Identification of Ectonucleotide Pyrophosphatase/Phosphodiesterase 3 (ENPP3) as a Regulator of N-Acetylglucosaminyltransferase GnT-IX (GnT-Vb)
Identification of Ectonucleotide Pyrophosphatase/Phosphodiesterase 3 (ENPP3) as a Regulator of N-Acetylglucosaminyltransferase GnT-IX (GnT-Vb)
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DOI：
10.1074/jbc.m113.474304
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发表时间：
2013-09-27
影响因子：
4.8
通讯作者：
Taniguchi, Naoyuki
中科院分区：
文献类型：
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作者：
Korekane, Hiroaki;Park, Jong Yi;Taniguchi, Naoyuki
我们先前对β 1，6-N-乙酰葡糖胺转移酶GnT-V的同系物GnT-IX（GnT-Vb）的研究表明，该酶对N-连接和O-甘露糖基聚糖核心结构具有广泛的GlcNAc转移活性，并且其脑特异性基因表达受表观遗传组蛋白修饰的调节。在这项研究中，我们证明了GnT-IX的内源性抑制因子的存在，其作为Neuro 2a（N2 a）细胞中GnT-IX酶活性的关键调节因子发挥作用。我们从N2 a细胞中纯化了该因子，并发现它与外核苷酸焦磷酸酶/磷酸二酯酶3（ENPP 3）相同，这一点通过质谱法以及培养细胞中ENPP 3的敲低和过表达来证明。动力学分析表明，ENPP 3引起的GnT-IX抑制的机制是ENPP 3介导的核苷酸糖供体底物UDP-GlcNAc的水解，从而产生UMP，一种有效的竞争性GnT-IX抑制剂。事实上，ENPP 3敲除细胞具有显著增加的细胞内核苷酸糖水平，并显示总细胞糖基化谱的变化。除了糖基转移酶的分子伴侣或其他已知的调节剂之外，ENPP 3介导的核苷酸糖的水解将对糖基转移酶活性具有广泛和显著的影响，并且将负责改变总细胞糖基化谱和调节细胞功能。
Our previous studies on a beta 1,6-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 hydro-lysis 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.