Identification of Ectonucleotide Pyrophosphatase/ Phosphodiesterase 3 (ENPP3) as a Regulator of N-Acetylglucosaminyltransferase GnT-IX (GnT-Vb).

Our previous studies on a β1,6-JV-acetylglucosaminyltrans-ferase, GnT-IX (GnT-Vb), a homolog of GnT-V, indicated that the enzyme has a broad GlcNAc transfer activity toward -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/phosphodies-terase 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 gly-cosylation 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. [ABSTRACT FROM AUTHOR]