Discovery and characterization of a Ca2+-independent phosphatidylethanolamine N-acyltransferase generating the anandamide precursor and its congeners.
N-Acylphosphatidylethanolamines (NAPEs) are precursors of bioactive N-acylethanolamines, including the endocannabinoid anandamide. In animal tissues, NAPE is formed by transfer of a fatty acyl chain at the sn-1 position of glycerophospholipids to the amino group of phosphatidylethanolamine (PE), and this reaction is believed to be the principal rate-limiting step in N-acylethanolamine synthesis. However, the Ca2+-dependent, membrane-associated N-acyltransferase (NAT) responsible for this reaction has not yet been cloned. In this study, on the basis of the functional similarity of NAT to lecithin-retinol acyltransferase (LRAT), we examined a possible PE N-acylation activity in two rat LRAT homologous proteins. Upon overexpression in COS-7 cells, one protein, named rat LRAT-like protein (RLP)-1, catalyzed transfer of a radioactive acyl group from phosphatidylcholine (PC) to PE, resulting in the formation of radioactive NAPE. However, the RLP-1 activity was detected mainly in the cytosolic rather than membrane fraction and was little stimulated by Ca2+. Moreover, RLP-1 did not show selectivity with respect to the sn-1 and sn-2 positions of PC as an acyl donor and therefore could generate N-arachidonoyl-PE (anandamide precursor) from 2-arachidonoyl-PC and PE. In contrast, under the same assay conditions, partially purified NAT from rat brain was highly Ca2+-dependent, membrane-associated, and specific for the sn-1-acyl group of PC. RLP-1 mRNA was expressed predominantly in testis among various rat tissues, and the testis cytosol exhibited an RLP-1-like activity. These results reveal that RLP-1 can function as a PE N-acyltransferase, catalytically distinguishable from the known Ca2+-dependent NAT.
Jin
Xing-Hua
XH
Department of Biochemistry, Kagawa University School of Medicine, 1750-1 Ikenobe, Miki, Kagawa 761-0793, Japan.
Okamoto
Yasuo
Y
Morishita
Jun
J
Tsuboi
Kazuhito
K
Tonai
Takeharu
T
Ueda
Natsuo
N
D000217:Acyltransferases / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001095:Arachidonic Acids / Q000737:chemistry* / Q000378:metabolism; D019556:COS Cells; D002118:Calcium / Q000502:physiology*; D002522:Chlorocebus aethiops; D063388:Endocannabinoids; D008297:Male; D008969:Molecular Sequence Data; D010713:Phosphatidylcholines / Q000378:metabolism; D053284:Polyunsaturated Alkamides / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017386:Sequence Homology, Amino Acid; D013737:Testis / Q000201:enzymology
D001095:Arachidonic Acids; D063388:Endocannabinoids; D010713:Phosphatidylcholines; D053284:Polyunsaturated Alkamides; D000217:Acyltransferases; C042139:phosphatidylethanolamine N-acyltransferase; C518181:RLP-1 protein, rat; C056942:lecithin-retinol acyltransferase; D002118:Calcium; C078814:anandamide