Biochemical and Molecular Characterization of a Novel Choline-specific Glycerophosphodiester Phosphodiesterase Belonging to the Nucleotide Pyrophosphatase/Phosphodiesterase Family

Abstract

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.Nucleotide pyrophosphatases/phosphodiesterases (NPPs) 1 are ubiquitous membrane-associated or secreted ectoenzymes that have a role in regulating extracellular nucleotide metabolism (1, 2). They act by hydrolyzing a variety of nucleotides and nucleotide derivatives such as ATP and ADP. The mammalian NPP family has had five members (NPP1-5) (1, 2) that fall within two subgroups. NPP1-3 are type II transmembrane glycoproteins (ϳ900 amino acids) that have similar modular structures composed of a short amino-terminal intracellular domain, a single transmembrane domain, two somatomedin B-like motifs, a conserved catalytic site, a nuclease-like sequence, and a putative carboxyl-terminal "EF-hand" motif (Fig. 1D). In contrast, NPP4 (1, 2) and NPP5 (1-3) have a shorter structure (ϳ450 amino acids), a predicted type I transmembrane orientation, a short intracellular carboxyl-terminal domain, and a conserved catalytic site. The extracellular domains of NPP4 and NPP5 contain only a phosphodiesterase motif (Fig. 1D). Recently two additional ...

Autotaxin–lysophosphatidic acid– LPA 3 signaling at the embryo‐epithelial boundary controls decidualization pathways

“…Phosphodiesterase activity was measured colorimetrically as previously described (Sakagami et al , 2005) with some modifications. Each mouse ENPP protein was transiently expressed in HEK293A cells using Lipofectamine 2000 as transfection reagent.…”

Cellular function and molecular structure of ecto-nucleotidases

“…Soluble forms have also been described for NPP1 [365,417], NPP3 [422], NPP6 [388], and NPP7 [423]. E-NPPs are found in human serum [152,424,425].…”

“…Because the WPG loop is present in all other E-NPPs, a different mode for lysophosphatidyl and sphingosyl binding must exist in NPP6 and NPP7. Compared to the lysophospholipase D activity of NPP2, these enzymes exhibit lysophospholipase C activity [388,391]. In addition, NPP6 shows high specificity for choline head groups.…”

“…The scheme is based on data presented in the following papers-NPP1: [351,353,412,453,625]; NPP2: [351,353,377,380,403,406]; NPP3: [351]; NPP6: [388]; NPP7: [389,626]. GPC glycerophosphorylcholine, LPC lysophosphatidylcholine, pNPPC p-nitrophenyl phosphorylcholine, SPC sphingosylphosphorylcholine substrate [354][355][356][357].…”

“…NPP6 is a glycerophosphodiester phosphodiesterase with a marked preference for choline containing phospholipids. It hydrolyzes lysophosphatidic acid to form monoacylglycerol and phosphorylcholine but not lysophosphatidic acid, identifying it as a lysophospholipase C. It also hydrolyzes efficiently glycerophosphorylcholine and sphingosylphosphorylcholine [388]. NPP7 was shown to possess alkaline sphingomyelin phosphodiesterase (sphingomyelinase) activity, generating ceramide from sphingomyelin in the intestinal tract [389,390].…”