title: cDNA cloning and characterization of human and mouse Ca(2+)-independent phosphatidylethanolamine N-acyltransferases.
abstract: The formation of N-acylphosphatidylethanolamine by N-acylation of phosphatidylethanolamine (PE) is the initial step in the biosynthetic pathway of bioactive N-acylethanolamines, including the endocannabinoid anandamide and the anti-inflammatory substance N-palmitoylethanolamine. We recently cloned a rat enzyme capable of catalyzing this reaction, and referred to the enzyme as Ca(2+)-independent N-acyltransferase (iNAT). Here we report cDNA cloning and characterization of human and mouse iNATs. We cloned iNAT-homologous cDNAs from human and mouse testes, and overexpressed them in COS-7 cells. The purified recombinant proteins abstracted an acyl group from both sn-1 and sn-2 positions of phosphatidylcholine, and catalyzed N-acylation of PE as well as phospholipase A(1)/A(2)-like hydrolysis. The iNAT activity was mainly detected in soluble rather than particulate fractions, and was only slightly increased by Ca(2+). These results demonstrated that the human and mouse homologues function as iNAT. As for the organ distribution of iNAT, human testis and pancreas and mouse testis exhibited by far the highest expression level, suggesting its physiological importance in the specific organs. Moreover, mutagenesis studies showed crucial roles of His-154 and Cys-241 of rat iNAT in the catalysis and a possible role of the N-terminal domain in membrane association or protein-protein interaction.
authors.affiliation: Department of Biochemistry, Kagawa University School of Medicine, 1750-1 Ikenobe, Miki, Kagawa 761-0793, Japan.
authors.forename: Xing-Hua
authors.initials: XH
authors.lastname: Jin
authors.forename: Toru
authors.initials: T
authors.lastname: Uyama
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Okamoto
authors.forename: Takeharu
authors.initials: T
authors.lastname: Tonai
authors.forename: Natsuo
authors.initials: N
authors.lastname: Ueda
mesh_terms: D000217:Acyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000494:pharmacology; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D051381:Rats; D016415:Sequence Alignment; D014018:Tissue Distribution
chemical_list: D018076:DNA, Complementary; D000217:Acyltransferases; C042139:phosphatidylethanolamine N-acyltransferase; D002118:Calcium