Glycine N-acyltransferase-like 3 is responsible for long-chain N-acylglycine formation in N18TG2 cells.

Long-chain fatty acid amides are signaling lipids found in mammals and other organisms; however， details of the metabolic pathways for the N-acylglycines and primary fatty acid amides (PFAMs) have remained elusive. Heavy-labeled precursor and subtraction lipidomic experiments in mouse neuroblastoma N18TG2 cells， a model cell line for the study of fatty acid amide metabolism， establish the biosynthetic pathways for the N-acylglycines and the PFAMs. We provide evidence that the N-acylglycines are formed by a long-chain specific glycine-conjugating enzyme， glycine N-acyltransferase-like 3 (GLYATL3). siRNA knockdown of GLYATL3 in the N18TG2 cells resulted in a decrease in the levels of the N-acylglycines and the PFAMs. This is the first report of an enzyme responsible for long-chain N-acylglycine production in cellula. The production of the PFAMs in N18TG2 cells was reported to occur by the oxidative cleavage of the N-acylglycines， as catalyzed by peptidylglycine α-amidating monooxygenase (PAM). siRNA knockdown of PAM resulted in an accumulation of [ C18]N-oleoylglycine and decreased levels of [ C18]oleamide when the N18TG2 cells were grown in the presence of [ C18]oleic acid. The addition of [1- C]palmitate to the N18TG2 cell growth media led to the production of a family of [1- C]palmitoylated fatty acid amides， consistent with the biosynthetic pathways detailed herein.