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 in NPP6 was in the and the and activity. NPP6 the but the nucleotide NPP6 to and but lysophosphatidic acid, a lysophospholipase activity. NPP6 showed a with and or and and NPP6 was in with a in and and in was in and The that NPP6 a the of or in these 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 in NPP6 was in the and the and activity. NPP6 the but the nucleotide NPP6 to and but lysophosphatidic acid, a lysophospholipase activity. NPP6 showed a with and or and and NPP6 was in with a in and and in was in and The that NPP6 a the of or in these Nucleotide pyrophosphatases/phosphodiesterases (NPPs) nucleotide glycerophosphodiester lysophospholipase lysophospholipase lysophosphatidic alkaline nucleotide glycerophosphodiester lysophospholipase lysophospholipase lysophosphatidic alkaline are ubiquitous membrane-associated or secreted ectoenzymes that have a in nucleotide act a variety of nucleotides and nucleotide and The mammalian NPP family members that are transmembrane that have of a a transmembrane a a and a and have a a transmembrane a and a The of and only a members of the NPP family, NPP6 and alkaline sphingomyelinase in the These have of the of NPP family of nucleotide and sequence of The and the nucleotide and the Nucleotide and are shown on The is shown in The are The is The the sequence of and members are shown in an are the of members of the was from The sequence of is to the of of the D, domain and of seven NPP are and are to have and nucleotide and of of of and in of and of of been in and and The of are the of the NPP family been the of lysophospholipase which hydrolyzes lysophosphatidylcholine (LPC) to produce lysophosphatidic The that a which the was recently that which is in is a member of NPP family the in These findings raised the possibility that other members of the NPP family act on phospholipids. we a novel member of the NPP family, NPP6, and show that is a choline-specific glycerophosphodiester acid, and from and from was from 5′-monophosphate and from was the of was from from of NPP6 in and NPP6 the the sequence of or the NPP6 was and and a the domain of was and and a the NPP6 was and and a the domain of with the was and and a in the the The and of was in the and to the The nucleotide sequence of the was an in with and an of with the to the the the the was the to The and the was an of of with or was a and with a of the to to the of was in a the with the protein The from the with The with and we and we and of NPP of NPP6 of NPP6 was NPP and The NPP6 protein was an with NPP6 in containing and and the of was the in a and was was was with NPP6 protein in containing alkaline and The was The of NPP6 and and the the of was the that to and and and the of or in the was a in which the of was The of was of of was was lysophospholipase and and was to and to the protein The with containing and with and with to the with an with on with in with with in the with and or the with or or The and the a was from to the of was a The was with a in The was in and in containing and the was an from was and the The of the in the an sequence The of was and was on the of was and in was the and the to the was with containing or with NPP6 or or protein the on with a of or the with the with and with of NPP6 and in the of the NPP family member of the NPP family, NPP6, was is on and is on and and the nucleotide with a with the nucleotide and with a nucleotide with a of and a sequence of the in the which is a of with other members of the NPP family NPP6 only a in domain and that NPP6 to the of the NPP family with NPP4, NPP5, and and The sequence of with with with with NPP4, with NPP5, and with The in showed that NPP6, NPP4, NPP5, and a the NPP in and in have been shown to the of these and are the NPP is that the is in and and and of NPP6 the and of the we to NPP6 in mammalian was in we a protein with an of on of the protein was in but a was in the The of that is in and is the of in we with the the of with of a that of the protein is to the of is the of and that is a transmembrane of we a of in which the sequence was shown in of the protein in was from that the sequence is of of NPP6 was in NPP6 is a and a is secreted from a is the with and of NPP6 protein in with domain of or and the of protein was with or with in and with of and in of with with with and with and or and are a the of NPP6, we we with the of or and the the we and is a The of these was the of the and to and the of NPP6 a and of with NPP6 The was in the from with NPP6 and but from with that NPP6 is and that NPP6 is secreted from other members of NPP family of NPP6 of to the of the that are The are and of NPP6 The protein was to the of NPP6 the shown in an of was in the and showed the in with domain of or domain of and the of the The are from three NPP6 we to an NPP6 a of lysophospholipase and we and shown in significant but was was a The was we alkaline in the we which is to the that was These show that NPP6 but alkaline in the and to and a The of NPP6 is of The the that are and The the of and in the of was with in the or of alkaline and the of and was The are from three a of NPP6 was we the of of and was to which hydrolyzes and to NPP6 only and to a NPP6 that NPP6 a choline-specific is a choline-specific phosphodiesterase. The of NPP6 and is of on the of The are and or of NPP6 phospholipids. The are and of the of on the of The of NPP6 with to the of was with was to the NPP6 and the The are and and The are from three we phospholipids. NPP6 act on or but and and These are to of the of the of on the was and with and and that have shown in NPP6 showed a or and to and and the was to the and was to the was that NPP6 that the of is the of The and the are shown in These show that NPP6 and and we that NPP6 is a and of NPP6 in a NPP6 the of other members of the NPP family, the was the and and that a is activity. the of NPP6 was the to the of NPP6 of the and the NPP6 of to which to an of the the activity. The of NPP6 and in the or of and The are from three and is that NPP6 the possibility we the in the of with or shown in in from which in The that NPP6 hydrolyzes in the and NPP6 the The of in the of with NPP6 or was The and and of NPP6 the of and NPP6 a other members of the NPP family, which showed ubiquitous NPP6 was in in was was in and These with in the to in and to to The and of in the and the The of in the the of protein The to NPP6 was only in in the and The that NPP6 is in the and of in the that are on the or of of NPP6 in and The NPP6 in and and are a to The are from three of NPP6 in of of NPP6 in the the in the the of in the the of D, the of and of a with to showed that a and that of is the was that in show a to recently is an NPP family member These raised the possibility that some members of the NPP family act on phospholipids. we a novel member to the NPP family, NPP6, and showed that a lysophospholipase NPP6 NPP6 is a a mammalian of a was a protein with of hydrolyzes that is in NPP6 show homology to that the have NPP6 is a of the NPP family with uncharacterized NPP4, NPP5, and recently NPP6 showed a or and and showed that show a a and and and NPP6 to show NPP6 a domain that a that was in the of NPP6 that NPP6 on the the and of is in to and that show a or in and or is the is a we the that but activity. NPP6 and and on of was that the recently to the NPP family is the of the NPP family of NPP4, NPP5, and on and the have is the member of the NPP showed that in the of and a have the hydrolyzes or The that other members of the NPP family have recently showed that and have activity. and show that and and show and The that of NPP6 is the and the of that with and to to other members of NPP of NPP6 in show that NPP6 is in and of NPP6 have in these NPP6 is to the of these and is that in these NPP6 is to NPP6 to have some in of and is the that is in but is in of NPP6 in are with and and is in the of in of from to on mammalian is an NPP6 the in NPP6 from which is or which is an mammalian is in a the of an an to The of and are in the of the in in the of have that the of is in the of are and in the are NPP6 have a in the of in and is that NPP6 is a showed that NPP6 and to and is that and are with and and the of these in are NPP6 have a in which the These are in Nucleotide pyrophosphatases/phosphodiesterases (NPPs) nucleotide glycerophosphodiester lysophospholipase lysophospholipase lysophosphatidic alkaline nucleotide glycerophosphodiester lysophospholipase lysophospholipase lysophosphatidic alkaline are ubiquitous membrane-associated or secreted ectoenzymes that have a in nucleotide act a variety of nucleotides and nucleotide and The mammalian NPP family members that are transmembrane that have of a a transmembrane a a and a and have a a transmembrane a and a The of and only a members of the NPP family, NPP6 and alkaline sphingomyelinase in the These have of the of NPP family to have and nucleotide and of of of and in of and of of been in and and The of are the of the NPP family been the of lysophospholipase which hydrolyzes lysophosphatidylcholine (LPC) to produce lysophosphatidic The that a which the was recently that which is in is a member of NPP family the in These findings raised the possibility that other members of the NPP family act on phospholipids. we a novel member of the NPP family, NPP6, and show that is a choline-specific glycerophosphodiester acid, and from and from was from 5′-monophosphate and from was the of was from from of NPP6 in and NPP6 the the sequence of or the NPP6 was and and a the domain of was and and a the NPP6 was and and a the domain of with the was and and a in the the The and of was in the and to the The nucleotide sequence of the was an in with and an of with the to the the the the was the to The and the was an of of with or was a and with a of the to to the of was in a the with the protein The from the with The with and we and we and of NPP of NPP6 of NPP6 was NPP and The NPP6 protein was an with NPP6 in containing and and the of was the in a and was was was with NPP6 protein in containing alkaline and The was The of NPP6 and and the the of was the that to and and and the of or in the was a in which the of was The of was of of was was lysophospholipase and and was to and to the protein The with containing and with and with to the with an with on with in with with in the with and or the with or or The and the a was from to the of was a The was with a in The was in and in containing and the was an from was and the The of the in the an sequence The of was and was on the of was and in was the and the to the was with containing or with NPP6 or or protein the on with a of or the with the with and with acid, and from and from was from 5′-monophosphate and from was the of was from from of NPP6 in and NPP6 the the sequence of or the NPP6 was and and a the domain of was and and a the NPP6 was and and a the domain of with the was and and a in the the The and of was in the and to the The nucleotide sequence of the was an in with and an of with the to the the the the was the to The and the was an of of with or was a and with a of the to to the of was in a the with the protein The from the with The with and we and we and of NPP of NPP6 of NPP6 was NPP and The NPP6 protein was an with NPP6 in containing and and the of was the in a and was was was with NPP6 protein in containing alkaline and The was The of NPP6 and and the the of was the that to and and and the of or in the was a in which the of was The of was of of was was lysophospholipase and and was to and to the protein The with containing and with and with to the with an with on with in with with in the with and or the with or or The and the a was from to the of was a The was with a in The was in and in containing and the was an from was and the The of the in the an sequence The of was and was on the of was and in was the and the to the was with containing or with NPP6 or or protein the on with a of or the with the with and with of NPP6 and in the of the NPP family member of the NPP family, NPP6, was is on and is on and and the nucleotide with a with the nucleotide and with a nucleotide with a of and a sequence of the in the which is a of with other members of the NPP family NPP6 only a in domain and that NPP6 to the of the NPP family with NPP4, NPP5, and and The sequence of with with with with NPP4, with NPP5, and with The in showed that NPP6, NPP4, NPP5, and a the NPP in and in have been shown to the of these and are the NPP is that the is in and and and of NPP6 the and of the we to NPP6 in mammalian was in we a protein with an of on of the protein was in but a was in the The of that is in and is the of in we with the the of with of a that of the protein is to the of is the of and that is a transmembrane of we a of in which the sequence was shown in of the protein in was from that the sequence is of of NPP6 was in NPP6 is a and a is secreted from a is the with a the of NPP6, we we with the of or and the the we and is a The of these was the of the and to and the of NPP6 a and of with NPP6 The was in the from with NPP6 and but from with that NPP6 is and that NPP6 is secreted from other members of NPP family of NPP6 of to the of the that are The are and of NPP6 The protein was to the of NPP6 the shown in an of was in the and showed the in with domain of or domain of and the of the The are from three NPP6 we to an NPP6 a of lysophospholipase and we and shown in significant but was was a The was we alkaline in the we which is to the that was These show that NPP6 but alkaline in the and to and a The of NPP6 is of The the that are and The the of and in the of was with in the or of alkaline and the of and was The are from three a of NPP6 was we the of of and was to which hydrolyzes and to NPP6 only and to a NPP6 that NPP6 a choline-specific is a choline-specific phosphodiesterase. The of NPP6 and is of on the of The are and or of NPP6 phospholipids. The are and of the of on the of The of NPP6 with to the of was with was to the NPP6 and the The are and and The are from three we phospholipids. NPP6 act on or but and and These are to of the of the of on the was and with and and that have shown in NPP6 showed a or and to and and the was to the and was to the was that NPP6 that the of is the of The and the are shown in These show that NPP6 and and we that NPP6 is a and of NPP6 in a NPP6 the of other members of the NPP family, the was the and and that a is activity. the of NPP6 was the to the of NPP6 of the and the NPP6 of to which to an of the the activity. The of NPP6 and in the or of and The are from three and is that NPP6 the possibility we the in the of with or shown in in from which in The that NPP6 hydrolyzes in the and NPP6 the The of in the of with NPP6 or was The and and of NPP6 the of and NPP6 a other members of the NPP family, which showed ubiquitous NPP6 was in in was was in and These with in the to in and to to The and of in the and the The of in the the of protein The to NPP6 was only in in the and The that NPP6 is in the and of in the that are on the or of of NPP6 in and The NPP6 in and and are a to The are from three of NPP6 in of of NPP6 in the the in the the of in the the of D, the of and of of NPP6 and in the of the NPP family member of the NPP family, NPP6, was is on and is on and and the nucleotide with a with the nucleotide and with a nucleotide with a of and a sequence of the in the which is a of with other members of the NPP family NPP6 only a in domain and that NPP6 to the of the NPP family with NPP4, NPP5, and and The sequence of with with with with NPP4, with NPP5, and with The in showed that NPP6, NPP4, NPP5, and a the NPP in and in have been shown to the of these and are the NPP is that the is in and and and of NPP6 the and of the we to NPP6 in mammalian was in we a protein with an of on of the protein was in but a was in the The of that is in and is the of in we with the the of with of a that of the protein is to the of is the of and that is a transmembrane of we a of in which the sequence was shown in of the protein in was from that the sequence is of of NPP6 was in NPP6 is a and a is secreted from a is the with NPP6 a the of NPP6, we we with the of or and the the we and is a The of these was the of the and to and the of NPP6 a and of with NPP6 The was in the from with NPP6 and but from with that NPP6 is and that NPP6 is secreted from other members of NPP family NPP6 NPP6 we to an NPP6 a of lysophospholipase and we and shown in significant but was was a The was we alkaline in the we which is to the that was These show that NPP6 but alkaline in the and to and NPP6 a of NPP6 was we the of of and was to which hydrolyzes and to NPP6 only and to a NPP6 that NPP6 a choline-specific activity. we phospholipids. NPP6 act on or but and and These are to of the of the of on the was and with and and that have shown in NPP6 showed a or and to and and the was to the and was to the was that NPP6 that the of is the of The and the are shown in These show that NPP6 and and we that NPP6 is a NPP6 the of other members of the NPP family, the was the and and that a is activity. the of NPP6 was the to the of NPP6 of the and the NPP6 of to which to an of the NPP6 and is that NPP6 the possibility we the in the of with or shown in in from which in The that NPP6 hydrolyzes in the and of NPP6 the of and NPP6 a other members of the NPP family, which showed ubiquitous NPP6 was in in was was in and These with in the to in and to to The and of in the and the The of in the the of protein The to NPP6 was only in in the and The that NPP6 is in the and of in the that are on the or of a with to showed that a and that of is the was that in show a to recently is an NPP family member These raised the possibility that some members of the NPP family act on phospholipids. we a novel member to the NPP family, NPP6, and showed that a lysophospholipase NPP6 NPP6 is a a mammalian of a was a protein with of hydrolyzes that is in NPP6 show homology to that the have NPP6 is a of the NPP family with uncharacterized NPP4, NPP5, and recently NPP6 showed a or and and showed that show a a and and and NPP6 to show NPP6 a domain that a that was in the of NPP6 that NPP6 on the the and of is in to and that show a or in and or is the is a we the that but activity. NPP6 and and on of was that the recently to the NPP family is the of the NPP family of NPP4, NPP5, and on and the have is the member of the NPP showed that in the of and a have the hydrolyzes or The that other members of the NPP family have recently showed that and have activity. and show that and and show and The that of NPP6 is the and the of that with and to to other members of NPP of NPP6 in show that NPP6 is in and of NPP6 have in these NPP6 is to the of these and is that in these NPP6 is to NPP6 to have some in of and is the that is in but is in of NPP6 in are with and and is in the of in of from to on mammalian is an NPP6 the in NPP6 from which is or which is an mammalian is in a the of an an to The of and are in the of the in in the of have that the of is in the of are and in the are NPP6 have a in the of in and is that NPP6 is a showed that NPP6 and to and is that and are with and and the of these in are NPP6 have a in which the These are in NPP6, a with to showed that a and that of is the was that in show a to recently is an NPP family member These raised the possibility that some members of the NPP family act on phospholipids. we a novel member to the NPP family, NPP6, and showed that a lysophospholipase NPP6 NPP6 is a a mammalian of a was a protein with of hydrolyzes that is in NPP6 show homology to that the have NPP6 is a of the NPP family with uncharacterized NPP4, NPP5, and recently NPP6 showed a or and and showed that show a a and and and NPP6 to show NPP6 a domain that a that was in the of NPP6 that NPP6 on the the and of is in to and that show a or in and or is the is a we the that but activity. NPP6 and and on of was that the recently to the NPP family is the of the NPP family of NPP4, NPP5, and on and the have is the member of the NPP showed that in the of and a have the hydrolyzes or The that other members of the NPP family have recently showed that and have activity. and show that and and show and The that of NPP6 is the and the of that with and to to other members of NPP of NPP6 in show that NPP6 is in and of NPP6 have in these NPP6 is to the of these and is that in these NPP6 is to NPP6 to have some in of and is the that is in but is in The of NPP6 in are with and and is in the of in of from to on mammalian is an NPP6 the in NPP6 from which is or which is an mammalian is in a the of an an to The of and are in the of the in in the of have that the of is in the of are and in the are NPP6 have a in the of in and is that NPP6 is a showed that NPP6 and to and is that and are with and and the of these in are NPP6 have a in which the These are in