title: 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.
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Sakagami
authors.forename: Junken
authors.initials: J
authors.lastname: Aoki
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Natori
authors.forename: Kiyotaka
authors.initials: K
authors.lastname: Nishikawa
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Kakehi
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Natori
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Arai
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D002412:Cations; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement; D005227:Fatty Acids / Q000737:chemistry; D005997:Glycerylphosphorylcholine / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D007150:Immunohistochemistry; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008244:Lysophosphatidylcholines / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology; D009399:Nephrons / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000737:chemistry; D010802:Phylogeny; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011755:Pyrophosphatases / Q000737:chemistry* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013108:Sphingomyelin Phosphodiesterase / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000737:chemistry; D013552:Swine; D013997:Time Factors; D014018:Tissue Distribution; D014162:Transfection; D010738:Type C Phospholipases / Q000737:chemistry
chemical_list: C033523:4-nitrophenylphosphorylcholine; D000911:Antibodies, Monoclonal; D002412:Cations; D005227:Fatty Acids; D008244:Lysophosphatidylcholines; D010743:Phospholipids; D012333:RNA, Messenger; D011994:Recombinant Proteins; C005356:sphingosine phosphorylcholine; D010767:Phosphorylcholine; D005997:Glycerylphosphorylcholine; D010727:Phosphoric Diester Hydrolases; D010738:Type C Phospholipases; D013108:Sphingomyelin Phosphodiesterase; C000601493:alkaline sphingomyelinase, mouse; C502079:ENPP6 protein, human; D011755:Pyrophosphatases; C405782:nucleotide pyrophosphatase - phosphodiesterase I; D013110:Sphingosine