Biochemical and pharmacological characterization of human α/β-hydrolase domain containing 6 (ABHD6) and 12 (ABHD12)
2012; Elsevier BV; Volume: 53; Issue: 11 Linguagem: Inglês
10.1194/jlr.m030411
ISSN1539-7262
AutoresDina Navia‐Paldanius, Juha R. Savinainen, Jarmo T. Laitinen,
Tópico(s)Diet, Metabolism, and Disease
ResumoIn the central nervous system, three enzymes belonging to the serine hydrolase family are thought to regulate the life time of the endocannabinoid 2-arachidonoylglycerol (C20:4) (2-AG). From these, monoacylglycerol lipase (MAGL) is well characterized and, on a quantitative basis, is the main 2-AG hydrolase. The postgenomic proteins α/β-hydrolase domain containing (ABHD)6 and ABHD12 remain poorly characterized. By applying a sensitive fluorescent glycerol assay, we delineate the substrate preferences of human ABHD6 and ABHD12 in comparison with MAGL. We show that the three hydrolases are genuine MAG lipases; medium-chain saturated MAGs were the best substrates for hABHD6 and hMAGL, whereas hABHD12 preferred the 1 (3)- and 2-isomers of arachidonoylglycerol. Site-directed mutagenesis of the amino acid residues forming the postulated catalytic triad (ABHD6: S148-D278-H306, ABHD12: S246-D333-H372) abolished enzymatic activity as well as labeling with the active site serine-directed fluorophosphonate probe TAMRA-FP. However, the role of D278 and H306 as residues of the catalytic core of ABHD6 could not be verified because none of the mutants showed detectable expression. Inhibitor profiling revealed striking potency differences between hABHD6 and hABHD12, a finding that, when combined with the substrate profiling data, should facilitate further efforts toward the design of potent and selective inhibitors, especially those targeting hABHD12, which currently lacks such inhibitors. In the central nervous system, three enzymes belonging to the serine hydrolase family are thought to regulate the life time of the endocannabinoid 2-arachidonoylglycerol (C20:4) (2-AG). From these, monoacylglycerol lipase (MAGL) is well characterized and, on a quantitative basis, is the main 2-AG hydrolase. The postgenomic proteins α/β-hydrolase domain containing (ABHD)6 and ABHD12 remain poorly characterized. By applying a sensitive fluorescent glycerol assay, we delineate the substrate preferences of human ABHD6 and ABHD12 in comparison with MAGL. We show that the three hydrolases are genuine MAG lipases; medium-chain saturated MAGs were the best substrates for hABHD6 and hMAGL, whereas hABHD12 preferred the 1 (3)- and 2-isomers of arachidonoylglycerol. Site-directed mutagenesis of the amino acid residues forming the postulated catalytic triad (ABHD6: S148-D278-H306, ABHD12: S246-D333-H372) abolished enzymatic activity as well as labeling with the active site serine-directed fluorophosphonate probe TAMRA-FP. However, the role of D278 and H306 as residues of the catalytic core of ABHD6 could not be verified because none of the mutants showed detectable expression. Inhibitor profiling revealed striking potency differences between hABHD6 and hABHD12, a finding that, when combined with the substrate profiling data, should facilitate further efforts toward the design of potent and selective inhibitors, especially those targeting hABHD12, which currently lacks such inhibitors. The human serine hydrolases comprises a large family of enzymes with a predicted number of ∼240 that fall into two subfamilies: the serine proteases (∼125 members) and the metabolic serine hydrolases (∼115 members) (1Simon G.M. Cravatt B.F. Activity-based proteomics of enzyme superfamilies: serine hydrolases as a case study.J. Biol. Chem. 2010; 285: 11051-11055Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 2Long J.Z. Cravatt B.F. The metabolic serine hydrolases and their functions in mammalian physiology and disease.Chem. Rev. 2011; 111: 6022-6063Crossref PubMed Scopus (268) Google Scholar). The metabolic serine hydrolases include lipases and amidases and use a conserved serine nucleophile to hydrolyze amide, ester, and thioester bonds. The majority of serine hydrolases contain an α/β-hydrolase domain (ABHD) fold and use a Ser-His-Asp (SHD) triad for catalysis. Although several members of the metabolic serine hydrolase family are relatively well defined, the majority remain poorly characterized with respect to their physiological substrates and functions. Members of the metabolic serine family are also intimately involved in the generation and degradation of the endocannabinoid 2-arachidonoylyglycerol (C20:4) (2-AG). In brain regions endowed with 2-AG signaling, "on demand" biosynthesis of 2-AG occurs through phospholipase Cβ-catalyzed cleavage of the membrane phospholipid phoshatidylinositol bisphosphate to generate sn-2-arachidonoyl-containing diacylglycerol (DAG) species, which are subsequently hydrolyzed by sn-1-specific lipases (DAGLα and DAGLβ) to generate 2-AG (3Bisogno T. Howell F. Williams G. Minassi A. Cascio M.G. Ligresti A. Matias I. Schiano-Moriello A. Paul P. Williams E.J. et al.Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid signaling in the brain.J. Cell Biol. 2003; 163: 463-468Crossref PubMed Scopus (839) Google Scholar). The endocannabinoids are involved in a broad range of (patho)physiological processes, including neurotransmission, appetite, nociception, addiction, inflammation, peripheral metabolism, and reproduction (4Piomelli D. The molecular logic of endocannabinoid signalling.Nat. Rev. Neurosci. 2003; 4: 873-884Crossref PubMed Scopus (1613) Google Scholar–5Di Marzo V. Bisogno T. De Petrocellis L. Endocannabinoids and related compounds: walking back and forth between plant natural products and animal physiology.Chem. Biol. 2007; 14: 741-756Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 6Kano M. Ohno-Shosaku T. Hashimotodani Y. Uchigashima M. Watanabe M. Endocannabinoid-mediated control of synaptic transmission.Physiol. Rev. 2009; 89: 309-380Crossref PubMed Scopus (1101) Google Scholar). The biological actions of 2-AG are mediated via two G protein-coupled receptors (CB1R and CB2R) that show unique and tissue-specific distribution. CB1R is highly enriched in the brain. The major enzymatic route for 2-AG inactivation is via hydrolysis, generating arachidonic acid (AA) and glycerol. In the CNS, three serine hydrolases, namely monoacylglycerol lipase (MAGL) and the α/β-hydrolase domain (ABHD)-containing proteins ABHD6 and ABHD12, account for ∼99% of 2-AG hydrolysis (7Blankman J.L. Simon G.M. Cravatt B.F. A comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol.Chem. Biol. 2007; 14: 1347-1356Abstract Full Text Full Text PDF PubMed Scopus (870) Google Scholar, 8Savinainen J.R. Saario S.M. Laitinen J.T. The serine hydrolases MAGL, ABHD6 and ABHD12 as guardians of 2-arachidonoylglycerol signalling through cannabinoid receptors.Acta Physiol. (Oxf.). 2012; 204: 267-276Crossref PubMed Scopus (203) Google Scholar). From these, MAGL is relatively well characterized and on a quantitative basis appears to be the main 2-AG hydrolase. MAGL is a ∼33 kDa protein that was originally purified and cloned from adipose tissue (9Karlsson M. Contreras J.A. Hellman U. Tornqvist H. Holm C. cDNA cloning, tissue distribution, and identification of the catalytic triad of monoglyceride lipase. Evolutionary relationship to esterases, lysophospholipases, and haloperoxidases.J. Biol. Chem. 1997; 272: 27218-27223Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar), where it catalyzes the final step in lipolysis (9Karlsson M. Contreras J.A. Hellman U. Tornqvist H. Holm C. cDNA cloning, tissue distribution, and identification of the catalytic triad of monoglyceride lipase. Evolutionary relationship to esterases, lysophospholipases, and haloperoxidases.J. Biol. Chem. 1997; 272: 27218-27223Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar, 10Zechner R. Zimmermann R. Eichmann T.O. Kohlwein S.D. Haemmerle G. Lass A. Madeo F. FAT SIGNALS: lipases and lipolysis in lipid metabolism and signaling.Cell Metab. 2012; 15: 279-291Abstract Full Text Full Text PDF PubMed Scopus (704) Google Scholar). MAGL shows a wide tissue distribution (9Karlsson M. Contreras J.A. Hellman U. Tornqvist H. Holm C. cDNA cloning, tissue distribution, and identification of the catalytic triad of monoglyceride lipase. Evolutionary relationship to esterases, lysophospholipases, and haloperoxidases.J. Biol. Chem. 1997; 272: 27218-27223Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar) and is therefore generally thought to serve "house-keeping" functions in lipid metabolism (11Dinh T.P. Freund T.F. Piomelli D. A role for monoglyceride lipase in 2-arachidonoylglycerol inactivation.Chem. Phys. Lipids. 2002; 121: 149-158Crossref PubMed Scopus (269) Google Scholar). In addition, recent studies have illuminated pathophysiological roles for MAGL (12Nomura D.K. Long J.Z. Niessen S. Hoover H.S. Ng S.W. Cravatt B.F. Monoacylglycerol lipase regulates a fatty acid network that promotes cancer pathogenesis.Cell. 2010; 140: 49-61Abstract Full Text Full Text PDF PubMed Scopus (726) Google Scholar, 13Nomura D.K. Morrison B.E. Blankman J.L. Long J.Z. Kinsey S.G. Marcondes M.C. Ward A.M. Hahn Y.K. Lichtman A.H. Conti B. et al.Endocannabinoid hydrolysis generates brain prostaglandins that promote neuroinflammation.Science. 2011; 334: 809-813Crossref PubMed Scopus (528) Google Scholar). Methylarachidonoylfluorophosphonate (MAFP) is among the most potent MAGL inhibitors identified to date (14Goparaju S.K. Ueda N. Taniguchi K. Yamamoto S. Enzymes of porcine brain hydrolyzing 2-arachidonoylglycerol, an endogenous ligand of cannabinoid receptors.Biochem. Pharmacol. 1999; 57: 417-423Crossref PubMed Scopus (199) Google Scholar–15Saario S.M. Savinainen J.R. Laitinen J.T. Järvinen T. Niemi R. Monoglyceride lipase-like enzymatic activity is responsible for hydrolysis of 2-arachidonoylglycerol in rat cerebellar membranes.Biochem. Pharmacol. 2004; 67: 1381-1387Crossref PubMed Scopus (154) Google Scholar, 16Savinainen J.R. Yoshino M. Minkkilä A. Nevalainen T. Laitinen J.T. Characterization of binding properties of monoglyceride lipase inhibitors by a versatile fluorescence-based technique.Anal. Biochem. 2010; 399: 132-134Crossref PubMed Scopus (28) Google Scholar). MAFP inhibits MAGL irreversibly but lacks selectivity because it inhibits most members of the metabolic serine hydrolase family. The post-genomic proteins ABHD6 and ABHD12 remain poorly characterized regarding their physiological substrates and functions. ABHD6 is a ∼30 kDa integral membrane protein with high expression reported in certain forms of tumors (17Li F. Fei X. Xu J. Ji C. An unannotated alpha/beta hydrolase superfamily member, ABHD6 differentially expressed among cancer cell lines.Mol. Biol. Rep. 2009; 36: 691-696Crossref PubMed Scopus (33) Google Scholar, 18Max D. Hesse M. Volkmer I. Staege M.S. High expression of the evolutionarily conserved alpha/beta hydrolase domain containing 6 (ABHD6) in Ewing tumors.Cancer Sci. 2009; 100: 2383-2389Crossref PubMed Scopus (24) Google Scholar). Based on hydropathy analysis and biochemical studies, ABHD6 appears to be an integral membrane protein whose active site is predicted to face the cell interior (7Blankman J.L. Simon G.M. Cravatt B.F. A comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol.Chem. Biol. 2007; 14: 1347-1356Abstract Full Text Full Text PDF PubMed Scopus (870) Google Scholar). Such an orientation suggests that ABHD6 might be well suited to guard the intracellular pool of 2-AG. Recent evidence suggests that ABHD6 can indeed control the levels and signaling efficacy of 2-AG in neurons (19Marrs W.R. Blankman J.L. Horne E.A. Thomazeau A. Lin Y.H. Coy J. Bodor A.L. Muccioli G.G. Hu S.S. Woodruff G. et al.The serine hydrolase ABHD6 controls the accumulation and efficacy of 2-AG at cannabinoid receptors.Nat. Neurosci. 2010; 13: 951-957Crossref PubMed Scopus (347) Google Scholar, 20Marrs W.R. Horne E.A. Ortega-Gutierrez S. Cisneros J.A. Xu C. Lin Y.H. Muccioli G.G. Lopez-Rodriguez M.L. Stella N. Dual inhibition of alpha/beta-hydrolase domain 6 and fatty acid amide hydrolase increases endocannabinoid levels in neurons.J. Biol. Chem. 2011; 286: 28723-28728Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), but it is not known whether this enzyme uses other substrates as well. Few inhibitors have been identified that selectively target ABHD6. ABHD12 is a ∼45 kDa glycoprotein whose potential role as a brain 2-AG hydrolase was disclosed using activity-based protein profiling (ABPP) with mouse brain proteome, and it was estimated that at the bulk brain level ABHD12 accounts for ∼9% of total 2-AG hydrolase activity (7Blankman J.L. Simon G.M. Cravatt B.F. A comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol.Chem. Biol. 2007; 14: 1347-1356Abstract Full Text Full Text PDF PubMed Scopus (870) Google Scholar). 2-AG is the only recognized substrate for ABHD12, and 2-AG hydrolase activity is the only feature potentially linking ABHD12 to the endocannabinoid system. Based on hydropathy analysis and biochemical data, ABHD12 is an integral membrane protein whose active site is predicted to face the lumen/extracellular space (7Blankman J.L. Simon G.M. Cravatt B.F. A comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol.Chem. Biol. 2007; 14: 1347-1356Abstract Full Text Full Text PDF PubMed Scopus (870) Google Scholar). Inactivating mutations in the ABHD12 gene have been causally linked to the neurodegenerative disease called PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract) (21Fiskerstrand T. H'mida-Ben Brahim D. Johansson S. M'zahem A. Haukanes B.I. Drouot N. Zimmermann J. Cole A.J. Vedeler C. Bredrup C. et al.Mutations in ABHD12 cause the neurodegenerative disease PHARC: an inborn error of endocannabinoid metabolism.Am. J. Hum. Genet. 2010; 87: 410-417Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). ABHD12 transcripts are abundant in various brain regions as well as in microglia and related peripheral cell types, such as macrophages and osteoclasts (21Fiskerstrand T. H'mida-Ben Brahim D. Johansson S. M'zahem A. Haukanes B.I. Drouot N. Zimmermann J. Cole A.J. Vedeler C. Bredrup C. et al.Mutations in ABHD12 cause the neurodegenerative disease PHARC: an inborn error of endocannabinoid metabolism.Am. J. Hum. Genet. 2010; 87: 410-417Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Further characterization of ABHD12 is hampered by the lack of selective inhibitors. Further research on the novel 2-AG hydrolases would greatly benefit from a versatile activity assay allowing profiling of a wide repertoire of substrates by a simple readout such as monitoring the end product glycerol. Although glycerol assays have been in routine use for decades in lipolysis research, as far as we are aware, the potential of this methodology has not been realized in the field of endocannabinoid hydrolases. By applying a sensitive fluorescent assay to kinetically monitor glycerol production "on line," we delineate here the substrate profiles of human ABHD6 and ABHD12 in comparison with MAGL. After transient transfections in HEK293 cells, the three hydrolases degraded a variety of MAGs, each with distinct substrate and isomer preferences. The enzymes exhibited no detectable fatty acid amide hydrolase or lysophospholipase activity; nor did they hydrolyze di- or triacylglycerols. By site-directed mutagenesis, we identified the catalytic triad of ABHD12 as S246-D333-H372 and verified S148 as the catalytic nucleophile of ABHD6. Preliminary inhibitor profiling revealed striking potency differences between hABHD6 and hABHD12. All reagents for the glycerol assay mix (Fig. 1b), and the following substrates were obtained from Sigma (St. Louis, MO): MAG-C8:0 (1-capryloyl-rac-glycerol [C8:0]); MAG-C10:0 (1-decanoyl-rac-glycerol [C10:0]); MAG-C12:0 (1-lauroyl-rac-glycerol [C12:0]); MAG-C14:0 (1-myristoyl-rac-glycerol [C14:0]); MAG-C16:0 (2-palmitoyl-rac-glycerol [C16:0]); MAG-C18:0 (1-stearoyl-rac-glycerol [C18:0]); the 1 (3)- and 2-isomers of MAG-C18:1; 1,2,3-trioleoyl-rac-glycerol; and 1-oleoyl(C18:1)-sn-glycero-3-phosphate (LPA). 1,2-dioleoyl-rac-glycerol (DAG), 1-palmitoyl-2-oleoyl-3-linoleoyl-rac-glycerol, and the 1 (3)- and 2-isomers of MAG-C18:2 and MAG-C20:4 were from Cayman Chemicals (Ann Arbor, MI). Hydrolase inhibitors were from the following sources: methyl arachidonoylfluorophosphonate (MAFP), isopropyl dodecylfluorophosphonate (IDFP), and WWL70 from Cayman Chemicals; THL (orlistat) and pristimerin from Sigma; RHC-80267 from Biomol (Enzo Life Sciences); and hexadecane-1-sulfonyl fluoride (HDSF) from Calbiochem. HEK293-cells were cultured as monolayers in DMEM (Euroclone, Milan, Italy) containing 10% FBS (Euroclone) under antibiotics (penicillin/streptomycin, Euroclone) at 37°C in a humidified atmosphere of 5% CO2/95% air. Plasmids containing wild-type (WT) or mutant cDNA were introduced to cells by a standard transient transfection procedure using X-tremeGENE Hp DNA Transfection reagent (Roche, Mannheim, Germany). HEK293 and/or Mock cells (cells transfected with an empty vector) were cultured in parallel for controlling expression and activity in later experiments. Cell lysates were prepared by washing cells two times with ice-cold PBS. Cells were scraped and pelleted at 250 g for 10 min at 4°C. Cell pellets were freeze-thawed three times, resuspended in ice-cold PBS, briefly sonicated, and aliquoted for storage at −80°C. Membranes were prepared by resuspending the cell pellet in PBS (pH 7.40), followed by brief sonication and centrifugation at 100,000 g for 45 min at 4°C. The pellet was resuspended in PBS by brief sonication and aliquoted for storage at −80°C. Protein concentrations were measured by using Pierce BCA Protein Assay Kit (Pierce, Rockford, IL) using BSA as a standard. The set-up and validation of the fluorometric 96-well-plate glycerol assay is detailed in Fig. 1. Briefly, glycerol production was coupled via a three-step enzymatic cascade to a hydrogen peroxide (H2O2)-dependent generation of resorufin whose fluorescence (λex 530; λem 590 nm) was monitored using a Tecan Infinite M200 plate reader (Tecan Group Ltd., Männedorf, Switzerland). The assay protocol is detailed in Fig. 1b. Activity-based protein profiling (ABPP) was conducted using the fluorophosphonate probe TAMRA-FP (ActivX Fluorophosphonate Probes, Thermo Fisher Scientific Inc., Rockford, IL) following outlines of previously published methodology (7Blankman J.L. Simon G.M. Cravatt B.F. A comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol.Chem. Biol. 2007; 14: 1347-1356Abstract Full Text Full Text PDF PubMed Scopus (870) Google Scholar). Briefly, 25 µl of cellular lysates (5–10 µg protein) diluted in PBS were preincubated with 0.5 µl of the indicated inhibitors or a vehicle (DMSO) for 1 h at room temperature. Then, serine hydrolases were labeled with 0.5 µl of 100 µM TAMRA-FP for 1 h at RT. The reaction was stopped by adding 2× SDS-loading buffer, and proteins were separated in SDS-electrophoresis gel (10%) with molecular weight standards and compared with the pattern of labeled serine hydrolases in HEK293 or mock-transfected cells by following in-gel fluorescent gel scanning with Fujifilm FLA-3000 laser fluorescence scanner (Fujifilm, Tokyo, Japan) (Fluor. 532 nm; Filter: O580 nm). The substrate preferences of the endocannabinoid hydrolases were determined by monitoring glycerol production in lysates of HEK293 cells after transient expressing of each of the enzymes in assay mixes containing mono-, di-, or triglycerides or LPA (25 µM final concentration) with varying acyl chain length and saturation, as detailed in Fig. 2. To facilitate comparison between the three hydrolases under identical assay conditions, each substrate was tested in parallel using the three hydrolase preparations in the same experiment. For each tested substrate, assay blanks without enzyme, cellular background (HEK293/Mock cell lysates), as well as a glycerol quality control sample was included to monitor assay performance. Fluorescence of the assay blank was subtracted before calculation of the final results. Plasmids (pCMV6-AC-hABHD6, pCMV6-XL4-hABHD12, and pCMV6-XL5-hMAGL transcript variant 1 [313 amino acid residues]) were purchased from Origene Technologies Inc. (Rockville, MD). Site-targeted mutagenesis to generate hABHD6 mutants S148A, D278A/E/N, and H306A/S/Y and hABHD12 mutants S246A, D333N, and H372A was performed by using a QuikChange® Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) following the manufacturer's instructions. Primers for the mutagenesis were purchased from Oligomer (www.oligomer.fi). After transformation of mutated material into competent bacterial cells, DNA was isolated, purified, and fully sequenced for confirmation of correct constructs. The expression level of WT and mutant hABHD6/12 in cells was analyzed by Western blot as follows. Samples of lysates from each HEK293 population carrying WT or mutant enzymes (20 µg protein of hABHD6 lysates and 50 µg protein of hABHD12 lysates), together with molecular weight markers, were fractionated by SDS-PAGE (10%) and transferred to a nitrocellulose membrane (Protran; Schleicher and Schell, Dassel, Germany). To block nonspecific binding, membranes were incubated with 0.5% (w/v) BSA and Tris-buffered saline containing 0.1% Tween (TBS-T) solution for 1 h at room temperature. Next, membranes were incubated overnight at 4°C with a rabbit anti-hABHD6 antibody (HPA017283, 1:1,000; Sigma) or with a mouse anti-hABHD12 antibody (ab68949, 1:200; Abcam) diluted in 0.5% BSA-TBS-T. After washing for 4 × 10 min in TBS-T, hABHD6 membranes were incubated with a goat anti-rabbit IgG secondary antibody ([H+L] 800, 1:30,000; Thermo Scientific) and hABHD12 membranes with a goat anti-mouse IgG secondary antibody (35521, Lot# MJ164339, [H+L] 800, 1:10,000; Thermo Scientific) diluted in 0.5% BSA-TBS-T for 1 h at 20°C, followed by washing 4 × 10 min with TBS-T. For the normalization of enzyme expression between separate WT and mutant cell transfections, expression of β-actin was determined from hABHD6 lysates by incubating membranes with a mouse anti-β-actin primary antibody (A5441, 1:2,000; Sigma) overnight at 4°C. After washing for 4 × 10 min in TBS-T, membranes were incubated with a secondary goat anti-mouse IgG antibody (cat#35521, DyLight 800 Conjugated, 1:10,000; Thermo Scientific). In the case of hABHD12, relative expression was calculated against expression of β-tubulin by using a mouse anti-β-tubulin primary antibody (1:500, cat#T4026; Sigma,) and goat anti-Mouse IgG secondary antibody (cat#35521, DyLight 800 Conjugated, 1:10,000; Thermo Scientific) with otherwise equal incubation conditions used with hABHD6 membranes. Immunoblots were visualized by Odyssey (Li-Cor Biosciences Inc., Lincoln, NB) and quantified by using ImageJ, a freely available Java-based image analysis software system developed in the National Institutes of Health (http://rsb.info.nih.gov/ij/). For hABHD6 and hABHD12, transient transfections were repeated independently four times (twice for hMAGL). For each enzyme batch, the relative hydrolysis rate of selected substrates was assessed to monitor the behavior and reproducibility of enzyme preparations between different transfections (supplementary Fig. I). A more detailed substrate profiling (Fig. 2) was performed using one particular batch of each enzyme and always so that that the three hydrolases were evaluated in parallel in the same experiment. With the exception of data presented in Fig. 1 and supplementary Figs. II and III, all numerical data are mean ± SEM from at least three independent experiments. The SHD mutants were analyzed from two to three separate transfections with similar outcome, and the numerical data presented in Fig. 3 are pooled from these experiment. The Km and Vmax values, inhibitor dose-response curves, and IC50 values derived thereof were calculated from nonlinear regressions using GraphPad Prism 5.0 for Windows. Statistical comparison between the MAG 1 (3)- and 2-isomers (Fig. 2) was done using paired t-test (* P < 0.05, **P < 0.01, and ***P < 0.001). The set-up and validation of the fluorometric endocannabinoid hydrolase assay is presented in Fig. 1a–c. Lysates were prepared from HEK293 cells 48 h after transient transfections with the cDNAs encoding hABHD6 and hABHD12. For comparative purposes, HEK293 cells were transfected also with the cDNA encoding hMAGL (313 amino acid residues). ABPP with the active site serine-targeting TAMRA-FP probe indicated that the three hydrolases were successfully expressed after transient transfections (Fig. 1d). TAMRA-FP recognized hMAGL migrating in SDS-PAGE gels as two protein bands (∼33 and ∼35 kDa). Similarly, hABHD6 migrated as a doublet with a molecular weight of ∼36 kDa. In contrast, hABHD12 migrated as a single band with a molecular weight of ∼46 kDa. In lysates pretreated with MAFP (10−6 M), TAMRA-FP labeling was fully prevented indicating that the fluorophosphonates shared a common target (i.e., the catalytic serine residues in each of the three hydrolases) (Fig. 1d). The ABPP also indicated that HEK293 cells express negligible levels of endogenous MAGL, ABHD6, or ABHD12 because there was low-to-undetectable TAMRA-FP labeling of endogenous proteins migrating at the position of these enzymes. Moreover, TAMRA-FP labeling of the weak endogenous band with a size approximating that of ABHD12 was not prevented by MAFP, indicating that this band did not correspond to endogenous ABHD12. Transient expression of the three hydrolases resulted in robust time- and protein-dependent increase in 2-AG hydrolase activity as compared with the cellular background (Fig. 1e, f). With the tested protein concentrations, enzymatic activity increased linearly for the three hydrolases. Comparison of activities between HEK cell lysates and membranes indicated modest enrichment of hABHD6 and hABHD12 activity in membrane preparations (supplementary Fig. II), consistent with previous evidence implicating that the two hydrolases are integral membrane proteins (7Blankman J.L. Simon G.M. Cravatt B.F. A comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol.Chem. Biol. 2007; 14: 1347-1356Abstract Full Text Full Text PDF PubMed Scopus (870) Google Scholar). However, because cellular background was generally lower in lysates than in membranes and, on the other hand, hABHD6 and hABHD12 activities were readily detectable also in lysates, we used lysates instead of membranes in these studies. This was justified also from the economical point of view that only a fraction of cellular material was required to produce equal amounts of protein from lysates than from membranes. The high sensitivity of the fluorescent method allowed us to routinely perform activity assays in a 96-well format using only 0.3 µg cellular lysate per well; at this protein concentration hydrolase activity due to cellular background was negligible. The pH dependence of 2-AG hydrolysis was tested at pH values ranging from 5.3 to 9.1 (supplementary Fig. III). These experiments indicated that hABHD6 and hABHD12 exhibited a broad pH optimum between 7.2 and 9.1. Because the TEMN-BSA buffer (pH 7.4) (Fig. 1b) has been used in our previous studies exploring the signaling capacity and degradation of 2-AG in native cellular membranes (15Saario S.M. Savinainen J.R. Laitinen J.T. Järvinen T. Niemi R. Monoglyceride lipase-like enzymatic activity is responsible for hydrolysis of 2-arachidonoylglycerol in rat cerebellar membranes.Biochem. Pharmacol. 2004; 67: 1381-1387Crossref PubMed Scopus (154) Google Scholar, 22Savinainen J.R. Järvinen T. Laine K. Laitinen J.T. Despite substantial degradation, 2-arachidonoylglycerol is a potent full efficacy agonist mediating CB1 receptor-dependent G-protein activation in rat cerebellar membranes.Br. J. Pharmacol. 2001; 134: 664-672Crossref PubMed Scopus (146) Google Scholar), we used this buffer also in the present study. Collectively, these experiments indicate that HEK293 cells serve a convenient host to express the three endocannabinoid hydrolases for further characterization. We delineated the substrate preferences of the three hydrolases by monitoring glycerol production in HEK293 cell lysates individually overexpressing each of the enzymes in assay mixes containing mono-, di-, and triglycerols (25 µM final concentration) with varying acyl chain length and saturation (Fig. 2). To facilitate comparison between the three hydrolases under identical assay conditions, each substrate was tested using the three hydrolase preparations in the same experiment. Cellular background activity was similar in HEK293 and mock-transfected cells (Fig. 3a, d and data not shown). Fig. 2a illustrates background activity with the tested substrates. This represented in most cases C8:0 ≈ C10:0 ≈ C14:0 >> C16:0 > C18:0 (marginal). The preference for 1 (3)- vs. 2-acylglycerols was tested using oleoyl (C18:1), linoleoyl (C18:2), and arachidonoyl (C20:4) glycerols; in each case, hABHD6 preferred the 1 (3)-isomer with the relative activity order C20:4 >> C18:2 ≈ C18:1. hABHD6 exhibited negligible activity toward di- and triglycerides; nor did it hydrolyze 1-oleoyl-lysophosphatidic acid (LPA). hABHD12 had a clearly different substrate profile (Fig. 1c). The preferred MAG substrate was 1 (3)-AG, followed by 2-AG and MAG(C14:0), which were hydrolyzed to the same extent. hABHD12 clearly preferred 1 (3)-AG over 2-AG, and a similar, although weaker, trend was observed for t
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