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, 54 (15), 2539-49

ABHD4 Regulates Multiple Classes of N-acyl Phospholipids in the Mammalian Central Nervous System

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ABHD4 Regulates Multiple Classes of N-acyl Phospholipids in the Mammalian Central Nervous System

Hyeon-Cheol Lee et al. Biochemistry.

Abstract

N-Acyl phospholipids are atypical components of cell membranes that bear three acyl chains and serve as potential biosynthetic precursors for lipid mediators such as endocannabinoids. Biochemical studies have implicated ABHD4 as a brain N-acyl phosphatidylethanolamine (NAPE) lipase, but in vivo evidence for this functional assignment is lacking. Here, we describe ABHD4(-/-) mice and their characterization using untargeted lipidomics to discover that ABHD4 regulates multiple classes of brain N-acyl phospholipids. In addition to showing reductions in brain glycerophospho-NAEs (GP-NAEs) and plasmalogen-based lyso-NAPEs (lyso-pNAPEs), ABHD4(-/-) mice exhibited decreases in a distinct set of brain lipids that were structurally characterized as N-acyl lysophosphatidylserines (lyso-NAPSs). Biochemical assays confirmed that NAPS lipids are direct substrates of ABHD4. These findings, taken together, designate ABHD4 as a principal regulator of N-acyl phospholipid metabolism in the mammalian nervous system.

Figures

Figure 1
Figure 1. N-acyl Phospholipids
Structures of representative N-acyl phosphatidylethanolamine (NAPE) and N-acyl phosphatidylserine (NAPS) lipids.
Figure 2
Figure 2. Generation and initial characterization of ABHD4−/− mice
(A) The genomic structure of the Abhd4 genomic locus on chromosome 14 is shown, along with the targeting construct with regions of homology flanking exons 3 and 4 (which contains the catalytic serine; asterisk) and the final recombined locus. Only relevant restriction sites are designated. The positions of probes for Southern hybridization and the gene fragments expected upon Southern blotting with the indicated probes and restriction enzymes are also shown. (B) Southern blot of BamHI-digested ES cell DNA showing a clone with targeted disruption of the Abhd4 gene. (C) Southern blot of NdeI/XhoI-digested tail-DNA from ABHD4+/+ and ABHD4+/− mice demonstrating germ-line transmission. (D) PCR genotyping in which the ABHD4+/+ locus is identified by a 220 bp band and the ABHD4−/− locus is identified by a 399 bp band. (E) RT-PCR analysis confirmed loss of Abhd4 mRNA expression in brain and testis tissues from ABHD4−/− mice. (F) ABPP of soluble and membrane fractions of brain tissue from ABHD4+/+ and ABHD4−/− mice using the serine hydrolase-directed probe FP-rhodamine shows that active ABHD4 protein is not observed in ABHD4−/− mice. (G) Lack of ABHD4 activity in ABHD4−/− brains was confirmed by the MS-based proteomic method ABPP-MudPIT using the probe FP-biotin. The activity of representative serine hydrolases is shown. See Table S1 for a complete list of detected serine hydrolases. (H) Soluble fractions of brain tissue from ABHD4+/+ and ABHD4−/− mice were incubated with NAPE (1,2-dioleoyl-sn-glycero-3-phospho (N-arachidonoyl) ethanolamine) or lyso-NAPE (1-oleoyl-2-hydroxy-sn-glycero-3-phospho (N-palmitoyl) ethanolamine) and hydrolytic activity was quantified by measuring lyso-NAPE and oleic acid release, respectively. Data represent mean values ± SEM (n = 5). **, P < 0.01; ***, P < 0.001. Unpaired, two-tailed t test was used.
Figure 3
Figure 3. GP-NAEs and lyso-pNAPEs are decreased in brain tissue from ABHD4−/− mice
(A–B) Targeted mass-spectrometry-based estimates of GP-NAE (A) and lyso-pNAPE (B) abundance in brain tissue from ABHD4+/+ and ABHD4−/− mice. Data represent mean values ± SEM (n = 8 for A, n = 5 for B). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Unpaired, two-tailed t test was used. (C, D) Soluble fractions of mock- and ABHD4-transfected COS-7 cell extracts (C) or brain tissue from ABHD4+/+ and ABHD4−/− mice (D) were incubated with plasmenyl PE (1-O-1'-(Z)-octadecenyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) or pNAPE (1-O-1'-(Z)-octadecenyl-2-oleoyl-sn-glycero-3-phospho (N-palmitoyl) ethanolamine), and hydrolytic activity was quantified by measuring oleic acid release. Data represent mean values ± SEM (n = 4). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Unpaired, two-tailed t test was used. See also Figure S1.
Figure 4
Figure 4. Lipidomic analysis of ABHD4−/− mice brains
(A) Changes in brain metabolites in ABHD4+/+ versus ABHD4−/− brains were measured by untargeted LC-MS. A metabolite with m/z 380.8 and 762.5 was profoundly decreased in brains from ABHD4−/− mice compared with ABHD4+/+ mice. (B) Structure of N-16:0/sn-1-O-18:0 lyso-NAPS (1-stearoyl-2-oleoyl-sn-glycero-3-phospho (N-palmitoyl) serine). (C, D) The m/z 762.5 metabolite (red trace) in brain tissue from ABHD4+/+ mice exhibited the same LC elution time (C) and MS/MS fragmentation pattern (D) as a synthetic N-16:0 O-18:0 lyso-NAPS standard (black trace). The decrease of the m/z 762.5 metabolite in ABHD4−/− mice is also evident (blue trace). Key daughter ions in the MS/MS analysis include 153.0 (dehydrated glycerophosphate) and 437.3 [loss of N-palmitoyl serine (O-18:0 lysophosphatidic acid)].
Figure 5
Figure 5. Lyso-NAPSs are decreased in brain tissue from ABHD4−/− mice
Targeted MS-based estimates of lyso-NAPS (A) and NAPS (B) abundance in brain tissue from ABHD4+/+ and ABHD4−/− mice. Lyso-NAPS measurements are reported in arbitrary units. Data represent mean values ± SEM (n = 8 for A, n = 5 for B). **, P < 0.01; ***, P < 0.001. Unpaired, two-tailed t test was used.
Figure 6
Figure 6. ABHD4 exhibits NAPS-lipase activity
Soluble fractions of mock- and ABHD4-transfected COS-7 cell extracts (A) or brain tissue from ABHD4+/+ and ABHD4−/− mice (B) were incubated with PS (1-stearoyl-2-oleoyl-sn-glycero-3-phosphoserine) or NAPS (1-stearoyl-2-oleoyl-sn-glycero-3-phospho (N-palmitoyl) serine) and hydrolytic activity was quantified by measuring oleic acid release in the absence or presence of 0.1% of Triton X-100. Also see Figure S3 for measurement of NAPS hydrolysis by quantifying release of lyso-NAPS product. Data represent mean values ± SEM (n = 4 for A, n = 5 for B). *, P < 0.05; **, P < 0.01. Unpaired, two-tailed t test was used.

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