Cloning, genomic organization, and characterization of a human cholinephosphotransferase

A cholinephosphotransferase activity catalyzes the final step in the de nov o synthesis of phosphatidylcholine Dia the transfer of a phosphocholine moi ety from CDP choline to diacylglycerol. Ethanolaminephosphotransferase acti vity catalyzes a similar reaction substituting CDP ethanolamine as the phos phobase donor. We report the identification and cloning of a human cDNA (hu man cholinephosphotransferase (hCPT1)) that codes for a cholinephosphotrans ferase-specific enzyme. This was demonstrated using in vitro enzyme assays and in vivo measurement of the reconstitution of the phosphatidylcholine an d phosphatidylethanolamine biosynthetic pathways in yeast cells devoid of t heir own endogenous cholinephosphotransferase and ethanolaminephosphotransf erase activities. This contrasted with our previously cloned human choline/ ethanolaminephosphotransferase cDNA that was demonstrated to code for a dua l specificity choline/ethanolaminephosphotransferase. The hCPT1 and human c holine/ethanolaminephosphotransferase (hCEPT1) predicted amino acid sequenc es possessed 60% overall identity and had only one variation in the amino a cid residues within the CDP-alcohol phosphotransferase catalytic motif, In vitro assessment of hCPT1 and hCEPT1 derived cholinephosphotransferase acti vities also revealed differences in diradylglycerol specificities including their capacity to synthesize platelet-activating factor and platelet-activ ating factor precursor. Expression of the hCPT1 mRNA varied greater than 10 0-fold between tissues and was most abundant in testis followed by colon, s mall 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 ch olinephosphotransferase; however, only hCEPT1-derived activity was able to complement the yeast CPT1 gene in its interaction with SEC14 and affect cel l growth.