Portal de Periódicos da CAPES

Cloning, Genomic Organization, and Characterization of a Human Cholinephosphotransferase

2000; Elsevier BV; Volume: 275; Issue: 38 Linguagem: Inglês

10.1074/jbc.m005786200

1083-351X

Annette L. Henneberry, Graeme Wistow, Christopher R. McMaster,

Mitochondrial Function and Pathology

A cholinephosphotransferase activity catalyzes the final step in the de novo synthesis of phosphatidylcholine via the transfer of a phosphocholine moiety from CDP choline to diacylglycerol. Ethanolaminephosphotransferase activity catalyzes a similar reaction substituting CDP ethanolamine as the phosphobase donor. We report the identification and cloning of a human cDNA (human cholinephosphotransferase (hCPT1)) that codes for a cholinephosphotransferase-specific enzyme. This was demonstrated usingin vitro enzyme assays and in vivo measurement of the reconstitution of the phosphatidylcholine and phosphatidylethanolamine biosynthetic pathways in yeast cells devoid of their own endogenous cholinephosphotransferase and ethanolaminephosphotransferase activities. This contrasted with our previously cloned human choline/ethanolaminephosphotransferase cDNA that was demonstrated to code for a dual specificity choline/ethanolaminephosphotransferase. The hCPT1 and human choline/ethanolaminephosphotransferase (hCEPT1) predicted amino acid sequences possessed 60% overall identity and had only one variation in the amino acid residues within the CDP-alcohol phosphotransferase catalytic motif. In vitro assessment of hCPT1 and hCEPT1 derived cholinephosphotransferase activities also revealed differences in diradylglycerol specificities including their capacity to synthesize platelet-activating factor and platelet-activating factor precursor. Expression of the hCPT1 mRNA varied greater than 100-fold between tissues and was most abundant in testis followed by colon, small intestine, heart, prostate, and spleen. This was in marked contrast to the hCEPT1 mRNA, which has been found in similar abundance in all tissues tested to date. Both the hCPT1 and hCEPT1 enzymes were able to reconstitute the synthesis of PC in yeast to levels provided by the endogenous yeast cholinephosphotransferase; however, only hCEPT1-derived activity was able to complement the yeastCPT1 gene in its interaction with SEC14 and affect cell growth. A cholinephosphotransferase activity catalyzes the final step in the de novo synthesis of phosphatidylcholine via the transfer of a phosphocholine moiety from CDP choline to diacylglycerol. Ethanolaminephosphotransferase activity catalyzes a similar reaction substituting CDP ethanolamine as the phosphobase donor. We report the identification and cloning of a human cDNA (human cholinephosphotransferase (hCPT1)) that codes for a cholinephosphotransferase-specific enzyme. This was demonstrated usingin vitro enzyme assays and in vivo measurement of the reconstitution of the phosphatidylcholine and phosphatidylethanolamine biosynthetic pathways in yeast cells devoid of their own endogenous cholinephosphotransferase and ethanolaminephosphotransferase activities. This contrasted with our previously cloned human choline/ethanolaminephosphotransferase cDNA that was demonstrated to code for a dual specificity choline/ethanolaminephosphotransferase. The hCPT1 and human choline/ethanolaminephosphotransferase (hCEPT1) predicted amino acid sequences possessed 60% overall identity and had only one variation in the amino acid residues within the CDP-alcohol phosphotransferase catalytic motif. In vitro assessment of hCPT1 and hCEPT1 derived cholinephosphotransferase activities also revealed differences in diradylglycerol specificities including their capacity to synthesize platelet-activating factor and platelet-activating factor precursor. Expression of the hCPT1 mRNA varied greater than 100-fold between tissues and was most abundant in testis followed by colon, small intestine, heart, prostate, and spleen. This was in marked contrast to the hCEPT1 mRNA, which has been found in similar abundance in all tissues tested to date. Both the hCPT1 and hCEPT1 enzymes were able to reconstitute the synthesis of PC in yeast to levels provided by the endogenous yeast cholinephosphotransferase; however, only hCEPT1-derived activity was able to complement the yeastCPT1 gene in its interaction with SEC14 and affect cell growth. phosphatidylcholine phosphatidylethanolamine diacylglycerol platelet-activating factor dithiothreitol human choline/ethanolaminephosphotransferase human cholinephosphotransferase rapid amplification of cDNA ends kilobase polymerase chain reaction cholinephosphotransferase of S. cerevisiae choline/ethanolaminephosphotransferase of S. cerevisiae Phosphatidylcholine (PC)1 and phosphatidylethanolamine (PE) are the two most abundant phospholipids found in eukaryotic cells, generally comprising 50 and 25% of cellular phospholipid mass, respectively (1Raetz C.R.H. Annu. Rev. Genet. 1986; 20: 253-295Crossref PubMed Google Scholar). PC synthesis via the CDP choline pathway is responsible for essentially all de novo PC biosynthetic activity in all eukaryotic cell types thus far examined except (i) the liver where the methylation of phosphatidylethanolamine is predicted to contribute to 30% of net synthesis (2Ridgway N.D. Yao Z. Vance D.E. J. Biol. Chem. 1989; 264: 1203-1207Abstract Full Text PDF PubMed Google Scholar, 3DeLong C. Shen Y.-J. Thomas M.J. Cui Z. J. Biol. Chem. 1999; 274: 29683-29688Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar) and (ii) yeast where the supply of exogenous choline dictates the relative contribution of the two PC biosynthetic pathways (4McMaster C.R. Bell R.M. J. Biol. Chem. 1994; 269: 14776-14783Abstract Full Text PDF PubMed Google Scholar, 5McGee T.P. Skinner H.B. Bankaitis V.A. J. Bacteriol. 1994; 176: 6861-6868Crossref PubMed Google Scholar). PE can also be synthesized by alternate routes with the CDP ethanolamine pathway supplemented by the decarboxylation of phosphatidylserine to PE, with the relative contribution of each route dependent on cell type (6Stone S.J. Cui Z. Vance J.E. J. Biol. Chem. 1998; 273: 7293-7302Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 7Kuge O. Hasegawa K. Saito K. Nishijima M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4199-4203Crossref PubMed Scopus (52) Google Scholar, 8McMaster C.R. Choy P.C. J. Biol. Chem. 1992; 267: 14586-14591Abstract Full Text PDF PubMed Google Scholar, 9Trotter P.J. Pedretti J. Yates R. Voelker D.R. J. Biol. Chem. 1995; 270: 6071-6080Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar).The final step in the CDP alcohol pathways for the synthesis of PC and PE is catalyzed by cholinephosphotransferase and ethanolaminephosphotransferase enzyme activities, respectively. Cholinephosphotransferase catalyzes the transfer of phosphocholine from CDP choline to diacylglycerol (DAG) with the release of CMP and the formation of PC (10McMaster C.R. Bell R.M. Biochim. Biophys. Acta. 1997; 1348: 100-110Crossref PubMed Scopus (66) Google Scholar, 11McMaster C.R. Bell R.M. J. Biol. Chem. 1994; 269: 28010-28016Abstract Full Text PDF PubMed Google Scholar, 12Kennedy E.P. Weiss S.B. J. Biol. Chem. 1956; 222: 193-214Abstract Full Text PDF PubMed Google Scholar, 13Weiss S.B. Smith S.W. Kennedy E.P. J. Biol. Chem. 1958; 231: 53-64Abstract Full Text PDF PubMed Google Scholar), whereas the ethanolaminephosphotransferase reaction catalyzes a similar transfer substituting CDP ethanolamine as the phosphobase donor resulting in the formation of PE (14McMaster C.R. Bell R.M. Biochim. Biophys. Acta. 1997; 1348: 117-123Crossref PubMed Scopus (31) Google Scholar, 15Mancini A. Del Rosso F. Roberti R. Orvietani P. Coletti L. Binaglia L. Biochim. Biophys. Acta. 1999; 1437: 80-92Crossref PubMed Scopus (17) Google Scholar, 16Ford D.A. Rosenbloom K.B. Gross R.W. J. Biol. Chem. 1992; 267: 11222-11228Abstract Full Text PDF PubMed Google Scholar). A cholinephosphotransferase has also been implicated in de novo synthesis of platelet-activating factor (PAF) (17Snyder F. Biochim. Biophys. Acta. 1997; 1348: 111-116Crossref PubMed Scopus (33) Google Scholar, 18Woodard D.S. Lee T.-C. Snyder F. J. Biol. Chem. 1987; 262: 2520-2527Abstract Full Text PDF PubMed Google Scholar). This putative PAF-specific cholinephosphotransferase was identified based on DTT susceptibility of cholinephosphotransferase activities measured in microsomal membranes, with PAF-specific activity being DTT resistant and PC-specific activity being DTT sensitive (18Woodard D.S. Lee T.-C. Snyder F. J. Biol. Chem. 1987; 262: 2520-2527Abstract Full Text PDF PubMed Google Scholar).The relative contribution of the CDP choline versus PE methylation pathways for the synthesis of PC have been implicated in regulating cell growth in the mammalian liver. PC synthesis through the CDP choline pathway favored proliferation, whereas PC synthesized by PE methylation inhibited cell growth and was negatively associated with the induction of liver tumor formation (19Cui Z. Houweling M. Vance D.E. J. Biol. Chem. 1994; 269: 24531-24533Abstract Full Text PDF PubMed Google Scholar, 20Walkey C.J., Yu, L. Agellon L.B. Vance D.E. J. Biol. Chem. 1998; 273: 27043-27046Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 21Tessitore L. Sesca E. Vance D.E. Int. J. Cancer. 2000; 86: 362-367Crossref PubMed Google Scholar). PC molecules synthesized by these two pathways have different fatty acyl chain compositions and this has been postulated to be the major contributor to the observed effects on cell growth (3DeLong C. Shen Y.-J. Thomas M.J. Cui Z. J. Biol. Chem. 1999; 274: 29683-29688Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). The diacylglycerol specificity of cholinephosphotransferase regulates the fatty acyl composition of de novo PC synthesized through the CDP choline pathway and thus would be predicted to participate in the regulation of PC pathway-dependent liver tumor development. We recently cloned and characterized a human dual specificity choline/ethanolaminephosphotransferase (hCEPT1) capable of using both CDP choline and CDP ethanolamine as substrates in vitro(22Henneberry A.L. McMaster C.R. Biochem. J. 1999; 339: 291-298Crossref PubMed Scopus (90) Google Scholar). Metabolic labeling experiments demonstrated this enzyme was able to reconstitute the synthesis of both PC and PE when expressed in a yeast strain in which the endogenous cholinephosphotransferase and ethanolaminephosphotransferase genes were inactivated. In the work presented here we de novo cloned two cDNAs, each coding for a splice variant of a human cholinephosphotransferase (hCPT1). The cDNA product was expressed for characterization in vitroand in vivo and was found to code for a cholinephosphotransferase-specific enzyme.DISCUSSIONThe final steps in the Kennedy pathways for the synthesis of PC and PE are catalyzed by cholinephosphotransferase and ethanolaminephosphotransferase activities, respectively (10McMaster C.R. Bell R.M. Biochim. Biophys. Acta. 1997; 1348: 100-110Crossref PubMed Scopus (66) Google Scholar, 11McMaster C.R. Bell R.M. J. Biol. Chem. 1994; 269: 28010-28016Abstract Full Text PDF PubMed Google Scholar, 12Kennedy E.P. Weiss S.B. J. Biol. Chem. 1956; 222: 193-214Abstract Full Text PDF PubMed Google Scholar, 13Weiss S.B. Smith S.W. Kennedy E.P. J. Biol. Chem. 1958; 231: 53-64Abstract Full Text PDF PubMed Google Scholar, 14McMaster C.R. Bell R.M. Biochim. Biophys. Acta. 1997; 1348: 117-123Crossref PubMed Scopus (31) Google Scholar, 15Mancini A. Del Rosso F. Roberti R. Orvietani P. Coletti L. Binaglia L. Biochim. Biophys. Acta. 1999; 1437: 80-92Crossref PubMed Scopus (17) Google Scholar, 16Ford D.A. Rosenbloom K.B. Gross R.W. J. Biol. Chem. 1992; 267: 11222-11228Abstract Full Text PDF PubMed Google Scholar). The hCPT1 cDNA described in this study codes for an enzyme that demonstrated cholinephosphotransferase activity in in vitroenzyme assays, and in yeast devoid of their endogenous cholinephosphotransferase activity, hCPT1 was able to reconstitute the PC biosynthetic pathway in vivo. The hCPT1 enzyme was unable to use CDP ethanolamine as a substrate in vitro and was unable to reconstitute the PE biosynthetic pathway in vivo. This is in contrast to our previously isolated human hCEPT1 cDNA, which encoded an activity capable of using both CDP choline and CDP ethanolamine as substrates in vitro and in vivo(22Henneberry A.L. McMaster C.R. Biochem. J. 1999; 339: 291-298Crossref PubMed Scopus (90) Google Scholar). It has been presumed that Kennedy pathways for the synthesis of PC and PE are completely separate in mammalian cells (49Kent C. Annu. Rev. Biochem. 1995; 64: 315-343Crossref PubMed Scopus (310) Google Scholar, 50Vance D.E. Vance D.E. Vance J.E. Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier, Amsterdam1996: 153-182Google Scholar); however, the redundancy of cholinephosphotransferase activity in both the hCPT1 and hCEPT1 enzymes and the dual specificity of hCEPT for the synthesis of PC and PE imply that these two pathways may not be metabolically distinct. Indeed, the hCEPT1 mRNA distribution was similar in all tissues tested, whereas the hCPT1 mRNA varied greater than 100-fold between the same tissues, implying tissue-specific expression of hCPT1. Consistent with different functions for hCPT1 and hCEPT1 was the ability of hCEPT1, and not hCPT1, to complement the yeastCPT1 protein in mediatingSEC14-dependent cell growth, even though both hCEPT1 and hCPT1 both reconstituted in vivo PC synthesis to levels similar to those provided by the yeast CPT1 gene product. In plants there is evidence that PC and PE are synthesized by a dual specificity choline/ethanolaminephosphotransferase (51Dewey R.E. Wilson R.F. Novitzky W.P. Goode J.H. Plant Cell. 1994; 6: 1495-1507PubMed Google Scholar, 52Justin A.M. Demandue C. Tremolieres A. Mazliak P. Biochim. Biophys. Acta. 1985; 836: 1-7Crossref Scopus (26) Google Scholar), and it will be interesting to determine if hCEPT1 can perform both functions in mammalian cells. A Chinese hamster ovary cell line had been produced by random mutagenesis with a specific defect in ethanolaminephosphotransferase activity (53Polokoff M.A. Wing D.C. Raetz C.R.H. J. Biol. Chem. 1981; 256: 7687-7690Abstract Full Text PDF PubMed Google Scholar), and it would have been illuminating to express the hCPT1 and hCEPT1 products in this cell line; unfortunately, this cell line is no longer available.Consistent with our hypothesis that hCPT1 and hCEPT1 functions may not be metabolically distinct was an examination of the expressed sequence tag data bases, which failed to uncover other mammalian cDNAs with similarity to hCPT1 or hCEPT1. This implies the two cDNAs isolated to date likely comprise the total complement of human cholinephosphotransferase- and ethanolaminephosphotransferase-encoded activities. This would be similar to the demonstrated gene complement in S. cerevisiae (CPT1 andEPT1) (29Hjelmstad R.H. Morash S.C. McMaster C.R. Bell R.M. J. Biol. Chem. 1994; 269: 20995-21002Abstract Full Text PDF PubMed Google Scholar), and an examination of the completed C. elegans data base also revealed only two genes coding for enzymes with similarity to hCPT1 and hCEPT1 (54Stein L.D. Trends Genet. 1999; 15: 425-427Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). Inconsistent with the hypothesis that hCEPT1 may function in both the CDP choline and CDP ethanolamine pathways in mammalian cells is the report of an ethanolaminephosphotransferase-specific enzyme purified to near homogeneity from bovine liver (15Mancini A. Del Rosso F. Roberti R. Orvietani P. Coletti L. Binaglia L. Biochim. Biophys. Acta. 1999; 1437: 80-92Crossref PubMed Scopus (17) Google Scholar). It will be interesting to determine if the purified ethanolaminephosphotransferase is indeed coded for by the bovine CEPT1 gene, but confirmation awaits peptide sequencing of the purified protein.A PAF-specific cholinephosphotransferase activity had been previously distinguished from the PC-specific cholinephosphotransferase enzyme based on DTT sensitivity (17Snyder F. Biochim. Biophys. Acta. 1997; 1348: 111-116Crossref PubMed Scopus (33) Google Scholar, 18Woodard D.S. Lee T.-C. Snyder F. J. Biol. Chem. 1987; 262: 2520-2527Abstract Full Text PDF PubMed Google Scholar). Microsomal membranes contained a PAF-specific cholinephosphotransferase activity that was moderately stimulated by DTT and a PC-specific cholinephosphotransferase activity that was inhibited by DTT. We found that the cloned hCPT1 and hCEPT1 were both able to synthesize PAF in vitro. The hCPT1 PAF-synthesizing activity was stimulated and dependent on DTT, whereas the hCEPT1 PAF synthesizing activity was unaffected by DTT. Neither hCPT1 nor hCEPT1 PC biosynthetic activity was inhibited by DTT. Hence, neither the hCPT1 nor the hCEPT1-encoded cholinephosphotransferase activities reflected those previously observed in mammalian microsomes for the synthesis of PAF and PC. As yeast do not contain ether linked lipids, the precise in vivo substrate specificity for the synthesis of ether-linked PC molecules by hCPT1 and hCEPT1 awaits the development of mammalian systems for subsequent analyses. However, we predict simple overexpression systems are unlikely to provide insight into hCPT1 and hCEPT1 substrate specificities as the cholinephosphotransferase step is not rate-limiting in the pathway, and thus more complicated knockout or antisense strategies will likely have to evolve; these are currently under development. Phosphatidylcholine (PC)1 and phosphatidylethanolamine (PE) are the two most abundant phospholipids found in eukaryotic cells, generally comprising 50 and 25% of cellular phospholipid mass, respectively (1Raetz C.R.H. Annu. Rev. Genet. 1986; 20: 253-295Crossref PubMed Google Scholar). PC synthesis via the CDP choline pathway is responsible for essentially all de novo PC biosynthetic activity in all eukaryotic cell types thus far examined except (i) the liver where the methylation of phosphatidylethanolamine is predicted to contribute to 30% of net synthesis (2Ridgway N.D. Yao Z. Vance D.E. J. Biol. Chem. 1989; 264: 1203-1207Abstract Full Text PDF PubMed Google Scholar, 3DeLong C. Shen Y.-J. Thomas M.J. Cui Z. J. Biol. Chem. 1999; 274: 29683-29688Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar) and (ii) yeast where the supply of exogenous choline dictates the relative contribution of the two PC biosynthetic pathways (4McMaster C.R. Bell R.M. J. Biol. Chem. 1994; 269: 14776-14783Abstract Full Text PDF PubMed Google Scholar, 5McGee T.P. Skinner H.B. Bankaitis V.A. J. Bacteriol. 1994; 176: 6861-6868Crossref PubMed Google Scholar). PE can also be synthesized by alternate routes with the CDP ethanolamine pathway supplemented by the decarboxylation of phosphatidylserine to PE, with the relative contribution of each route dependent on cell type (6Stone S.J. Cui Z. Vance J.E. J. Biol. Chem. 1998; 273: 7293-7302Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 7Kuge O. Hasegawa K. Saito K. Nishijima M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4199-4203Crossref PubMed Scopus (52) Google Scholar, 8McMaster C.R. Choy P.C. J. Biol. Chem. 1992; 267: 14586-14591Abstract Full Text PDF PubMed Google Scholar, 9Trotter P.J. Pedretti J. Yates R. Voelker D.R. J. Biol. Chem. 1995; 270: 6071-6080Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The final step in the CDP alcohol pathways for the synthesis of PC and PE is catalyzed by cholinephosphotransferase and ethanolaminephosphotransferase enzyme activities, respectively. Cholinephosphotransferase catalyzes the transfer of phosphocholine from CDP choline to diacylglycerol (DAG) with the release of CMP and the formation of PC (10McMaster C.R. Bell R.M. Biochim. Biophys. Acta. 1997; 1348: 100-110Crossref PubMed Scopus (66) Google Scholar, 11McMaster C.R. Bell R.M. J. Biol. Chem. 1994; 269: 28010-28016Abstract Full Text PDF PubMed Google Scholar, 12Kennedy E.P. Weiss S.B. J. Biol. Chem. 1956; 222: 193-214Abstract Full Text PDF PubMed Google Scholar, 13Weiss S.B. Smith S.W. Kennedy E.P. J. Biol. Chem. 1958; 231: 53-64Abstract Full Text PDF PubMed Google Scholar), whereas the ethanolaminephosphotransferase reaction catalyzes a similar transfer substituting CDP ethanolamine as the phosphobase donor resulting in the formation of PE (14McMaster C.R. Bell R.M. Biochim. Biophys. Acta. 1997; 1348: 117-123Crossref PubMed Scopus (31) Google Scholar, 15Mancini A. Del Rosso F. Roberti R. Orvietani P. Coletti L. Binaglia L. Biochim. Biophys. Acta. 1999; 1437: 80-92Crossref PubMed Scopus (17) Google Scholar, 16Ford D.A. Rosenbloom K.B. Gross R.W. J. Biol. Chem. 1992; 267: 11222-11228Abstract Full Text PDF PubMed Google Scholar). A cholinephosphotransferase has also been implicated in de novo synthesis of platelet-activating factor (PAF) (17Snyder F. Biochim. Biophys. Acta. 1997; 1348: 111-116Crossref PubMed Scopus (33) Google Scholar, 18Woodard D.S. Lee T.-C. Snyder F. J. Biol. Chem. 1987; 262: 2520-2527Abstract Full Text PDF PubMed Google Scholar). This putative PAF-specific cholinephosphotransferase was identified based on DTT susceptibility of cholinephosphotransferase activities measured in microsomal membranes, with PAF-specific activity being DTT resistant and PC-specific activity being DTT sensitive (18Woodard D.S. Lee T.-C. Snyder F. J. Biol. Chem. 1987; 262: 2520-2527Abstract Full Text PDF PubMed Google Scholar). The relative contribution of the CDP choline versus PE methylation pathways for the synthesis of PC have been implicated in regulating cell growth in the mammalian liver. PC synthesis through the CDP choline pathway favored proliferation, whereas PC synthesized by PE methylation inhibited cell growth and was negatively associated with the induction of liver tumor formation (19Cui Z. Houweling M. Vance D.E. J. Biol. Chem. 1994; 269: 24531-24533Abstract Full Text PDF PubMed Google Scholar, 20Walkey C.J., Yu, L. Agellon L.B. Vance D.E. J. Biol. Chem. 1998; 273: 27043-27046Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 21Tessitore L. Sesca E. Vance D.E. Int. J. Cancer. 2000; 86: 362-367Crossref PubMed Google Scholar). PC molecules synthesized by these two pathways have different fatty acyl chain compositions and this has been postulated to be the major contributor to the observed effects on cell growth (3DeLong C. Shen Y.-J. Thomas M.J. Cui Z. J. Biol. Chem. 1999; 274: 29683-29688Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). The diacylglycerol specificity of cholinephosphotransferase regulates the fatty acyl composition of de novo PC synthesized through the CDP choline pathway and thus would be predicted to participate in the regulation of PC pathway-dependent liver tumor development. We recently cloned and characterized a human dual specificity choline/ethanolaminephosphotransferase (hCEPT1) capable of using both CDP choline and CDP ethanolamine as substrates in vitro(22Henneberry A.L. McMaster C.R. Biochem. J. 1999; 339: 291-298Crossref PubMed Scopus (90) Google Scholar). Metabolic labeling experiments demonstrated this enzyme was able to reconstitute the synthesis of both PC and PE when expressed in a yeast strain in which the endogenous cholinephosphotransferase and ethanolaminephosphotransferase genes were inactivated. In the work presented here we de novo cloned two cDNAs, each coding for a splice variant of a human cholinephosphotransferase (hCPT1). The cDNA product was expressed for characterization in vitroand in vivo and was found to code for a cholinephosphotransferase-specific enzyme. DISCUSSIONThe final steps in the Kennedy pathways for the synthesis of PC and PE are catalyzed by cholinephosphotransferase and ethanolaminephosphotransferase activities, respectively (10McMaster C.R. Bell R.M. Biochim. Biophys. Acta. 1997; 1348: 100-110Crossref PubMed Scopus (66) Google Scholar, 11McMaster C.R. Bell R.M. J. Biol. Chem. 1994; 269: 28010-28016Abstract Full Text PDF PubMed Google Scholar, 12Kennedy E.P. Weiss S.B. J. Biol. Chem. 1956; 222: 193-214Abstract Full Text PDF PubMed Google Scholar, 13Weiss S.B. Smith S.W. Kennedy E.P. J. Biol. Chem. 1958; 231: 53-64Abstract Full Text PDF PubMed Google Scholar, 14McMaster C.R. Bell R.M. Biochim. Biophys. Acta. 1997; 1348: 117-123Crossref PubMed Scopus (31) Google Scholar, 15Mancini A. Del Rosso F. Roberti R. Orvietani P. Coletti L. Binaglia L. Biochim. Biophys. Acta. 1999; 1437: 80-92Crossref PubMed Scopus (17) Google Scholar, 16Ford D.A. Rosenbloom K.B. Gross R.W. J. Biol. Chem. 1992; 267: 11222-11228Abstract Full Text PDF PubMed Google Scholar). The hCPT1 cDNA described in this study codes for an enzyme that demonstrated cholinephosphotransferase activity in in vitroenzyme assays, and in yeast devoid of their endogenous cholinephosphotransferase activity, hCPT1 was able to reconstitute the PC biosynthetic pathway in vivo. The hCPT1 enzyme was unable to use CDP ethanolamine as a substrate in vitro and was unable to reconstitute the PE biosynthetic pathway in vivo. This is in contrast to our previously isolated human hCEPT1 cDNA, which encoded an activity capable of using both CDP choline and CDP ethanolamine as substrates in vitro and in vivo(22Henneberry A.L. McMaster C.R. Biochem. J. 1999; 339: 291-298Crossref PubMed Scopus (90) Google Scholar). It has been presumed that Kennedy pathways for the synthesis of PC and PE are completely separate in mammalian cells (49Kent C. Annu. Rev. Biochem. 1995; 64: 315-343Crossref PubMed Scopus (310) Google Scholar, 50Vance D.E. Vance D.E. Vance J.E. Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier, Amsterdam1996: 153-182Google Scholar); however, the redundancy of cholinephosphotransferase activity in both the hCPT1 and hCEPT1 enzymes and the dual specificity of hCEPT for the synthesis of PC and PE imply that these two pathways may not be metabolically distinct. Indeed, the hCEPT1 mRNA distribution was similar in all tissues tested, whereas the hCPT1 mRNA varied greater than 100-fold between the same tissues, implying tissue-specific expression of hCPT1. Consistent with different functions for hCPT1 and hCEPT1 was the ability of hCEPT1, and not hCPT1, to complement the yeastCPT1 protein in mediatingSEC14-dependent cell growth, even though both hCEPT1 and hCPT1 both reconstituted in vivo PC synthesis to levels similar to those provided by the yeast CPT1 gene product. In plants there is evidence that PC and PE are synthesized by a dual specificity choline/ethanolaminephosphotransferase (51Dewey R.E. Wilson R.F. Novitzky W.P. Goode J.H. Plant Cell. 1994; 6: 1495-1507PubMed Google Scholar, 52Justin A.M. Demandue C. Tremolieres A. Mazliak P. Biochim. Biophys. Acta. 1985; 836: 1-7Crossref Scopus (26) Google Scholar), and it will be interesting to determine if hCEPT1 can perform both functions in mammalian cells. A Chinese hamster ovary cell line had been produced by random mutagenesis with a specific defect in ethanolaminephosphotransferase activity (53Polokoff M.A. Wing D.C. Raetz C.R.H. J. Biol. Chem. 1981; 256: 7687-7690Abstract Full Text PDF PubMed Google Scholar), and it would have been illuminating to express the hCPT1 and hCEPT1 products in this cell line; unfortunately, this cell line is no longer available.Consistent with our hypothesis that hCPT1 and hCEPT1 functions may not be metabolically distinct was an examination of the expressed sequence tag data bases, which failed to uncover other mammalian cDNAs with similarity to hCPT1 or hCEPT1. This implies the two cDNAs isolated to date likely comprise the total complement of human cholinephosphotransferase- and ethanolaminephosphotransferase-encoded activities. This would be similar to the demonstrated gene complement in S. cerevisiae (CPT1 andEPT1) (29Hjelmstad R.H. Morash S.C. McMaster C.R. Bell R.M. J. Biol. Chem. 1994; 269: 20995-21002Abstract Full Text PDF PubMed Google Scholar), and an examination of the completed C. elegans data base also revealed only two genes coding for enzymes with similarity to hCPT1 and hCEPT1 (54Stein L.D. Trends Genet. 1999; 15: 425-427Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). Inconsistent with the hypothesis that hCEPT1 may function in both the CDP choline and CDP ethanolamine pathways in mammalian cells is the report of an ethanolaminephosphotransferase-specific enzyme purified to near homogeneity from bovine liver (15Mancini A. Del Rosso F. Roberti R. Orvietani P. Coletti L. Binaglia L. Biochim. Biophys. Acta. 1999; 1437: 80-92Crossref PubMed Scopus (17) Google Scholar). It will be interesting to determine if the purified ethanolaminephosphotransferase is indeed coded for by the bovine CEPT1 gene, but confirmation awaits peptide sequencing of the purified protein.A PAF-specific cholinephosphotransferase activity had been previously distinguished from the PC-specific cholinephosphotransferase enzyme based on DTT sensitivity (17Snyder F. Biochim. Biophys. Acta. 1997; 1348: 111-116Crossref PubMed Scopus (33) Google Scholar, 18Woodard D.S. Lee T.-C. Snyder F. J. Biol. Chem. 1987; 262: 2520-2527Abstract Full Text PDF PubMed Google Scholar). Microsomal membranes contained a PAF-specific cholinephosphotransferase activity that was moderately stimulated by DTT and a PC-specific cholinephosphotransferase activity that was inhibited by DTT. We found that the cloned hCPT1 and hCEPT1 were both able to synthesize PAF in vitro. The hCPT1 PAF-synthesizing activity was stimulated and dependent on DTT, whereas the hCEPT1 PAF synthesizing activity was unaffected by DTT. Neither hCPT1 nor hCEPT1 PC biosynthetic activity was inhibited by DTT. Hence, neither the hCPT1 nor the hCEPT1-encoded cholinephosphotransferase activities reflected those previously observed in mammalian microsomes for the synthesis of PAF and PC. As yeast do not contain ether linked lipids, the precise in vivo substrate specificity for the synthesis of ether-linked PC molecules by hCPT1 and hCEPT1 awaits the development of mammalian systems for subsequent analyses. However, we predict simple overexpression systems are unlikely to provide insight into hCPT1 and hCEPT1 substrate specificities as the cholinephosphotransferase step is not rate-limiting in the pathway, and thus more complicated knockout or antisense strategies will likely have to evolve; these are currently under development. The final steps in the Kennedy pathways for the synthesis of PC and PE are catalyzed by cholinephosphotransferase and ethanolaminephosphotransferase activities, respectively (10McMaster C.R. Bell R.M. Biochim. Biophys. Acta. 1997; 1348: 100-110Crossref PubMed Scopus (66) Google Scholar, 11McMaster C.R. Bell R.M. J. Biol. Chem. 1994; 269: 28010-28016Abstract Full Text PDF PubMed Google Scholar, 12Kennedy E.P. Weiss S.B. J. Biol. Chem. 1956; 222: 193-214Abstract Full Text PDF PubMed Google Scholar, 13Weiss S.B. Smith S.W. Kennedy E.P. J. Biol. Chem. 1958; 231: 53-64Abstract Full Text PDF PubMed Google Scholar, 14McMaster C.R. Bell R.M. Biochim. Biophys. Acta. 1997; 1348: 117-123Crossref PubMed Scopus (31) Google Scholar, 15Mancini A. Del Rosso F. Roberti R. Orvietani P. Coletti L. Binaglia L. Biochim. Biophys. Acta. 1999; 1437: 80-92Crossref PubMed Scopus (17) Google Scholar, 16Ford D.A. Rosenbloom K.B. Gross R.W. J. Biol. Chem. 1992; 267: 11222-11228Abstract Full Text PDF PubMed Google Scholar). The hCPT1 cDNA described in this study codes for an enzyme that demonstrated cholinephosphotransferase activity in in vitroenzyme assays, and in yeast devoid of their endogenous cholinephosphotransferase activity, hCPT1 was able to reconstitute the PC biosynthetic pathway in vivo. The hCPT1 enzyme was unable to use CDP ethanolamine as a substrate in vitro and was unable to reconstitute the PE biosynthetic pathway in vivo. This is in contrast to our previously isolated human hCEPT1 cDNA, which encoded an activity capable of using both CDP choline and CDP ethanolamine as substrates in vitro and in vivo(22Henneberry A.L. McMaster C.R. Biochem. J. 1999; 339: 291-298Crossref PubMed Scopus (90) Google Scholar). It has been presumed that Kennedy pathways for the synthesis of PC and PE are completely separate in mammalian cells (49Kent C. Annu. Rev. Biochem. 1995; 64: 315-343Crossref PubMed Scopus (310) Google Scholar, 50Vance D.E. Vance D.E. Vance J.E. Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier, Amsterdam1996: 153-182Google Scholar); however, the redundancy of cholinephosphotransferase activity in both the hCPT1 and hCEPT1 enzymes and the dual specificity of hCEPT for the synthesis of PC and PE imply that these two pathways may not be metabolically distinct. Indeed, the hCEPT1 mRNA distribution was similar in all tissues tested, whereas the hCPT1 mRNA varied greater than 100-fold between the same tissues, implying tissue-specific expression of hCPT1. Consistent with different functions for hCPT1 and hCEPT1 was the ability of hCEPT1, and not hCPT1, to complement the yeastCPT1 protein in mediatingSEC14-dependent cell growth, even though both hCEPT1 and hCPT1 both reconstituted in vivo PC synthesis to levels similar to those provided by the yeast CPT1 gene product. In plants there is evidence that PC and PE are synthesized by a dual specificity choline/ethanolaminephosphotransferase (51Dewey R.E. Wilson R.F. Novitzky W.P. Goode J.H. Plant Cell. 1994; 6: 1495-1507PubMed Google Scholar, 52Justin A.M. Demandue C. Tremolieres A. Mazliak P. Biochim. Biophys. Acta. 1985; 836: 1-7Crossref Scopus (26) Google Scholar), and it will be interesting to determine if hCEPT1 can perform both functions in mammalian cells. A Chinese hamster ovary cell line had been produced by random mutagenesis with a specific defect in ethanolaminephosphotransferase activity (53Polokoff M.A. Wing D.C. Raetz C.R.H. J. Biol. Chem. 1981; 256: 7687-7690Abstract Full Text PDF PubMed Google Scholar), and it would have been illuminating to express the hCPT1 and hCEPT1 products in this cell line; unfortunately, this cell line is no longer available. Consistent with our hypothesis that hCPT1 and hCEPT1 functions may not be metabolically distinct was an examination of the expressed sequence tag data bases, which failed to uncover other mammalian cDNAs with similarity to hCPT1 or hCEPT1. This implies the two cDNAs isolated to date likely comprise the total complement of human cholinephosphotransferase- and ethanolaminephosphotransferase-encoded activities. This would be similar to the demonstrated gene complement in S. cerevisiae (CPT1 andEPT1) (29Hjelmstad R.H. Morash S.C. McMaster C.R. Bell R.M. J. Biol. Chem. 1994; 269: 20995-21002Abstract Full Text PDF PubMed Google Scholar), and an examination of the completed C. elegans data base also revealed only two genes coding for enzymes with similarity to hCPT1 and hCEPT1 (54Stein L.D. Trends Genet. 1999; 15: 425-427Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). Inconsistent with the hypothesis that hCEPT1 may function in both the CDP choline and CDP ethanolamine pathways in mammalian cells is the report of an ethanolaminephosphotransferase-specific enzyme purified to near homogeneity from bovine liver (15Mancini A. Del Rosso F. Roberti R. Orvietani P. Coletti L. Binaglia L. Biochim. Biophys. Acta. 1999; 1437: 80-92Crossref PubMed Scopus (17) Google Scholar). It will be interesting to determine if the purified ethanolaminephosphotransferase is indeed coded for by the bovine CEPT1 gene, but confirmation awaits peptide sequencing of the purified protein. A PAF-specific cholinephosphotransferase activity had been previously distinguished from the PC-specific cholinephosphotransferase enzyme based on DTT sensitivity (17Snyder F. Biochim. Biophys. Acta. 1997; 1348: 111-116Crossref PubMed Scopus (33) Google Scholar, 18Woodard D.S. Lee T.-C. Snyder F. J. Biol. Chem. 1987; 262: 2520-2527Abstract Full Text PDF PubMed Google Scholar). Microsomal membranes contained a PAF-specific cholinephosphotransferase activity that was moderately stimulated by DTT and a PC-specific cholinephosphotransferase activity that was inhibited by DTT. We found that the cloned hCPT1 and hCEPT1 were both able to synthesize PAF in vitro. The hCPT1 PAF-synthesizing activity was stimulated and dependent on DTT, whereas the hCEPT1 PAF synthesizing activity was unaffected by DTT. Neither hCPT1 nor hCEPT1 PC biosynthetic activity was inhibited by DTT. Hence, neither the hCPT1 nor the hCEPT1-encoded cholinephosphotransferase activities reflected those previously observed in mammalian microsomes for the synthesis of PAF and PC. As yeast do not contain ether linked lipids, the precise in vivo substrate specificity for the synthesis of ether-linked PC molecules by hCPT1 and hCEPT1 awaits the development of mammalian systems for subsequent analyses. However, we predict simple overexpression systems are unlikely to provide insight into hCPT1 and hCEPT1 substrate specificities as the cholinephosphotransferase step is not rate-limiting in the pathway, and thus more complicated knockout or antisense strategies will likely have to evolve; these are currently under development. We thank Harold Cook, David Byers, and Neale Ridgway for helpful discussions during the course of this study.