N-acyl phosphatidylethanolamine-specific phospholipase D

N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) is an enzyme that catalyzes the release of N-acylethanolamine (NAE) from N-acyl-phosphatidylethanolamine (NAPE). This is a major part of the process that converts ordinary lipids into chemical signals like anandamide and oleoylethanolamine. In humans, the NAPE-PLD protein is encoded by the NAPEPLD gene.[1][2][3][4]

N-acyl phosphatidylethanolamine phospholipase D
Identifiers
Symbol	NAPEPLD
NCBI gene	222236
HGNC	21683
OMIM	612334
PDB	4QN9
RefSeq	NM_001122838
UniProt	Q6IQ20
Other data
EC number	3.1.4.54
Locus	Chr. 7 q22.1

Discovery

NAPE-PLD is an enzyme activity - a phospholipase, acting on phospholipids found in the cell membrane. It is not homology but the chemical outcome of its activity that classes it as phospholipase D. The enzymatic activity was discovered and characterized in a series of experiments culminating in the 2004 publication of a biochemical purification scheme from which peptide sequencing could be accomplished.[2] Researchers homogenized (finely ground) hearts from 150 rats and subjected the resulting crude lysate to sucrose sedimentation at 105,000 x g to separate out the cell membranes from the remainder of the cell. The integral membrane proteins were then solubilized using octyl glucoside and subjected to four column chromatography steps (HiTrap SP HP cation-exchange column, HiTrap Q anion-exchange column, HiTrap Blue affinity column, Bio-Gel HTP hydroxyapatite column). Each of these separates the different types of membrane proteins into different sample containers when the proteins are eluted from the column over time, and by measuring the activity of samples in each container it was possible to track which ones received the active enzyme. Measurement of the enzyme activity was done by thin layer chromatography of a radioactive substrate sensitive to the NAPE-PLD enzymatic activity: Cleavage of the substrate affected where it appeared on the plate when the radiation was detected on a bioimaging analyzer.

The result of this extensive procedure was still not a pure protein, but it produced a limited number of bands by SDS-PAGE, and one band of 46 kilodaltons was found to correlate in intensity with the enzymatic activity. This band was cut out from the gel and digested with trypsin, and peptides from it were separated from one another by reverse phase high performance liquid chromatography. The resulting fragments were then microsequenced by an automated Edman degradation.[5] Three corresponded to vimentin, an intermediate filament protein of 56 kDa believed to be a contaminant, and the other two matched the cDNA clone subsequently identified as NAPE-PLD.

Once this clue had been obtained, the identification could be confirmed by a less onerous procedure: Overexpression of the putative NAPE-PLD cDNA in COS-7 cells yielded a strong NAPE-PLD enzymatic activity, whose characteristics were shown to be similar to those of the original heart extract.[2]

Characteristics

The NAPEPLD cDNA sequence predicts 396 amino acid sequences in both mice and rats, which are 89% and 90% identical to that of humans.[2] NAPE-PLD was found to have no homology to the known phospholipase D genes, but can be classed by homology to fall into the zinc metallohydrolase family of the beta-lactamase fold. In particular, the highly conserved motif HX(E/H)XD(C/R/S/H)X_50–70HX_15–30(C/S/D)X_30–70H was observed, which is, in general, associated with zinc binding and hydrolysis reaction in this class of proteins, leading the authors to propose that activity should be correlated with zinc content.

When recombinant NAPE-PLD was tested in COS cells in vitro it had similar activity toward several radiolabeled substrates: N-palmitoylphosphatidylethanolamine, N-arachidonoylphosphatidylethanolamine, N-oleoylphosphatidylethanolamine, and N-stearoylphosphatidylethanolamine all reacted with a K_m between 2–4 micromolar and a V_max between 73 and 101 nanomole per milligram per minute as calculated by Lineweaver–Burk plot.[2] (These generate N-palmitoylethanolamine, anandamide, N-oleoylethanolamine, and N-stearoylethanolamine, respectively) The enzyme also reacted N-palmitoyl-lyso-phosphatidylethanolamine and N-arachidonoyl-lyso-phosphatidylethanolamine with similar K_m but at one-third to one-fourth the V_max. These activities are consistent with the observation that many tissues produce a range of N-acylethanolamines.

However, NAPE-PLD had no ability to produce detectable phosphatidic acid from phosphatidylcholine or phosphatidylethanolamine as is catalyzed by other phospholipase D enzymes. It also lacks the transphosphatidylation activity of phospholipase D that allows the creation of phosphatidyl alcohols rather than phosphatidic acid in the presence of ethanol or butanol.

Pathway

This enzyme acts as the second step of a biochemical pathway initiated by the creation of N-acylphosphatidylethanolamine, by means of the transfer of an acyl group from the sn-1 position of glycerophospholipid onto the amino group of phosphatidylethanolamine.[2] While NAPE-PLD contributes to the biosynthesis of several NAEs in the mammalian central nervous system, it is not clear if this enzyme is not responsible for the formation of the endocannabinoid anandamide, since NAPE-PLD knockout mice have been reported to have wild-type levels or very reduced levels of anandamide.[6]

The N-acylethanolamines released by this enzyme become potential substrates for fatty acid amide hydrolase (FAAH), which hydrolyzes the free fatty acids from ethanolamine. Defects in this enzyme can cause NAPE-PLD products such as anandamide to build up to levels 15-fold higher than normally observed.[7]

Structure

This membrane enzyme forms homodimers, partly separated by an internal ∼9-Å-wide channel.[8] The metallo beta-lactamase protein fold is adapted to associate with membrane phospholipids. A hydrophobic cavity provides an entry way for the substrate NAPE into the active site, where a binuclear zinc center catalyzes its hydrolysis. Bile acids bind with high affinity to selective pockets in this cavity, enhancing dimer assembly and enabling catalysis. NAPE-PLD facilitates crosstalk between bile acid signals and lipid amide signals.[8][9] [10]

References

1. See "Entrez Gene". for in-depth coverage.
2. Okamoto Y, Morishita J, Tsuboi K, Tonai T, Ueda N (Feb 2004). "Molecular characterization of a phospholipase D generating anandamide and its congeners". The Journal of Biological Chemistry. 279 (7): 5298–305. doi:10.1074/jbc.M306642200. PMID 14634025.
3. Curtiss NP, Bonifas JM, Lauchle JO, Balkman JD, Kratz CP, Emerling BM, Green ED, Le Beau MM, Shannon KM (May 2005). "Isolation and analysis of candidate myeloid tumor suppressor genes from a commonly deleted segment of 7q22". Genomics. 85 (5): 600–7. doi:10.1016/j.ygeno.2005.01.013. PMID 15820312.
4. Egertová M, Simon GM, Cravatt BF, Elphick MR (Feb 2008). "Localization of N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) expression in mouse brain: A new perspective on N-acylethanolamines as neural signaling molecules". The Journal of Comparative Neurology. 506 (4): 604–15. doi:10.1002/cne.21568. PMID 18067139.
5. "Amino Acid Sequencing". W.M. Keck Facility at Yale. 2006-10-23. Retrieved 2009-01-12. The Procise 494 cLC is described from the end user's perspective
6. Tsuboi K, Okamoto Y, Ikematsu N, Inoue M, Shimizu Y, Uyama T, Wang J, Deutsch DG, Burns MP, Ulloa NM, Tokumura A, Ueda N (Oct 2011). "Enzymatic formation of N-acylethanolamines from N-acylethanolamine plasmalogen through N-acylphosphatidylethanolamine-hydrolyzing phospholipase D-dependent and -independent pathways". Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids. 1811 (10): 565–77. doi:10.1016/j.bbalip.2011.07.009. PMID 21801852.
7. Cravatt BF, Demarest K, Patricelli MP, Bracey MH, Giang DK, Martin BR, Lichtman AH (Jul 2001). "Supersensitivity to anandamide and enhanced endogenous cannabinoid signaling in mice lacking fatty acid amide hydrolase". Proceedings of the National Academy of Sciences of the United States of America. 98 (16): 9371–6. doi:10.1073/pnas.161191698. PMC 55427. PMID 11470906.
8. Magotti P, Bauer I, Igarashi M, Babagoli M, Marotta R, Piomelli D, Garau G (Dec 2014). "Structure of Human N-Acylphosphatidylethanolamine-Hydrolyzing Phospholipase D: Regulation of Fatty Acid Ethanolamide Biosynthesis by Bile Acids". Structure. 23 (3): 598–604. doi:10.1016/j.str.2014.12.018. PMC 4351732. PMID 25684574.
9. Kostic M (2015). "Bile Acids as Enzyme Regulators". Chemistry & Biology. 22 (4): 427–428. doi:10.1016/j.chembiol.2015.04.007.
10. Margheritis E, Castellani B, Magotti P, Peruzzi S, Romeo E, Natali F, Mostarda S, Gioiello A, Piomelli D, Garau G (Oct 2016). "Bile Acid Recognition by NAPE-PLD". ACS Chem Biol. 11 (10): 2908–2914. doi:10.1021/acschembio.6b00624. PMC 5074845. PMID 27571266.