The hydrolysis of monoacylglycerides, and 2-AG in particular, has been ascribed as an activity to many enzymes in vitro
, including several that are expressed in the nervous system (e.g., MAGL, FAAH, HSL, and NTE). These findings starkly contrast with the degradation of AEA, which is principally dictated by a single brain enzyme, FAAH, and raise the pertinent question – which enzymes in the nervous system make the most significant contribution to 2-AG hydrolysis in vivo
? To answer this question, a more complete and quantitative understanding of the brain enzymes that exhibit 2-AG hydrolase activity is required. Recognizing that virtually all 2-AG hydrolase activity in brain tissue is sensitive to inhibition by general serine hydrolase inhibitors, such as MAFP or PMSF [29
], we adopted a functional proteomic approach to fully inventory the 2-AG hydrolases expressed in mouse brain. This approach took advantage of the activity-based probe FP-biotin [30
] coupled with advanced LC-MS methods [31
] to assemble a list of 32 serine hydrolases expressed in brain. Recombinant expression of these enzymes identified seven proteins that hydrolyzed 2-AG. Using ‘isotope-free’, spectral counting methods to quantify the relative expression levels of these enzymes in brain tissue, we were able to estimate their respective contributions to total 2-AG hydrolase activity. These estimates were confirmed by pharmacological studies with inhibitors that showed distinct selectivity profiles for the brain 2-AG hydrolases.
MAGL was found to mediate ~85% of total brain membrane 2-AG hydrolase activity. This value matches remarkably well previous reports that used the MAGL inhibitor NAM to block 80–85% of cerebellar membrane 2-AG hydrolase activity [24
]. These data, in conjunction with our competitive ABPP studies (e.g., see ), further argue that NAM, despite containing a highly reactive maleimide group, exhibits rather high selectivity for MAGL relative to other brain serine hydrolases. MAGL has been modeled to contain a non-catalytic cysteine residue in its active site [24
], which could account for its unusual sensitivity to maleimide reagents. We initially anticipated that the remaining 15% of brain 2-AG hydrolase activity might be due to other enzymes known to hydrolyze 2-AG, such as FAAH, HSL, and NTE. However, our data indicate that none of these enzymes makes substantial contributions to total brain 2-AG hydrolase activity. Instead, the ‘MAGL-independent’ 2-AG hydrolase activity is largely mediated by two enzymes of previously uncharacterized function – ABHD12 (~9%) and ABHD6 (~4%), with the remaining ~2% activity presumably being performed by FAAH and/or other enzymes. These percent contribution values were determined at near physiologic pH (pH 7.5), and it is important to note that they may be pH-dependent.
When contemplating why the brain might contain multiple enzymes with 2-AG hydrolase activity, we consider the following points. First, these enzymes could exhibit distinct cellular or subcellular distributions, or undergo different forms of regulated expression. Indeed, the metabolism of other classes of bioactive molecules, including acetylcholine [41
], monoamines [42
], and prostaglandins [43
], has been shown to be regulated by multiple enzymes or multiple isoforms of the same enzyme. This “redundancy” presumably offers cells greater versatility to tailor the magnitude and duration of small-molecule signaling events to meet specific physiological objectives. For example, neurons that express membrane-bound versus secreted acetylcholinesterase isoforms display differences in synaptic signal strength due to distinct rates of acetylcholine degradation at the synapse [41
]. In this context, it is noteworthy that MAGL, ABHD6, and ABHD12 each displayed a distinct subcellular distribution. We speculate that these enzymes may have preferred access to distinct pools of 2-AG in vivo
, which could in turn shape the signaling activity of this endocannabinoid at different synapses throughout the nervous system. Recent work from the Parsons group also argues for the existence of distinct pools of 2-AG in the brain. These authors determined by in vivo
microdialysis that extracellular levels of 2-AG are approximately 200-fold lower than total brain levels of this lipid [44
], indicating that only a small fraction of total 2-AG may be “signaling-competent”. Of course, elucidating the respective roles of MAGL, ABHD12, and ABHD6 as regulators of 2-AG signaling in vivo
will require selective genetic and/or pharmacological tools to perturb their individual functions. In this regard, it is noteworthy that both ABHD6 and ABHD12 were inactivated by the lipase inhibitor THL. This finding indicates that ABHD6 and ABHD12 likely share active site structural similarity, despite showing very low sequence homology (< 20%). Active site relatedness among enzymes from the serine hydrolase family that lack sequence identity has been noted previously [39
It is also possible that ABHD6 and/or ABHD12 may play a more dominant role in 2-AG hydrolysis in cells that lack MAGL. It will be interesting, for example, to determine whether these enzymes contribute to 2-AG hydrolysis in microglial cells, which have recently been shown to possess this activity despite lacking MAGL [28
]. Finally, it is also possible that ABHD6 and ABHD12 metabolize endogenous substrates that are distinct from 2-AG. Such has proven to be the case with FAAH, which despite hydrolyzing 2-AG in vitro
], is primarily responsible for degrading fatty acid amide substrates in vivo
]. On this subject, however, we do believe it is instructive to place the 2-AG hydrolase activities of ABHD6 and ABHD12 in perspective by noting that they exceed the rate of FAAH-catalyzed hydrolysis of AEA in brain tissue by ~10–20-fold (2.6 and 5.9 nmol/min/mg vrs 0.3 nmol/min-mg [13
]). Thus, although ABHD12 and ABHD6 only contribute to ~15% of the total 2-AG hydrolysis in brain, their activities are still quite high compared to other pathways for lipid transmitter degradation.
In summary, we have performed herein the first comprehensive characterization of brain enzymes that hydrolyze 2-AG. These studies both confirm the role of established 2-AG hydrolases, such as MAGL, and designate the previously uncharacterized enzymes ABHD12 and ABHD6 as potential regulators of endocannabinoid signaling pathways. Assuming that one or more of these enzymes is confirmed to regulate 2-AG degradation in vivo, they might constitute useful therapeutic targets for a range of nervous system disorders. More generally, we suggest that the functional proteomic strategy put forth in this manuscript could be employed to comprehensively inventory enzymes that possess other hydrolytic activities of relevance to mammalian signaling and physiology, including, for example, diacyl- and triacylglyceride metabolism and the production and/or degradation of bioactive lipids such as lysophosphatidic acid.