Publication Types

• Research Support, Non-U.S. Gov’t

MeSH Terms

• Acylation
• Animals
• COS Cells
• Carbon Radioisotopes
• Cercopithecus aethiops
• Ethanolamine/metabolism
• Ethanolamines/metabolism*
• Gene Expression Regulation
• HEK293 Cells
• Humans
• Isoenzymes/antagonists & inhibitors
• Isoenzymes/genetics
• Isoenzymes/metabolism
• Membrane Transport Proteins/genetics
• Membrane Transport Proteins/metabolism*
• Mice
• Palmitic Acid/metabolism
• Peroxisomes/metabolism
• Phosphatidylethanolamines/biosynthesis*
• Phospholipases A/antagonists & inhibitors
• Phospholipases A/genetics
• Phospholipases A/metabolism*
• RNA, Small Interfering/genetics
• RNA, Small Interfering/metabolism
• Rats
• Signal Transduction

Substances

• Carbon Radioisotopes
• Ethanolamines
• Isoenzymes
• Membrane Transport Proteins
• N-acylethanolamines
• Phosphatidylethanolamines
• RNA, Small Interfering
• SLC33A1 protein, human
• Palmitic Acid
• Ethanolamine
• Phospholipases A

Full Text Sources

• Elsevier Science
• 

Highlights

•

Bioactive N-acylethanolamines are formed from N-acylphosphatidylethanolamine (NAPE).

•

N-acyltransferase responsible for NAPE formation is poorly characterized.

•

We previously showed that the protein PLA/AT-1 has this N-acyltransferase activity.

•

The expression or knockdown of PLA/AT-1 in cells increased or decreased NAPE levels.

•

Our present results suggest that PLA/AT-1 is involved in NAPE generation in vivo.

Figures and tables from this article:

Fig. 1.

Major biosynthetic and degradative pathways of NAPE and pNAPE. *PC is shown as a representative acyl donor phospholipid. Abh4, α/β-hydrolase 4; Ca-NAT, Ca^2 +-dependent N-acyltransferase; GDE1, glycerophosphodiesterase 1.

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Fig. 2.

The phylogenetic tree of PLA/AT family members. The tree was constructed using the program GENETYX-MAC (version 15). h, human; m, mouse; r, rat.

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Fig. 3.

Generation of NAPE and NAE in COS-7 cells transiently expressing PLA/AT-1. COS-7 cells were transiently transfected with the insert-free vector (lanes (–)) or the expression vector harboring PLA/AT-1 from human (h), mouse (m) or rat (r). A, cell homogenates (10 μg protein) were analyzed by Western blotting with anti-FLAG antibody. B, cell homogenates (30 μg protein) were incubated with 40 μM 1,2-[1-¹⁴C]dipalmitoyl-PC and 75 μM 1,2-dioleoyl-PE, and the resultant products were separated by TLC. C, in the experiment of B, N-palmitoyl-PE-forming activity (N-acyltransferase activity, black column) and palmitic acid-releasing activity (PLA_1/2 activity, gray column) were quantified (mean values ± S.D. (error bars), n = 3). Intact cells were incubated with [¹⁴C]ethanolamine (D–F) or [¹⁴C]palmitic acid (G and H), and their total lipids were extracted and separated by TLC (D and G). Relative radioactivities of NAPE (E and H) and NAE (F) are shown (mean values ± S.D. (error bars), n = 3). Asterisks indicate significant differences from control cells (p < 0.01). In B, D and G, the positions of authentic compounds on the TLC plate are indicated. NPPE, N-palmitoyl-PE; C16:0, palmitic acid.

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Fig. 4.

Requirement of the catalytic activity of PLA/AT-1 for intracellular NAPE generation. COS-7 cells were transiently transfected with the insert-free vector (Mock) or expression vector harboring human wild-type PLA/AT-1 (WT) or its mutant C119S. Homogenates (30 μg protein) of cells were subjected to Western blotting with the anti-FLAG antibody (A) and N-acyltransferase assay (B) as described under Materials and methods. C, intact cells were incubated with [¹⁴C]ethanolamine and relative radioactivity of NAPE was examined. In B and C, mean values ± S.D. (error bars) are shown (n = 3). Asterisks indicate significant differences from control cells (p < 0.001).

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Fig. 5.

Generation of NAPE in HEK293 cells stably expressing PLA/AT-1. HEK-PLA/AT-1 cells were transfected with a control siRNA (siControl) or two different PLA/AT-1 siRNAs (siPLA/AT-1-A and -B). A, total RNAs were isolated from the cells and analyzed by RT-PCR using specific primers for the mRNAs of PLA/AT-1 and GAPDH (a control). B, intact cells were radiolabeled with [¹⁴C]ethanolamine, and relative radioactivity of NAPE was examined (mean values ± S.D. (error bars), n = 3). * and ^# indicate significant differences from siControl cells (p < 0.02 and p < 0.004, respectively).

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Fig. 6.

LC–MS/MS analysis of N-acylated ethanolamine phospholipids and their metabolites in HEK293 cells stably expressing human PLA/AT-1 (HEK-PLA/AT-1 cells). Various species of NAPEs (A), pNAPEs (B), NAEs (C) and GP-NAEs (D) in HEK-PLA/AT-1 cells (black) and control cells (white) were analyzed by LC–MS/MS as described in Materials and methods. Results are shown as pmol/μmol of total phospholipids (mean values ± S.D., n = 2). R₁CO + R_NCO and R₁(CH)₂ + R_NCO represent the total number of carbon atoms and double bonds in sn − 1 O-acyl (or sn − 1 O-alkenyl) and N-acyl chains.

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Fig. 7.

LC–MS/MS analysis of plasmenylethanolamines and PEs and Western blot analysis of peroxisomal proteins in HEK-PLA/AT-1 cells. Various species of plasmenylethanolamines (A) and diacyl-type PEs (B) in HEK-PLA/AT-1 cells (black) and control cells (white) were analyzed by LC–MS/MS as described in Materials and methods. Results are shown as nmol/μmol of total phospholipids (mean values ± S.D., n = 2). C, postnuclear supernatant (PNS), particulate (P) and supernatant (S) fractions of control cells (Mock), HEK-PLA/AT-1 cells (indicated as PLA/AT-1) and HEK293 cells overexpressing PLA/AT-2 or PLA/AT-3 were analyzed by Western blotting with antibodies against PMP70 and catalase. D, total RNAs isolated from the indicated cells were analyzed by RT-PCR using specific primers for PMP70, catalase and GAPDH (a control).

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Fig. 8.

Effect of additional NAPE-PLD expression on NAPE metabolism in PLA/AT-1-expressing cells. A and B, control HEK293 cells transfected with the insert-free vector (lanes 1), HEK-PLA/AT-1 cells (lanes 2) and HEK-PLA/AT-1 cells stably expressing mouse NAPE-PLD (lanes 3 and 4) were radiolabeled with [¹⁴C]ethanolamine in the absence (lanes 1–3) or presence (lanes 4) of 0.2 μM URB597. Radiolabeled lipids were separated by TLC (A) and relative radioactivities of NAPE and NAE were examined (mean values ± S.D. (error bars), n = 3) (B). The positions of authentic compounds on the TLC plate are indicated. Asterisks indicate significant differences from NAPE and NAE levels in HEK-PLA/AT-1 cells (p < 0.001). C, HEK-PLA/AT-1 cells stably expressing NAPE-PLD were transfected with a control siRNA (siControl) or NAAA siRNA (siNAAA). Total RNAs were isolated from the cells and analyzed by RT-PCR using specific primers for NAAA and GAPDH (a control). D, HEK-PLA/AT-1 cells stably expressing NAPE-PLD were treated with siControl (lane 5) or siNAAA (lane 6) and radiolabeled with [¹⁴C]ethanolamine in the presence of 0.2 μM URB597. Relative radioactivity of NAE was then examined (mean values ± S.D. (error bars), n = 3). The cells were also radiolabeled in the presence of 0.2 μM URB597 alone (lane 7) or the combination of 0.2 μM URB597 and 10 μM pentadecylamine (lane 8). ^# and ^##indicate significant differences (p < 0.005 and p < 0.002, respectively).

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Fig. 9.

LC–MS/MS analysis of N-acylated ethanolamine phospholipids in ATDC5 cells. ATDC5 cells were transfected with control siRNA (siControl) or two different PLA/AT-1 siRNAs (siPLA/AT-1-A or -B). A, total RNAs were isolated from the cells and analyzed by RT-PCR using specific primers for PLA/AT-1, PLA/AT-3 and GAPDH (a control). Various species of NAPEs (B) and pNAPEs (C) in ATDC5 cells treated with siControl (white), siPLA/AT-1-A (black) or siPLA/AT-1-B (gray) were analyzed by LC–MS/MS as described in Materials and methods. Results are shown as pmol/μg of protein (mean values ± S.D., n = 3). R₁CO + R_NCO and R₁(CH)₂ + R_NCO represent the total number of carbon atoms and double bonds in sn − 1 O-acyl (or sn − 1 O-alkenyl) and N-acyl chains. The experiments were performed twice and the representative data were shown.

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