Abstract
N-Acyl ethanolamines (NAEs) are a large class of signaling lipids implicated in diverse physiological processes, including nociception, cognition, anxiety, appetite, and inflammation. It has been proposed that NAEs are biosynthesized from their corresponding N-acyl phosphatidylethanolamines (NAPEs) in a single enzymatic step catalyzed by a phospholipase D (NAPE-PLD). The recent generation of NAPE-PLD(-/-) mice has revealed that these animals possess lower brain levels of saturated NAEs but essentially unchanged concentrations of polyunsaturated NAEs, including the endogenous cannabinoid anandamide. These findings suggest the existence of additional enzymatic routes for the production of NAEs in vivo. Here, we report evidence for an alternative pathway for NAE biosynthesis that proceeds through the serine hydrolase-catalyzed double-deacylation of NAPE to generate glycerophospho-NAE, followed by the phosphodiesterase-mediated cleavage of this intermediate to liberate NAE. Furthermore, we describe the functional proteomic isolation and identification of a heretofore uncharacterized enzyme alpha/beta-hydrolase 4 (Abh4) as a lysophospholipase/phospholipase B that selectively hydrolyzes NAPEs and lysoNAPEs. Abh4 accepts lysoNAPEs bearing both saturated and polyunsaturated N-acyl chains as substrates and displays a distribution that closely mirrors lysoNAPE-lipase activity in mouse tissues. These results support the existence of an NAPE-PLD-independent route for NAE biosynthesis and suggest that Abh4 plays a role in this metabolic pathway by acting as a (lyso)NAPE-selective lipase.
Highlights
1 (CB1),2 inspired the search for a natural ligand and, soon after, the lipid N-arachidonoyl ethanolamine, or anandamide, was identified as an endogenous CB1 agonist [2]
It has been generally assumed that N-acyl ethanolamines (NAEs) are produced from N-acyl phosphatidylethanolamines (NAPEs) in a single enzymatic step catalyzed by a type D phospholipase [18]
A Role for Abh4 in Endocannabinoid Biosynthesis respectively. These results suggest the existence of multiple biosynthetic pathways for NAEs in vivo, with NAPE-PLD being principally responsible for generating very long chain saturated NAEs and other, as of yet, unidentified enzymes contributing to the production of long chain saturated and polyunsaturated NAEs
Summary
Materials—[1Ј-14C]Palmitic acid was purchased from Moravek Biochemicals (Brea, CA). 1,2-Dioleoyl-sn-glycero3-phosphoethanolamine, 1-oleoyl-2-hydroxy-sn-glycero-3phosphoethanolamine, 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine, 1-oleoyl-2-hydroxy-sn-glycero-3-phosphoserine, and 1,2dioleyl-sn-glycero-3-phosphoethanolamine (di-ether phosphatidylethanolamine) were purchased from Avanti Polar Lipids (Alabaster, AL). To investigate whether serine hydrolases are involved in NAE biosynthesis, we measured the conversion of NAPE to NAE in NAPE-PLD(ϩ/ϩ) and (Ϫ/Ϫ) brains in the presence of methoxy arachidonyl FP (MAFP), a general inhibitor of this enzyme class (Fig. 2A) [28, 29]. Phospholipase-mediated conversion of NAPE to NAE could occur by removing either one or both O-acyl chains to produce lysoNAPE (Fig. 1, reaction II) or GP-NAE (Fig. 1, reaction III), respectively To distinguish between these two routes, we synthesized radiolabeled C16:0-lysoNAPE and measured its conversion to C16:0-NAE in the presence or absence of MAFP (Fig. 2A). Treatment with MAFP (5 M in Me2SO) blocked greater than 80% of the conversion of lysoNAPE to NAE in both NAPEPLD(ϩ/ϩ) and (Ϫ/Ϫ) brains, suggesting that both O-acyl chains of NAPEs must be hydrolyzed for NAPE-PLD-independent NAE biosynthesis to occur. Considering further that the extent of hydrolysis of lysoNAPE was equivalent between NAPE-PLD(ϩ/ϩ) and (Ϫ/Ϫ) brains, these results support previous studies indicating that NAPE-PLD does not possess sig-
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