Sakagami Hideki, Aoki Junken, Natori Yumiko, Nishikawa Kiyotaka, Kakehi Yoshiyuki, Natori Yasuhiro, Arai Hiroyuki
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Japan.
J Biol Chem. 2005 Jun 17;280(24):23084-93. doi: 10.1074/jbc.M413438200. Epub 2005 Mar 23.
Nucleotide pyrophosphatases/phosphodiesterases (NPPs) are ubiquitous membrane-associated or secreted ectoenzymes that release nucleoside 5'-monophosphate from a variety of nucleotides and nucleotide derivatives. The mammalian NPP family comprises seven members, but only three of these (NPP1-3) have been studied in some detail. Previously we showed that lysophospholipase D, which hydrolyzes lysophosphatidylcholine (LPC) to produce lysophosphatidic acid, is identical to NPP2. More recently an uncharacterized novel NPP member (NPP7) was shown to have alkaline sphingomyelinase activity. These findings raised the possibility that other members of the NPP family act on phospholipids. Here we show that the sixth member of the NPP family, NPP6, is a choline-specific glycerophosphodiester phosphodiesterase. The sequence of NPP6 encodes a transmembrane protein containing an NPP domain with significant homology to NPP4, NPP5, and NPP7/alkaline sphingomyelinase. When expressed in HeLa cells, NPP6 was detected in both the cells and the cell culture medium as judged by Western blotting and by enzymatic activity. Recombinant NPP6 efficiently hydrolyzed the classical substrate for phospholipase C, p-nitrophenyl phosphorylcholine, but not the classical nucleotide phosphodiesterase substrate, p-nitrophenyl thymidine 5'-monophosphate. In addition, NPP6 hydrolyzed LPC to form monoacylglycerol and phosphorylcholine but not lysophosphatidic acid, showing it has a lysophospholipase C activity. NPP6 showed a preference for LPC with short (12:0 and 14:0) or polyunsaturated (18:2 and 20:4) fatty acids. It also hydrolyzed glycerophosphorylcholine and sphingosylphosphorylcholine efficiently. In mice, NPP6 mRNA was predominantly detected in kidney with a lesser expression in brain and heart, and in human it was detected in kidney and brain. The present results suggest that NPP6 has a specific role through the hydrolysis of polyunsaturated LPC, glycerophosphorylcholine, or sphingosylphosphorylcholine in these organs.
核苷酸焦磷酸酶/磷酸二酯酶（NPPs）是普遍存在的与膜相关或分泌的胞外酶，可从多种核苷酸和核苷酸衍生物中释放核苷5'-单磷酸。哺乳动物NPP家族由七个成员组成，但其中只有三个成员（NPP1-3）得到了较为详细的研究。此前我们发现，水解溶血磷脂酰胆碱（LPC）生成溶血磷脂酸的溶血磷脂酶D与NPP2相同。最近，一个未被鉴定的新型NPP成员（NPP7）被证明具有碱性鞘磷脂酶活性。这些发现增加了NPP家族其他成员作用于磷脂的可能性。在此我们表明，NPP家族的第六个成员NPP6是一种胆碱特异性甘油磷酸二酯磷酸二酯酶。NPP6的序列编码一种跨膜蛋白，该蛋白含有一个与NPP4、NPP5和NPP7/碱性鞘磷脂酶具有显著同源性的NPP结构域。当在HeLa细胞中表达时，通过蛋白质印迹法和酶活性检测发现，NPP6在细胞和细胞培养基中均有存在。重组NPP6能有效水解磷脂酶C的经典底物对硝基苯基磷酰胆碱，但不能水解经典核苷酸磷酸二酯酶底物对硝基苯基胸苷5'-单磷酸。此外，NPP6水解LPC生成单酰甘油和磷酰胆碱，但不生成溶血磷脂酸，表明它具有溶血磷脂酶C活性。NPP6对含有短链（12:0和14:0）或多不饱和脂肪酸（18:2和20:4）的LPC表现出偏好。它还能有效水解甘油磷酰胆碱和鞘氨醇磷酰胆碱。在小鼠中，NPP6 mRNA主要在肾脏中检测到，在脑和心脏中的表达较少，而在人类中，在肾脏和脑中均检测到该基因。目前的结果表明，NPP6通过在这些器官中水解多不饱和LPC、甘油磷酰胆碱或鞘氨醇磷酰胆碱发挥特定作用。
