediated hydrolysis of the nucleotide sugar donor substrate, UDP-GlcNAc, with the resulting generation of UMP, a potent and competitive inhibitor of GnT-IX. Indeed, ENPP3 knockdown cells had significantly increased levels of intracellular nucleotide sugars and displayed changes in the total cellular glycosylation profile. In addition to chaperones or other known regulators of glycosyltransferases, the ENPP3-mediated hydrolysis of nucleotide sugars would have widespread and significant impacts on glycosyltransferase activities and would be responsible for altering the total cellular glycosylation profile and modulating cellular functions.
Highlights
The regulatory mechanisms of glycosyltransferase activity are still poorly understood
We report on a novel regulatory mechanism for glycosyltransferase activity, based on purifying, characterizing, and identifying a cellular proteinaceous inhibitor for GnT-IX as ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3), which catalyzes the hydrolysis of the nucleotide sugar donor substrate UDP-GlcNAc, which results in a reduction in its cellular level and simultaneously generates UMP, which exerts a strong inhibitory activity
We reported an important link of ENPP3 to the regulatory system for dictating the level of intracellular nucleotide sugars (Fig. 6) that would be possible to modulate the broad spectrum glycosyltransferase activities and to alter the total cellular glycosylation profile (Figs. 7 and 8 and Table 1)
Summary
The regulatory mechanisms of glycosyltransferase activity are still poorly understood. A series of glycosyltransferases [6, 7] that reside in the endoplasmic reticulum, Golgi apparatus, nucleus, and cytoplasm are principally involved in the construction of glycans and play a role in generating the structurally diverse glycans that are found in nature Such structural diversity is regulated by various factors encompassing the cell type-specific expression of glycosyltransferases, the availability of acceptor and nucleotide sugar donor substrates, and competition between glycosyltransferases for common acceptor substrates [2, 3, 8, 9]. We report on a novel regulatory mechanism for glycosyltransferase activity, based on purifying, characterizing, and identifying a cellular proteinaceous inhibitor for GnT-IX as ENPP3 (ectonucleotide pyrophosphatase/phosphodiesterase 3), which catalyzes the hydrolysis of the nucleotide sugar donor substrate UDP-GlcNAc, which results in a reduction in its cellular level and simultaneously generates UMP, which exerts a strong inhibitory activity
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