Biochemical and pharmacological characterization of human α/β-hydrolase domain containing 6 (ABHD6) and 12 (ABHD12)

Abstract

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.

Fig. 1.

A sensitive fluorometric glycerol assay for endocannabinoid hydrolases. a: The assay is based on the following principle: The endocannanabinoid 2-AG is degraded to AA and glycerol by the endocannabinoid hydrolases MAGL, ABHD6, and ABHD12. Glycerol is converted to glycerol-1-phosphate (G-1-P) by glycerol kinase (GK). Glycerol phosphate oxidase (GPO)-catalyzed oxidation of G-1-P generates H₂O₂, which in the presence of HRP converts Amplifu™ Red to the fluorescent product resorufin. Resorufin fluorescence is monitored kinetically using excitation and emission wavelengths of 530 and 590 nm, respectively. b: Our assay protocol for the 96-well plate. c: Linearity of glycerol standard plot (0–520 pmol/well) at various time points. Although the coupled enzyme reactions generate maximal fluorescence only after prolonged (60 and 90 min) incubation time, the assay linearly detects glycerol at every time-point tested. d: Competitive ABPP of HEK cell lysates after transient transfections of the endocannabinoid hydrolases using the active site serine targeting fluorescent probe TAMRA-FP. After separation in SDS-electrophoresis gel (10%), serine hydrolases were visualized by in-gel fluorescent gel scanning as detailed in Materials and Methods. Molecular weight markers (kDa) are indicated by the numbers at left. Lysates were pretreated with DMSO or the serine-nucleophile targeting irreversible inhibitor MAFP (10⁻⁶ M) to demonstrate that TAMRA-FP labels the active-site serine residues of the endocannabinoid hydrolases. hMAGL and hABHD6 migrate as doublets (MAGL ∼33 and ∼35 kDa; ABHD6 ∼36 kDa), whereas hABHD12 migrates as single band (∼46 kDa). In addition to the endocannabinoid hydrolases, MAFP-sensitive TAMRA-FP labeling of endogenous serine hydrolases (<25 kDa and >250 kDa) is also evident. Data are from one typical transfection; transfections were repeated independently four times (hABHD6 or hABHD12) or twice (hMAGL). e and f: Linear protein-dependent (e) and time-dependent (f) glycerol response in lysates of HEK293 cells (0.3 µg protein/well) overexpressing the three human endocannabinoid hydrolases in assay mix containing 25 µM final 2-AG concentration. In panel e, glycerol was determined at time-point 30 min. In panels c, e, and f, values are means ± SD of duplicate wells from a typical experiment.

Fig. 2.

Substrate profiles of the human endocannabinoid hydrolases. HEK293 cells were transiently transfected with the cDNAs encoding hMAGL, hABHD6, or hABHD12 as detailed in Materials and Methods. After 48 h, cells were harvested, and lysates were prepared for hydrolase activity measurements using a sensitive fluorescent glycerol assay as described in Fig. 1. The substrate panel included MAGs with the indicated acyl chain length, isomer and degree of saturation, one DAG 1,2-dioleoyl(C18:1)-rac-glycerol, two triacylglycerols (TAG1, 1,2,3-trioleoyl(C18:1)glycerol; TAG2, 1-palmitoyl(C16:0)-2-oleoyl(C18:1)-3-linoleoyl(C18:2)-rac-glycerol, and LPA. Cellular lysates (0.3 µg/well) were incubated together with the indicated substrates (25 µM final concentration, added from 10 mM stock solutions in ethanol into the glycerol assay mix containing 0.5% [w/v] BSA and 1% [v/v ethanol]). Glycerol production was determined at 90 min. a: Background activity for the tested substrates. Cellular background was similar between HEK and mock-transfected cells (data not shown). b: Substrate profile of hABHD6. c: Substrate profile of hABHD12. d: Substrate profile of hMAGL. Data are mean + SEM from three to seven independent experiments using lysates of each enzyme from one transfection. Transfections were repeated independently four times (hABHD6 or hABHD12) or twice (hMAGL), and the relative profile for selected substrates (1-AG, 2-AG, 1-lauroyl-rac-glycerol [C14:0], and 1-myristoyl-rac-glycerol [C16:0]) was similar between different transfections (supplementary Fig. I). Statistical comparison between the MAG 1 (3)- and 2-isomers was done using a paired t-test (*P < 0.05; **P < 0.01; ***P < 0.001). The 1 (3)- and 2-isomers of MAG(C20:4) are highlighted in each panel.

Fig. 3.

Analysis of hABHD6 and hABHD12 active site mutants. a: Raw fluorescence data of the hABHD12 SDH-mutants, analyzed using 2.8 µg cellular lysate per well. b: Western blots of the hABHD12 SDH-mutants (top) and the relative expression level of the mutants as compared with the wild-type enzyme (bottom), based on quantification of the protein bands indicated with an asterisk. The quantitative data are mean ± SEM from three separate transfections and were normalized against β-tubulin. c: TAMRA-FP labeling of the hABHD12 SDH-mutants. Molecular weight markers (kDa) are shown in the column at right. Data are from one set of transient transfections; transfections were repeated independently three times with identical outcome. d: Raw fluorescence data of the hABHD6 SDH-mutants analyzed using 2.8 µg cellular lysate per well. e: Western blots of the hABHD6 SDHmutants (top) and the relative expression level of the mutants as compared with the WT enzyme (bottom) based on quantification of the protein bands indicated with an asterisk. The quantitative data are mean ± SEM from three (WT, S148A, D278N, H306A) or two (all others) separate transfections and were normalized against β-actin. f: TAMRA-FP labeling of the hABHD6 SDH mutants. Molecular weight markers (kDa) are shown in the column at right. Data are from two sets of transient transfections; transfections were repeated independently three times (WT, S148A, D278N, H306A) or twice (all others) with identical outcome.

Fig. 4.

K_m and V_max values for 2-AG. a: HEK293 cells were transiently transfected with the cDNAs encoding hMAGL, hABHD6, or hABHD12 as detailed in Materials and Methods. After 48 h, cells were harvested, and lysates were prepared for hydrolase activity measurements as described in Fig. 1. Cellular lysates (0.3 µg/well) were incubated with the indicated concentrations of 2-AG, and the K_m and V_max values were determined at 60 min; substrate consumption was <10%. The K_m and V_max values are shown in the box and were calculated as nonlinear regressions using GraphPad Prism 5.0 for Windows. b: The Lineweaver-Burk plots of the data. Values are mean ± SEM from three independent experiments.