Microsomal epoxide hydrolase is not a 2-arachidonyl glycerol hydrolase

The endocannabinoid 2-arachidonyl glycerol (2-AG) is substantially hydrolysed by at least two enzymes, fatty acid amide hydrolase (FAAH) and monoarachidonyl glycerol lipase (MAGL), which thereby terminate its biological activity. In a recent report it has been claimed that microsomal epoxide hydrolase (mEH), hitherto known as a xenobiotic detoxifying enzyme, also rapidly catalyses the breakdown of 2-AG. However, the catalytic site architecture of mEH argues against an esterase activity. We therefore analysed the capacity of recombinant purified human, mouse and rat mEH to hydrolyse 2-AG. In contrast to the previous finding, we find only marginal 2-AG esterase activity ( ≤50 nmol/mg protein/min) associated with the purified enzymes that was resistant to inhibition by the potent mechanism-based mEH inhibitor 1,1,1-trichloropropene 2,3-oxide (TCPO). Likewise, 2-AG hydrolysis in mouse liver microsomes was resistant to TCPO inhibition while being efficiently blocked by methyl arachidonyl fluorophosphonate (MAFP). MAFP, on the other hand, failed to inhibit epoxide hydrolase activity of both, purified mEH and mouse liver microsomes. We therefore conclude that mEH lacks any appreciable 2-AG hydrolase activity. Introduction In a recent publication, Nithipatikom and colleagues [1] claimed that the xenobioticmetabolising enzyme microsomal epoxide hydrolase (mEH) [2] efficiently hydrolyses the endocannabinoid 2-arachidonyl glycerol (2-AG) [3]. Endocannabinoid signalling is important for a variety of physiological processes such as pain sensation [4], cognition and emotion [5] and appetite regulation [6]. Thus the capability of mEH to control 2-AG levels would have substantial implications, also because mEH is almost ubiquitously expressed throughout the human body [7]. The study by [1] was inspired by the fact that mEH belongs to the structural superfamily of α/β hydrolase fold enzymes [8] and therefore shares structural similarity to a wide range of esterases and lipases [9]. Two of these, ABHD6 and ABHD12, have recently been shown to possess 2-AG hydrolase activity [10]. In their experiments, the authors used microsomes of cell lines (over)expressing mEH. Microsomes aremembrane preparations obtained by differential centrifugation of cell or tissue homogenates and contain essentially all the membrane-associated proteins that are not confined to large organelles such as nuclei and mitochondria. Therefore, the contribution of mEH to the total protein content of these microsomes probably does not exceed 1%. Unfortunately, the authors do not give quantitative measures for this, but the fact that they use 30 μg microsomal protein for their western blot analyses is in agreement with the above estimate. In their turnover experiments, the authors observed 2-AG hydrolysis in microsomes that, after subtraction of the significant background reaction observed in non-transfected material, correlated with the degree of mEH expression and was responsive to siRNA mediated mEH down-regulation and inhibition by small molecules, the most potent of these being methyl arachidonyl fluorophosphonate (MAFP). From their results, they conclude that mEH is a novel 2-AG hydrolase. Some facts cast doubt on the interpretation of the study: 1) Although mEH is indeed a member of the α/β hydrolase fold enzyme superfamily, it belongs to a sub-class that differs in the most important component of its catalytic machinery from esterases and lipases: While the active site catalytic nucleophile of lipases/esterases is a serine that forms a covalent intermediate with the acid component of its ester substrates [11], epoxide hydrolases carry an aspartic acid at this position, forming an ester intermediate with the alcoholic component of its substrates [12] (Fig. 1A; Fig. S1 for a more detailed explanation). 2) The control membrane preparations used in the study possess already a high background activity for 2-AG hydrolysis that is not attributable to mEH, FAAH or Microsomal epoxide hydrolase is not a 2-arachidonyl glycerol hydrolase DOI: 10.19185/matters.201605000008 Matters (ISSN: 2297-8240) | 2 MAGL, and the increase in activity, where it can be taken from the authors’ presentation, is only a twoto three fold gain, 3) the strongest inhibitor identified in the study, MAFP, is known as a powerful inhibitor of PLA2-type lipases [13], yet no potency to inhibit mEH has so far been demonstrated, and finally 4) neither 2-AG hydrolytic activity nor sensitivity to MAFP inhibition of purified mEH has been tested by the author, which would be a definitive proof for their claim. Objective We therefore set out to re-assess the potential activity of mEH as a 2-AG hydrolase by directly testing this activity with purified mEH.

In their experiments, the authors used microsomes of cell lines (over)expressing mEH. Microsomes are membrane preparations obtained by differential centrifugation of cell or tissue homogenates and contain essentially all the membrane-associated proteins that are not confined to large organelles such as nuclei and mitochondria. Therefore, the contribution of mEH to the total protein content of these microsomes probably does not exceed 1%. Unfortunately, the authors do not give quantitative measures for this, but the fact that they use 30 µg microsomal protein for their Western blot analyses is in agreement with the above estimate. In their turnover experiments, the authors observed 2-AG hydrolysis in microsomes that, after subtraction of the significant background reaction observed in non-transfected material, correlated with the degree of mEH expression and was responsive to siRNA mediated mEH down regulation and inhibition by small molecules, the most potent of these being methyl arachidonyl fluorophosphonate (MAFP). From their results, they conclude that mEH is a novel 2-AG hydrolase. Some facts cast doubt on the interpretation of the study: 1) Although mEH is indeed a member of the α/β hydrolase fold enzyme superfamily, it belongs to a sub-class that differs  [11] ), epoxide hydrolases carry an aspartic acid at this position, forming an ester intermediate with the alcoholic component of its substrates (Lacourciere 1993 [12] ) ( Fig. 1A; Fig. S1 for a more detailed explanation).
2) The control membrane preparations used in the study posses already a high background activity for 2-AG hydrolysis that is not attributable to mEH, FAAH or MAGL, and the increase in activity, where it can be taken from the authors presentation, is only a 2 -3-fold gain, 3) the strongest inhibitor identified in the study, MAFP, is known as a powerful inhibitor of PLA2-type lipases (Lio 1996 [13] ), yet no potency to inhibit mEH has so far been demonstrated, and finally 4) neither 2-AG hydrolytic activity nor sensitivity to MAFP inhibition of purified mEH have been tested by the author, which would be a definitive proof for their claim.
(A) The separate types of α/β hydrolase fold enzymes use different catalytic residues to hydrolyse different types of substrates: while epoxide hydrolases cleave the ether bond in epoxides with an aspartic acid side chain, esterases hydrolyse esters using a serine side chain (for further details see Fig. S1). Epoxide hydrolase activity of purified human mEH (B) and of mouse liver microsomes (D) is sensitive to TCPO inhibition and resistant to MAFP treatment. In contrast, 2-AG hydrolase activity of purified human mEH (C) and of mouse liver microsomes (E) is resistant to TCPO treatment, while this activity in liver microsomes is strongly inhibited by MAFP. The column heights give the mean of 3 separate measurements. Error bars indicate the standard deviation. *** p < 0.001 NB: MAFP sensitivity of 2-AG hydrolase activity associated with the human mEH purified from bacteria was not tested because of the lack of relevance: the bacterial enzyme obviously contributing this activity (see our collective results) may or may not be inhibited by the compound. Either result would not have any impact on our conclusions.

Results & Discussion
Recombinant expression and His-tag-based metal chelate chromatography purification yielded purified mouse, rat and human mEH protein that displayed turnover rates with the prototypic substrate 11,12-epoxyeicosatrienoic acid (11,12-EET) of 0.1, 0.3 and 0.65 µmol per mg protein per minute, respectively. On SDS polyacrylamide gel electrophoresis and subsequent Coomassie staining the recombinant proteins displayed the expected dominant signal at 52 kDa ( Fig.  S2A), with some minor impurities amounting to 14, 18 and 27% for the rat, mouse and human enzyme, respectively, as determined by densitometric quantification.
2-AG turnover with purified rat, mouse and human mEH revealed relatively poor, apparently TCPO-resistant enzymatic activity ( Fig. S2B-G) that roughly correlated with the degree of impurity of the enzyme preparation. The most active preparation was that of human mEH with an estimated turnover rate of 50 nmol arachidonic acid formed per milligram protein per minute. Because the activity reported by (Nithipatikom 2014 [1] ) for human mEH was several orders of magnitudes higher (see last paragraph of this section for a comparison), these results already demonstrate that mEH does not possess substantial 2-AG hydrolase activity. However, to clarify whether the measurable 2-AG hydrolase activity in our recombinant purified enzymes might be attributed to any impurities of the preparation rather than to mEH itself, we refined the analysis of enzymatic turnover in the presence and absence of 1,1,1-trichloropropene 2,3oxide (TCPO), a potent mEH inhibitor. This compound acts as a suicide substrate, characterised by an extremely slow hydrolysis rate of the covalent intermediate formed in the first step. Therefore, any activity of the enzyme requiring the catalytic nucleophile is efficiently blocked by a sufficient TCPO concentration. In presence of 1 mM TCPO, 99% of the human mEH enzymatic activity were blocked using 4 uM 11,12-EET as substrate ( Fig. 1B; the Km for human mEH with 11,12-EET is reported to be 0.4 µM (Decker 2012 [14] )). By contrast, 2-AG hydrolysis by human mEH remained essentially unchanged with 100 µM 2-AG ( Fig. 1C; the Km with 2-AG is 40 µM according to (Nithipatikom 2014[1])). This shows that 2-AG hydrolytic activity is not associated with the mEH protein itself, but is most probably due to minor impurities of bacterial esterases in our enzyme preparation. In addition, we tested MAFP for its capability to block mEH hydrolase activity. With an IC50 of 7.9 nM this compound was identified as the most potent inhibitor by (Nithipatikom 2014[1]) to block what they proposed to be mEH-mediated 2-AG hydrolysis. However, 11,12-EET hydrolysis by purified human mEH remained unaffected in the presence of 10 µM MAFP (Fig. 1B).
To test whether the native membrane environment in the endoplasmic reticulum provides essential components for any mEH-mediated 2-AG hydrolytic activity which might explain the lack of the respective enzymatic activity with the purified enzyme, we next analysed 2-AG and 11,12-EET hydrolysis in microsomes prepared from the livers of sEH -/mice, lacking the mEH sister enzyme soluble epoxide hydrolase (sEH), the presence of which might otherwise interfere with the efficacy of TCPO to inhibit 11,12-EET hydrolysis. In the absence of any inhibitor, the microsomes showed the expected hydrolytic activities with 11,12-EET (0.66 nmol/mg/min; Fig 1D) and 2-AG (950 nmol/mg/min; Fig 1E). Again, EET hydrolysis was highly sensitive to TCPO inhibition and resistant to MAFP treatment (Fig. 1D), while the opposite effect was observed with 2-AG hydrolysis (Fig 1E). The 2-AG hydrolase activity reported by (Nithipatikom 2014[1]) is very high. From Fig. 4 of their manuscript, an enzymatic activity of around 300 nmol 2-AG being hydrolyzed per minute and milligram microsomal protein can be deduced. Given the above estimate that at best 1% of the microsomal enzyme can be attributed to mEH, this would imply that mEH has a specific 2-AG hydrolase activity of ≥ 30 µmol per milligram per minute. We do not find activity with purified mEH from either rat, mouse or human in excess of 50 nmol per milligram per minute. On top, the inhibitor sensitivity of both, the 2-AG hydrolysis by purified mEH enzyme as well as of mouse liver microsomes, is incompatible with its origin from mEH, but suggests that even this minor activity in our preparations originates from bacterial enzymes that have not completely separated from the mammalian proteins during purification.

Conclusions
Our results clearly demonstrate that mEH lacks any appreciable 2-AG hydrolase activity. With respect to the observations made by (Nithipatikom 2014 [1] ), we suggest as an alternative explanation that mEH might activate a so far uncharacterized MAFP-sensitive 2-AG hydrolase.

Conjectures
The next obvious step is to identify the 2-AG hydrolase that is apparently activated by mEH, as well as the mechanism by which this activation takes place. According to the report by Nithipatikom et al., this hydrolase can neither be FAAH nor MAGL. In our view, the potential candidates are ABHD6, ABHD12, CES1 and CES2, due to their reported significant capacity to hydrolyze 2-AG (Blankman 2007 [10] ) (Xie 2010 [15] ).

Funding Statement
This work was funded by the Swiss National Foundation, grant n°31003A_146635   Chromatographic separation was performed using procedure 3. The red arrows indicate the position of arachidfonic acid. E. -G. Ion chromatograms of 11,12-EET hydrolysis by the same enzyme preparation. E shows the substrate under starting conditions (4 µM) while F and G present the signal after substrate turnover in the absence (F) and presence (G) of TCPO. The strong signal reduction for the product 11,12-DHET in the presence of TCPO is obvious. Note, that also the scale has changed to higher sensitivity, due to the fact that the less sensitively detected substrate is now limiting the scale (note that, with the exception of 3C2 and 3D2, all chromatograms are presented as screen shots without adjustmet of the scale, for the sake of authenticity; only external rulers have been added to facilitate the reading of the scales). Chromatographic separation was performed using procedure 1. The red arrow indicates the position of 11,12-DHET.