Endocannabinoids anandamide and 2-arachidonoylglycerol are substrates for human CYP2J2 epoxygenase

Article abstract of DOI:10.1124/jpet.114.216598

The endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are arachidonic acid (AA) derivatives that are known to regulate human cardiovascular functions. CYP2J2 is the primary cytochrome P450 in the human heart and is most well known for the metabolism of AA to the biologically active epoxyeicosatrienoic acids. In this study, we demonstrate that both 2-AG and AEA are substrates for metabolism by CYP2J2 epoxygenase in the model membrane bilayers of nanodiscs. Reactions of CYP2J2 with AEA formed four AEA-epoxyeicosatrienoic acids, whereas incubations with 2-AG yielded detectable levels of only two 2-AG epoxides. Notably, 2-AG was shown to undergo enzymatic oxidative cleavage to form AA through a NADPH-dependent reaction with CYP2J2 and cytochrome P450 reductase. The formation of the predominant AEA and 2-AG epoxides was confirmed using microsomes prepared from the left myocardium of porcine and bovine heart tissues. The nuances of the ligand-protein interactions were further characterized using spectral titrations, stopped-flow small-molecule ligand egress, and molecular modeling. The experimental and theoretical data were in agreement, which showed that substitution of the AA carboxylic acid with the 2-AG ester-glycerol changes the binding interaction of these lipids within the CYP2J2 active site, leading to different product distributions. In summary, we present data for the functional metabolomics of AEA and 2-AG by a membrane-bound cardiovascular epoxygenase.

Full text of DOI:10.1124/jpet.114.216598

Supplemental material to this article can be found at:
http://jpet.aspetjournals.org/content/suppl/2014/10/02/jpet.114.216598.DC1.html
1521-0103/351/3/616–627$25.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
http://dx.doi.org/10.1124/jpet.114.216598
J Pharmacol Exp Ther 351:616–627, December 2014
Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics
Endocannabinoids Anandamide and 2-Arachidonoylglycerol Are
Substrates for Human CYP2J2 Epoxygenases
Daniel R. McDougle, Amogh Kambalyal, Daryl D. Meling, and Aditi Das
Department of Comparative Biosciences (D.R.M., A.D.), Department of Biochemistry (A.K., D.D.M., A.D.), and Medical Scholars
Program (D.R.M.), Beckman Institute for Advanced Science and Technology, and Department of Bioengineering (A.D.),
University of Illinois Urbana-Champaign, Urbana, Illinois
Received June 5, 2014; accepted October 1, 2014
ABSTRACT
The endocannabinoids, anandamide (AEA) and 2-arachidonoyl- and cytochrome P450 reductase. The formation of the pre-
glycerol (2-AG), are arachidonic acid (AA) derivatives that are dominant AEA and 2-AG epoxides was confirmed using micro-
known to regulate human cardiovascular functions. CYP2J2 is somes prepared from the left myocardium of porcine and bovine
the primary cytochrome P450 in the human heart and is most heart tissues. The nuances of the ligand-protein interactions
well known for the metabolism of AA to the biologically active were further characterized using spectral titrations, stopped-
epoxyeicosatrienoic acids. In this study, we demonstrate flow small-molecule ligand egress, and molecular modeling.
that both 2-AG and AEA are substrates for metabolism by The experimental and theoretical data were in agreement,
CYP2J2 epoxygenase in the model membrane bilayers of which showed that substitution of the AA carboxylic acid with
nanodiscs. Reactions of CYP2J2 with AEA formed four AEA- the 2-AG ester-glycerol changes the binding interaction of these
epoxyeicosatrienoic acids, whereas incubations with 2-AG lipids within the CYP2J2 active site, leading to different product
yielded detectable levels of only two 2-AG epoxides. Notably, distributions. In summary, we present data for the functional
2-AG was shown to undergo enzymatic oxidative cleavage to metabolomics of AEA and 2-AG by a membrane-bound cardio-
form AA through a NADPH-dependent reaction with CYP2J2 vascular epoxygenase.
Mechoulam, 2007). Therefore, a thorough understanding of
Introduction
all the potential metabolic pathways in the body is necessary
The human body contains endogenous cannabinoids (endo-
to potentially target and alter these pathophysiological
cannabinoids) that elicit similar effects as D9-tetrahydroca-
nabinol, the principal component of cannabis. The two most
conditions.
Endocannabinoids are derivatives of arachidonic acid (AA)
well characterized endocannabinoids are anandamide (AEA)
that are stored in the plasma membrane (Fig. 1). AA is the
and 2-arachidonylglycerol (2-AG), which primarily exert their
primary substrate for the eicosanoid-synthesizing cascade
effects by activating the cannabinoid receptors—cannabinoid
that includes three branches—the cyclooxygenase (COX),
receptor 1 (CB1) and cannabinoid receptor 2 (CB2). Dysregu-
lipoxygenase (LOX), and cytochrome P450 epoxygenase path-
lation of the endocannabinoids has been implicated in a wide
ways (Yang et al., 2011). Endocannabinoids provide substrate
range of pathologies, including anxiety disorders, neurode-
for the eicosanoid-synthesizing cascade through both direct
generative diseases, obesity, and heart disease (Kogan and
and indirect pathways. The indirect route is through the
cleavage of either the AEA ethanolamide motif by fatty acid
amide hydrolase or the 2-AG glycerol motif by monoacylglycerol
This work was supported in part through the American Heart Predoctoral
Fellowship [14PRE20130015] (to D.R.M.). The Roy J. Carver Biotechnology lipase, which produces AA that can enter one of the three
Center’s 5500 QTrap MS was funded by the National Institutes of Health
branches (Yang et al., 2011). Alternatively, AEA can directly
enter each of the pathways and produce a diverse range of
metabolites with different pharmacologic properties when
National Center for Research Resources [Grant S10-RR024516]. The School of
Chemical Sciences Q-TOF Ultima mass spectrometer was purchased in part
with a grant from the National Science Foundation, Division of Biological
Infrastructure [DBI-0100085].
compared with the endocannabinoid parent compound. Prior
dx.doi.org/10.1124/jpet.114.216598.
s
This article has supplemental material available at jpet.aspetjournals.org. to this work, 2-AG was only shown to be a direct substrate of
ABBREVIATIONS: AA, arachidonic acid; AEA, anandamide; 2-AG, 2-arachidonoylglycerol; BHT, butylated hydroxytoluene; CB1, cannabinoid
receptor 1; CB2, cannabinoid receptor 2; COX, cyclooxygenase; CPR, cytochrome P450 reductase; DTT, dithiothreitol; EET, epoxyeicosatrienoic
acid; EET-EA, epoxyeicosatrienoic ethanolamide; EET-G, epoxyeicosatrienoic glycerol; ESI, electrospray ionization; HETE, hydroxyeicosatetraeonic
acid; HPLC, reversed-phase liquid chromatography; HRP, horseradish peroxidase; LC-ESI-MS, liquid chromatography-electrospray ionization-
mass spectrometry; LC-MS, liquid chromatography-mass spectrometry; LC-MS/MS, liquid chromatography–tandem mass spectrometry; LOX,
lipoxygenase; MRM, multiple reaction monitoring; MS-PPOH, N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide; ND, nanodisc; P450,
cytochrome P450; PIP2, 1-heptadecanoyl-2-arachidonoyl-sn-glycero-3-[phosphoinositol-49,59-bisphosphate]; PMSF, phenylmethanesulfonylfluor-
ide; POPC, 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine; POPS, 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phos-
pho-L serine.
616

CYP2J2-Mediated Metabolism of Endocannabinoids
617
Fig. 1. Chemical structures of substrates that bind to
CYP2J2-ND. Endogenous CYP2J2 ligands include (A)
arachidonic acid, (B) anandamide, and (C) 2-arachido-
noyl glycerol. (D) The xenobiotic, ebastine, is a cardio-
toxic CYP2J2 substrate. (E) MS-PPOH is a nonspecific
P450 inhibitor. (F) Schematic of CYP2J2 and CPR in-
corporated into the membrane bilayers of nanodiscs.
The membrane scaffold protein is represented in cyan
surrounding a bilayer of phospholipids colored white
with gold phosphate head groups. CYP2J2 is red, and its
obligate redox partner cytochrome P450 reductase is
colored blue.
the COX and LOX pathways, although the 2-AG epoxide me- lipase (Rouzer and Marnett, 2011). However, inhibition of this
tabolites had been extracted from rat kidney, spleen, and brain enzyme does not completely block the 2-AG conversion to
tissues (Chen et al., 2008). Recently, it has been shown that AA (Blankman et al., 2007). This indicates the presence of
endocannabinoids regulate human cardiovascular functions and alternative pathways of attenuation. In this work, we demon-
are important for homeostasis (Pacher and Steffens, 2009). strate another potential pathway in which 2-AG is converted to
Thus, it is of interest to evaluate the metabolism of endocanna- AA by CYP2J2 and other P450s.
binoids by relevant heart enzymes. In this work, we reveal the
In summary, we elucidate the nuances of binding and
nuances of the metabolism of 2-AG and AEA by the predominant metabolism of AA and its derivatives, AEA and 2-AG, by CYP2J2
P450 in the heart, CYP2J2 epoxygenase. CYP2J2 is an ex- using liquid chromatography–tandem mass spectrometry
trahepatic cytochrome P450 that is highly expressed in the (LC-MS/MS), liquid chromatography-mass spectrometry
myocardium and surrounding aortic epithelium and has been (LC-MS), spectral titrations, small-molecule ligand egress stud-
shown to be an important regulator of cardiovascular function ies, and molecular modeling. For all studies with recombinant
(Delozier et al., 2007; Alghasham et al., 2012; Panigrahy et al., protein, we used the nanoscale lipid bilayers of nanodiscs (ND)
2012). CYP2J2 produces potent lipid epoxide mediators derived to solubilize and stabilize membrane-bound CYP2J2. These
from AA known as epoxyeicosatrienoic acids (EETs), which are model membranes have been previously used to functionally
involved in pain, inflammation, kidney, and cardiovascular dis- stabilize membrane protein in solution and on surfaces (Nath
ease (Imig and Hammock, 2009; Rouzer and Marnett, 2011). et al., 2007; Bayburt and Sligar, 2010; Denisov and Sligar,
Additionally, the epoxygenases also produce hydroxyeicosate- 2011; Das et al., 2014; Orlando et al., 2014). Importantly, the
traeonic acids (HETE) that are hydroxylated at the 19- or 20-V use of these model membranes significantly reduced scattering
position of AA.
and enabled spectroscopic characterization by increasing the
Currently, there are no reports of the direct oxygenation of solubility of the hydrophobic lipid substrates and membrane
2-AG by a cytochrome P450 (P450). Previous studies with proteins.
several recombinant P450 epoxygenases (CYP2C8, CYP2C11,
and CYP2C3) failed to produce 2-AG epoxide metabolites (Chen
et al., 2008). In this work, we show that CYP2J2 epoxygenase
metabolizes 2-AG to form 11,12-epoxyeicosatrienoic glycerol
(EET-G) and 14,15-EET-G. Several reports have demonstrated
the P450-mediated metabolism of AEA to 20-HETE-EA, 5(6)-,
8(9)-, 11(12)-, and 14(15)-epoxyeicosatrienoic ethanolamide
Materials and Methods
Reagents. NADPH and NADP were obtained from P212121 (Ann
Arbor, MI). The POPC [1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-
3-phosphocholine], 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine,
POPS [1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phospho-
(EET-EA) by CYP3A4, CYP4F2, CYP4X1, CYP2B6, and CYP2D6 L serine], and PIP2 (1-heptadecanoyl-2-arachidonoyl-sn-glycero-
3-[phosphoinositol-49,59-bisphosphate]) were purchased from Avanti
Polar Lipids (Alabaster, AL). AA, 2-AG, AEA, deuterated anandamide,
MS-PPOH [N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide],
14,15-EET, 11-12-EET, 8,9-EET, 5,6-EET, (6)16(17)-epoxy-4Z,7Z,10Z,
13Z,19Z-docosapentaenoic acid, 2-(14,15-epoxyeicosatrienoyl glycerol),
and 14,15-EET ethanolamide were obtained from Cayman Chemical
(Ann Arbor, MI). Whole cow and porcine hearts were obtained fresh
from the University of Illinois Meat Sciences Laboratory. Polyclonal
CYP2J2 antibody (catalog ABIN1529400) and horseradish peroxidase
(HRP)–conjugated donkey anti-rabbit antibody (catalog ABIN101948)
(Snider et al., 2007, 2008; Stark et al., 2008; Sridar et al., 2011).
Notably, it was demonstrated that these new metabolites
provide different pharmacological profiles when compared
with the parent endocannabinoid. In this study, we characterize
the oxidation of 2-AG and AEA with the heart epoxygenase,
CYP2J2, using both recombinantly expressed CYP2J2 and heart
microsomes.
Additionally, the activities of endocannabinoids are regulated
through various activation and inactivation pathways. 2-AG is
primarily inactivated through hydrolysis by monoacylglycerol were purchased from Antibodies-Online, Inc. (Atlanta, GA). Recombinant

618
McDougle et al.
CYP2C8 supersomes were purchased from BD Biosciences Gentest (San
Jose, CA). The antioxidant butylated hydroxytoluene (BHT) was ob-
tained from Sigma-Aldrich (St. Louis, MO). All other materials and
reagents used were purchased from Sigma-Aldrich and Fisher Scientific.
Milford, MA) coupled to a Waters 996 photodiode diode array detector
(Waters) and a semipreparative Superdex 10/200 column (GE Health-
care) column.
Recombinant CYP2J2 Metabolism Assays Containing
Recombinant Expression of CYP2J2 in Escherichia coli. The Arachidonic Acid, Anandamide, and 2-Arachidonoyl Glycerol.
recombinant CYP2J2 (construct D34G) was expressed and purified, as Incubation mixtures containing CYP2J2-ND (0.2 mM), CPR (0.6 mM),
published previously (McDougle et al., 2013; Zelasko et al., 2013). and 0.1% (w/v) BHT in 100 mM phosphate buffer (pH 7.4) and either
Briefly, DH5a cells containing the His-tagged and N-terminally AA, AEA, or 2-AG (500 ml total volume) were equilibrated at 37°C for
truncated CYP2J2 plasmid and pTGro7 chaperonin plasmid were
5 minutes before the reaction was initiated with the addition of 1 mM
cultured in 30 ml Luria Bertani media with chloroamphenicol (20 mg/ml) NADPH. Importantly, CPR was added to the P450-nanodisc in the
and ampicillin (100 mg/ml) at 37°C and 250 rpm overnight. The optimal 3:1 CPR to P450 ratio. Previous reports demonstrated the
resulting culture was used to inoculate 500 ml Terrific Broth con-
taining ampicillin (100 mg/ml) and chloroamphenicol (20 mg/ml). The
culture was grown at 37°C and 220 rpm until reaching an optical
density of 1.0, and then d-aminolevulinic acid (500 ml 0.5 mM) was
added and grown for 2 hours at 26°C and 160 rpm. A total of 1 mM
isopropyl b-D-1-thiogalactopyranoside and 2 g arabinose was added to
each 500 ml culture for induction and subsequently grown for an
additional 44 hours. The cells were harvested via centrifugation (2800g
for 10 minutes) using a JA-10 rotor (Beckman Coulter, Brea, CA) at 4°C.
The cells were then sonicated in lysis buffer [dithiothreitol (DTT),
0.2 mM phenylmethanesulfonylfluoride (PMSF), 5 mg DNase, and
RNase] before ultracentrifugation at 140,000g for 30 minutes with a
Ti-45 rotor (Beckman Coulter) for membrane fraction isolation. Protein
was extracted from the membrane fraction using 1.0% (w/v) cholate and
stirred at 4°C for 4 hours. The membrane fraction was removed by
ultracentrifugation at 140,000g for 30 minutes with a Ti-45 rotor
(Beckman Coulter). CYP2J2 was isolated from the supernatant by
running the solution through a Ni-NTA column and eluting in 0.1%
(w/v) cholate, 100 mM KPi, 200 mM imidazole, and 20% glycerol with
protein yield of ∼150 nmol/l.
incorporation of CPR and P450 into NDs as verified by size-exclusion
chromatography (Grinkova et al., 2010). Additionally, in a previous
work, we prepared discs containing both CYP2J2 and CPR NDs
(McDougle et al., 2013). Incubations for subsequent qualitative
LC-MS analysis contained saturating concentrations of each substrate,
whereas samples for quantitative kinetic analysis contained either 1, 5,
10, 20, 40, or 60 mM of each respective substrate. Incubations were
reacted for 30 minutes, unless otherwise stated in the figure legends.
Samples lacking NADPH were performed in parallel as a routine
control. For termination of the reaction, samples were quenched with
glacial acetic acid to pH of 3–4 and extracted (three times) with an equal
volume of ice-cold ethyl acetate, vortexed 1 minute, and centrifuged at
1590g for 3 minutes for phase separation (Stark et al., 2008). The
organic layer was removed and dried under a steady stream of N2
and then reconstituted with 100 ml methanol. AA, AEA, and 2-AG
incubations were qualitatively analyzed using LC-MS, as described
below. For quantitative analysis, LC-MS/MS was used to directly
quantify 14,15-, 11,12-, 8,9-, and 5,6-EET turnover when AA was
used as the substrates. For quantitative analysis of EET-G, extracted
samples were split into two pools as described in Saponification of
EET-G Regioisomers to EETs and Quantification.
Expression and Purification of Cytochrome P450 Reduc-
tase. Expression of the redox partner, cytochrome P450 reductase
(CPR), from Rattus norvegicus was described previously (McDougle
et al., 2013). Briefly, CPR starter cultures were used to inoculate 1 l
Luria Bertani supplemented with ampicillin (100 mg/ml) and riboflavin
(1 mg/l) and grown for 4 hours at 37°C and 220 rpm, before induction
with isopropyl b-D-1-thiogalactopyranoside (1 ml 1 mM), and then
grown for 18–20 hours at 33°C and 220 rpm. Upon harvesting the cells
by centrifugation (2800g for 10 minutes), the pellets were treated with
500 ml cold lysozyme buffer (75 mM Tris, pH 8.0, 0.25 M sucrose,
0.25 mM EDTA, and 0.02 mg/ml lysozyme) and allowed to stir at 4°C for
30 minutes. The resulting solution was centrifuged (2800g for
10 minutes) and resuspended in 40 ml lysis buffer (50 mM Tris,
pH 8.0, and 1 mM PMSF) at 4°C before sonication on ice (5 cycles ꢀ
30 seconds) with 1-minute rests. The membrane fraction was isolated
via ultracentrifugation and solubilized in column buffer [50 mM Tris,
pH 7.7, 0.1 mM EDTA, 0.1 mM DTT, and 20% (v/v) glycerol] containing
0.2% (v/v) Triton X-100 for 1 hour at 4°C. The solution was subjected to
ultracentrifugation (140,000g for 30 minutes), and the resulting
supernatant was loaded onto 29,59-ADP column, followed by de-
tergent removal using a DEAE Sepharose column.
Incorporation of CYP2J2 into Nanodiscs. Purified CYP2J2
was incorporated into the membrane bilayers of NDs as previously
described, with the only modification being the addition of POPS
(McDougle et al., 2013). POPS and POPC lipid stocks were combined
in chloroform in a 20:80 molar ratio and dried down under a steady
stream of N2. The lipid mixture was reconstituted with 200 mM
cholate in phosphate buffer (pH 7.4) and added to membrane scaffold
protein MSP1D1(2) in a 65:1 molar ratio and mixed at 4°C for 1 hour.
Purified CYP2J2 [100 mM KPi, pH 7.4, 20% (v/v) glycerol, and 0.1%
Preparation of Bovine and Porcine Microsomes and Incu-
bations. Fresh heart microsomes were prepared from the left
myocardium of female porcine and male bovine hearts using differential
centrifugation methods, as described previously (Pretorius et al., 1969).
Briefly, tissue from the left myocardium was dissected to remove the
endo- and epicardium and minced. Tissue was weighed and diluted
to 20% (w/v) using a buffer containing 250 mM sucrose, 10 mM Tris-Cl
(pH 7.5), and 1 mM PMSF, and a protease inhibitor cocktail for mammalian
tissue extraction (Nacalai Tesque 25955-11). Next, tissue was homog-
enized on ice for 1 minute at maximum speed using a metal Bio-
homogenizer (Biospec, Bartlesville, OK). A low-speed centrifugation
step was employed to remove large tissue debris at 3000g for 20 minutes
at 4°C. The supernatant was then centrifuged at 10,000g for 20 minutes
at 4°C using a Ti-45 rotor (Beckman) and then repeated once more. The
resulting supernatant was centrifuged at 100,000g for 90 minutes at
4°C to pellet the sarcoplasmic reticulum containing heart P450s. The
cell pellet was then resuspended in 50 mM Tris (pH 7.5), 1 mM DTT,
1 mM EDTA, and 20% glycerol with a Teflon homogenizer. The
resulting microsomal solution was aliquoted and stored at 280°C until
future use. Microsomal protein content was calculated using a bicin-
choninic acid assay kit from Thermo Fisher Scientific (Waltham, MA).
Incubations with both porcine and bovine microsomes were performed
with a concentration of 1 mg/ml microsomes with saturating concen-
trations of AA, AEA, and 2-AG and then processed, as described, with
recombinant CYP2J2-ND incubations.
Immunoblotting of CYP2J2 Located in Cow and Porcine
Heart Microsomes. Heart microsomes were prepared, as described
above, and used for CYP2J2 immunoblotting following a previously
described protocol (Zeldin et al., 1997). In short, samples were boiled for
cholate] was added to the solution in a CYP2J2:MSP molar ratio of 10 minutes in 5ꢀ Laemmli buffer supplemented with 8 M urea and run
1:10 and mixed for several hours before detergent removal with
Amberlite beads overnight. The CYP2J2-NDs were separated from
on a 12% SDS polyacrylamide gel. Proteins were transferred to the
nitrocellulose membrane and blocked with 5% low-fat milk in Tris-
empty NDs using a Ni-NTA column and eluted with 100 mM phosphate buffered saline with Tween 20 for 1 hour before adding the primary
buffer containing 200 mM imidazole. Homogenous CYP2J2-ND pop- polyclonal antibody raised against the 250–275 segment of human
ulations were further purified using size-exclusion chromatography
CYP2J2 (Antibodies-Online, Inc.). The membrane was washed three
utilizing an Alliance 2695 analytical separation module (Waters, times with Tris-buffered saline with Tween 20, and the HRP-conjugated

CYP2J2-Mediated Metabolism of Endocannabinoids
619
donkey anti-rabbit antibody (Antibodies-Online, Inc.) was used for
detection. Blots were visualized using a HRP development solution
(Thermo Fisher Scientific) and a ChemiDoc XRS1 system (Bio-Rad,
Hercules, CA).
injection volume was 5 ml. Mass spectra were acquired with negative ESI,
and the ion spray voltage was 24500 V. The source temperature was 450°C.
The curtain gas, ion source gas 1, and ion source gas 2 were 32, 50, and
65, respectively. Multiple reaction monitoring (MRM) was used to quantify
Saponification of EET-G Regioisomers to EETs and Quan- EETs, as follows: 5(6)-EET m/z 319.2→191.0; 8(9)-EET m/z 319.2→155.1;
tification. For the quantification of EET-G in solution, the extracted 11(12)-EET m/z 319.2→167.0; 14(15)-EET m/z 319.2→219.1; internal
fatty acids were cleaved from the glycerol backbone using methanolic
potassium hydroxide (KOH) hydrolysis, as previously described (Capdevila
et al., 1991). Extracted samples were hydrolyzed in 1 ml of methanol
containing 0.4 N KOH and incubated at 50°C for 1 hour. Next, 1.5 ml
Millipore water was added, and glacial acetic acid was added to achieve
pH 3–4. Samples were extracted with an equal volume of ice-cold ethyl
acetate (3ꢀ), vortexed 1 minute, and centrifuged at 1600g for 3 minutes
for phase separation (Stark et al., 2008). The organic layer was removed
and dried under a steady stream of N2 and then reconstituted with 100 ml
methanol. Thus, the saponified sample contained both free EETs
originally present in solution and EETs derived from EET-G hydrolysis.
EET-G quantification at the time of reaction quenching was calculated
using eq. 1:
standard (6)16(17)-epoxy-4Z,7Z,10Z,13Z,19Z-docosapentaenoic acid m/z
343.2→274.1.
Quantification of 14,15-EET-EA samples was analyzed using the
5500 QTRAP LC/MS/MS system with a LC separation method consisting
of 0–1 minute, 50% A; 5–17 minutes, 0% A; and then returned to initial
conditions. The autosampler was set at 5°C. The injection volume was
2 ml. Mass spectra were acquired with positive ESI, and the ion spray
voltage was 5500 V. The source temperature was 450°C. The curtain
gas, ion source gas 1, and ion source gas 2 were 32, 50, and 65,
respectively. MRM was used to quantify 14(15)-EET ethanolamide
(m/z 364.3→346.3) with spiked anandamide-d4 (m/z 352.3→287.2)
as the internal standard).
Direct quantification of the 14,15-EET-G regioisomer was analyzed
using the 5500 QTRAP LC/MS/MS system with a linear gradient, as
follows: 0–2 minutes, 90% A; 8 minutes, 55% A; 13–25 minutes, 40%
A; 30 minutes, 30% A; 35 minutes, 25% A; 40 minutes, 2% A; 45–47
minutes, 15% A; 48–56 minutes, 0% A; and then returned to initial
conditions. Mass spectra were acquired under positive (ion spray voltage
at 5500 V) ESI. The source temperature was 450°C. The 14,15-EET-G
(m/z 395.3→377.3) was measured in positive ESI with arachidonoyl-1-
thio-glycerol (m/z 395.3→287.3) as an internal standard.
EET‐G 5 EETðsaponifiedÞ–FreeꢀEET
(1)
Extracted EET-G regioisomers were converted to their corresponding
EETs using methanolic KOH (0.4 N) and quantified using LC-MS/MS,
as described below.
Liquid Chromatography-Electrospray Ionization-Mass
Spectrometry Product Analysis. AA, AEA, and 2-AG regioisomers
were resolved using a XTerra C18 column 2.1 ꢀ 150 mm, 3.5 mM
(Waters), and a Waters Alliance 2695 reversed-phase high performance
liquid chromatography (HPLC) coupled to an electrospray ionization
(ESI) source. For analysis of AA and AEA metabolites, the mobile
phases consisted of mobile phase A (acetonitrile/H2O/formic acid, 95:5:
0.1) and mobile phase B (acetonitrile/H2O/formic acid, 5:95:0.1) and
were run with a linear gradient as follows: 0–1 minute, 60% A;
41 minutes, 20% A; 42 minutes, 0% A; and then returned to initial
conditions. The same solvent system was used in the analysis of
2-AG metabolites with a linear gradient as follows: 0–1 minute, 70%
A; 51 minutes, 20% A; 52 minutes, 0% A; and then returned to initial
conditions. For ESI, a Q-TOF Ultima time-of-flight mass spectrom-
eter was used in positive ion mode for analysis of AEA, with a spray
voltage of 4.5 V and capillary temperature of 200°C. Data were
collected and processed using Mass Lynx software (version 4.1), initially
scanning from 200 to 800 m/z and processed for the selected AEA-EET
m/z of 364. The mass and elution time of the 14,15-EET-EA moiety
were confirmed using the commercially available authentic standard.
The detection of AA and 2-AG metabolites was detected with the same
system in negative ion mode, with a cone voltage of 35 V and a 200°C
desolvation temperature, scanning from 200 to 800 m/z and processed
for the selected EET m/z of 319 and EET-G m/z of 393–394. The mass
and elution times of the EET regioisomers were confirmed with com-
mercially available authentic standards. For 2-AG analysis, the mass
and elution time of the commercially available 14,15-EET-G, EETs, and
AA lipids were confirmed with authentic standards purchased from
Cayman Chemical.
Quantification of CYP2J2-ND Rate of 2-AG Ester Cleavage.
The rate of 2-AG ester cleavage was quantified using a HPLC system
consisting of an Alliance 2695 analytical separation module (Waters)
coupled to a Waters 996 photodiode array and a XTerra C18 column, 1.3 Å,
2.1 mm ꢀ 50 mm, 3.5 mm pore size column (Waters). The mobile system
was composed of two solutions, as follows: solvent A (H2O/ACN/AcOH 95:
5:0.1) and solvent B (H2O/ACN/AcOH 5:95:0.1). The linear gradient was
as follows: 0–1 minute, 60% A; 30 minutes, 20% A; 31 minutes, 0% A;
then returned to initial conditions. Elution times of 12.6 minutes for
2-AG and 19.7 minutes for AA were verified with authentic standards.
Quantification of free AA was calculated using AA absorbance at
204 nm. For kinetic analysis, samples containing CYP2J2-ND (50 pmol;
0.2 mM), CPR (150 pmol; 0.6 mM), and 0.1% BHT in 0.1 M phosphate
buffer (pH 7.4) were incubated at 37°C for 5 minutes with 1, 5, 10, 20,
40, and 80 mM 2-AG before initiation of reaction with 1 mM NADPH at
37°C for 20 minutes. Controls lacking either NADPH or CYP2J2 were
run simultaneously with 80 mM 2-AG.
CYP2J2 Molecular Operating Environment Homology Mod-
eling. A multiple sequence alignment was performed using the protein
data bank to cross-reference the CYP2J2 sequence against 2A1, 2D6,
2E1, 2R1, 2A6, 2C8, 2B4, 2C5, 2C9, and 2A13. For construction of the
CYP2J2 homology model, crystal structure Protein Data Bank coor-
dinates were used for the isozymes with the closest homology to 2J2 and
included CYP2B4 (backbone, 42% primary sequence homology), 2C8
(B region, 38% primary sequence homology), 2R1 (FG region, 40%
primary sequence homology), and 2C9 (b4 region, 26% primary
sequence homology). An open CYP2J2 conformation was initially
used to dock AA, AEA, and 2-AG following previously described
procedures (Baudry et al., 2003). Two apparent CYP2J2 active site
cavities exhibited adjacent/overlapping areas near the heme in agree-
ment with the highest propensity of ligand-binding number and were
both used for docking of each respective substrate. Next, we conducted an
Liquid Chromatography–Tandem Mass Spectrometry for
Quantitation of 14,15-, 11,12-, 8,9-, 5,6-EETs, 14,15-EET-EA, and
14,15-EET-G. Quantification EET regioisomers derived from in vitro
CYP2J2 incubations were analyzed with the 5500 QTRAP LC-MS/MS
system (AB Sciex, Foster City, CA) with a 1200 series HPLC system energy minimization step for each respective ligand conformation, and
(Agilent Technologies, Santa Clara, CA), including a degasser, an
the induced fitting resulted in the CYP2J2-substrate model used to
autosampler, and a binary pump. The LC separation was performed on examine heme and ligand distances as well as the predicted interacting
an Agilent Zorbax Eclipse XDB C18 column (4.6 ꢀ 150 mm, 5 mm; Agilent
Technologies) with mobile phase A (0.1% formic acid in water) and mobile
phase B (0.1% formic acid in acetonitrile). The flow rate was 0.4 ml/min.
active site residues. Lastly, interaction energies were calculated for each
substrate to examine the energetics and feasibility of substrate binding.
Spectral Titrations of CYP2J2-ND. Spectral binding studies
The linear gradient was as follows: 0–2 minutes, 90% A; 8 minutes, 50% were performed by titrating AA (both AA and sodium arachidonate),
A; 13–25 minutes, 25% A; 30 minutes, 20% A; 35 minutes, 15% A; and AEA, 2-AG, MS-PPOH, and ebastine against CYP2J2-NDs (8 mM) in
then returned to initial conditions. The autosampler was set at 5°C. The 100 mM phosphate buffer (pH 7.4). Importantly, substrate stocks

620
McDougle et al.
were prepared with dimethylsulfoxide (5 mM and 50 mM) after the
addition of ethanol and acetonitrile was determined to cause type I
binding. CYP2J2-ND were incrementally titrated with increasing
concentrations of each putative substrate (either 5 mM or 50 mM
stocks) at 22°C and allowed to equilibrate for 5 minutes before
measuring the spectra. The total dilution of the original CYP2J2
concentration was #1.5%. Absorbance spectra were collected from
800 to 200 nm using a Cary Bio 300 UV-visible spectrophotometer
(Agilent Technologies). Raw absorbance spectra were processed to the
corresponding subtraction spectra using a standard MATLAB sub-
routine (Mathworks, Natick, MA). The absolute absorbance difference
of the subtraction spectra (peak and trough) was plotted against the
corresponding substrate concentration in Origin Laboratory (North-
hampton, MA) and fitted using a single binding isotherm (eq. 2).
Stopped-flow data were analyzed for statistical significance using a
Student’s t test where *P , 0.05, **P , 0.01, and ***P , 0.001.
Results
CYP2J2-Mediated Metabolism of AA and AEA De-
termined Using Liquid Chromatography-Electrospray
Ionization-Mass Spectrometry. We first investigated
the metabolism of AA and AEA by CYP2J2. CYP2J2 was
recombinantly expressed in E. coli, purified, and assembled into
NDs as described in Materials and Methods. Due to increased
functional stability of the CYP2J2-CPR-nanodisc system, all of
the studies utilizing recombinant CYP2J2 were first incorpo-
rated into NDs. To examine the metabolism of AA and AEA,
a liquid chromatography-electrospray ionization-mass spectrom-
etry (LC-ESI-MS) method was developed to qualitatively assess
the product formation. Importantly, the use of a slow linear
gradient was necessary for separation of the regioisomers and
structural identification by mass and elution time. Product
ꢀ
ꢁ
DA 5 Amax½Sꢁ= Ks 1 ½Sꢁ
(2)
where ΔA is the absorbance difference at each difference spectra peak
and trough, Amax is the amplitude corresponding to maximal spin
shift, Ks is the spectral dissociation constant, and [S] is the substrate
concentration. None of the substrates investigated exhibited any analysis of CYP2J2-ND-CPR incubations with AA produced four
homotropic cooperativity, and hence the titration data were fitted to
a single binding isotherm.
regioisomers (m/z 319.5) corresponding to the mass and elution
times of 14,15-, 11,12-, 8,9-, and 5,6-EET (Fig. 2A). The ad-
ditional peaks (m/z 319), 19-HETE, was confirmed at earlier
elution times (11 minutes). Similarly, analysis of CYP2J2 and
AEA incubations produced the same olefin oxygenation pattern
as AA, with a regioisomer corresponding to the elution times
of 14,15-EET-EA, as confirmed using the authentic standard
(Fig. 2B). The identity of the 11,12-, 8,9-, and 5,6-EET-EA was
extrapolated based on literature describing the elution profile
Cyanide Binding and Equilibrium Constant (Ks) Determi-
nation for CYP2J2-ND. The steady-state cyanide spectral equilib-
rium constants were determined by titrating cyanide concentrations
(800 mM and 4 M stocks) against ∼3 mM CYP2J2-ND in the absence
and the presence of 40 mM AA, AEA, 2-AG, and MS-PPOH until
saturation was reached (Das et al., 2014). The total addition of
CYP2J2 concentration was #3% and did not affect Soret peak height.
The raw data collected were converted into the difference spectra
using a MATLAB subroutine (Mathworks). The DA (DA444–414nm) was and peak mass confirmation. Similar to AA, the presence of ad-
plotted against the corresponding cyanide concentration and fitted to
a single binding isotherm equation using Origin Pro 8.6 (San Clemente,
ditional HETE metabolites (19- and 20-HETE-EA) was observed.
These results show that both AA and AEA are substrates of
CYP2J2 and form four epoxide products similar to previous
reports with other P450s (Snider et al., 2010).
CYP2J2-Mediated Metabolism of 2-AG Determined
Using LC-ESI-MS. The in vitro incubations containing
CYP2J2-ND-CPR and 2-AG produced the following products:
CA) with eq. 2, where DA 5 DA444–414nm
.
Transient Cyanide-Binding Kinetics to CYP2J2 Active Site
Measured Using Stopped-Flow Spectroscopy. The cyanide-
binding kinetics to the CYP2J2 active site in the absence and pres-
ence of varying concentrations of AA, AEA, 2-AG, and MS-PPOH was
determined using stopped-flow spectroscopy. Raw data were collected
using an Applied Photophysics SX.18MV (Leatherhead, UK) spectro-
photometer to monitor the rate of cyanide binding. A dual syringe
system was prepared, with syringe I containing CYP2J2-ND in the
absence or presence of varying concentrations (10, 20, and 40 mM) of
substrate and syringe 2 containing 80 mM cyanide in the absence or
presence of varying concentrations (10, 20, and 40 mM) of substrate. The
relatively high cyanide concentration was selected based on steady-state
cyanide-binding measurements and was necessary to ensure pseudo
first-order binding kinetics (Denisov et al., 2007). The parameters for
rapid mixing and data collection were as follows: 1.5 ms of dead time,
external triggering mechanism, photodiode array detection, and collec-
tion obtained on a logarithmic scale over 20 seconds. The raw data were
converted to the difference spectra by subtracting each successive scan
from the time 5 0 scan using a MATLAB subroutine (Mathworks) that
produced the typical 446 and 415 nm cyanide-binding peaks. The
difference of absorbance was plotted against time and analyzed with
a double exponential equation using Origin Pro 8.6.
DA4442414nm 5 A0 1 A1exk 1 A2exk
;
2
(3)
1
where A1 and A2 are the amplitudes of the fast and slow phases of the
reaction, respectively, and k1 and k2 are the rates of fast and slow
phases of cyanide binding to the protein, determined from fitting data
obtained to eq. 3.
Data Analysis. Data were analyzed and presented as means 6 S.E.
For all experiments, data were collected in triplicate (n 5 3) or greater.
Fig. 2. Metabolism of AA and AEA by human CYP2J2-ND-CPR identified
qualitatively using LC-ESI-MS. The different regioisomers of (A) AA (m/z
319.5 of 14,15-EET at 22.5 minutes) and (B) AEA (m/z 363.4 of
14,15-EET-EA at 14 minutes) produced six different products, including four
epoxide regioisomers at the 5,6-, 8,9-, 11,12-, and 14,15-olefin positions
and two HETEs.

CYP2J2-Mediated Metabolism of Endocannabinoids
621
two detectable 2-AG epoxide metabolites (m/z 393.3), AA efficiency. Reaction linearity was demonstrated for each of
(m/z 303.3) and EETs (m/z 319.5), as shown in Fig. 3. Authentic the substrates at 15, 30, and 60 minutes with R2 of 0.98 or
standards were used to confirm the 14,15-EET-G, EETs greater. The advantages of a LC-MS/MS method are that it
(14,15-, 11,12-, 8,9-, 5,6-EET), and AA metabolites. Although provides structural elucidation, quantification, and efficiency
the 11,12-EET-G was not commercially available, the results with excellent sensitivity without the need of derivatization.
from EET-G saponification confirmed the production of the This method should be widely applicable for complex biologic
11,12-regioisomer.
samples with low levels of EETs. The identification of the
In contrast to AA and AEA, only two olefin sites of 2-AG metabolites of 2-AG and AEA was followed by quantification
were epoxidized to form 2-AG-EETs (m/z 393.3). Thus, the and measurement of the rate of formation using a LC-MS-MS
functional data suggest that the interaction of 2-AG within method, as described in Materials and Methods (Fig. 4, B and C).
the CYP2J2-binding pocket is influenced by the presence of
The rates of formation of the two EET-G regioisomers were
the bulky glycerol group of 2-AG (Fig. 1C). Previous in- determined by saponification of the EET-G to the corresponding
cubations with other P450s have failed to produce the EET-G EET regioisomers. The final value of EETs arising from EET-G
regioisomers (Chen et al., 2008). In this work, we report that was obtained by subtracting the free EETs, as determined in
CYP2J2 is identified as an isoform of P450 that produces Materials and Methods. The kinetics of EET-G formation were
epoxide 2-AG metabolites. As a control, we analyzed CYP2C8 as follows: 14,15-EET-G Vmax 5 265.4 6 12.3 pmol/min per
(supersomes) with 2-AG following the same methodology and nanomole and Km 5 32.6 6 1.7 mM; 11,12-EET-G Vmax 5 125.5 6
failed to observe any detectable EET-G formation (Supplemen- 5.2 pmol/min per nanomole and Km 5 13.6 6 1.3 mM (Table 1).
tal Fig. 1). This suggests that CYP2J2 may be unique in the
epoxidation of 2-AG as compared with other family 2 P450s.
In a separate experiment, the rate of metabolism of AA to
form the four EET regioisomers was measured in the pres-
Quantitative Analysis of CYP2J2-Mediated Epoxida- ence of saturating concentrations of AA. Catalytic rates were
tion of 2-AG and AEA Using LC-ESI-MS/MS. The de- calculated at 103 6 4.6, 61.5 6 5.6, 18.4 6 1.7, and 8.9 6
tection and quantification of fatty acids and their metabolites 0.1 pmol/min per nanomole CYP2J2 for 14,15-, 11,12-, 8,9-, and
have been most commonly examined using radio flow HPLC 5,6-EET, respectively.
due to the high sensitivity and ease of use (Wu et al., 1996).
Additionally, we examined the CYP2J2-mediated epoxida-
Alternatively, detection by mass spectrometry after derivati- tion of saturating amounts of AEA (60 mM) toward the
zation, coupled with gas chromatography or liquid chroma- production of 14,15-EET-EA, which was calculated to be 391
tography, has also been widely used for EET analysis (Zhu 6 12 pmol/min per nanomole CYP2J2. Thus, the production of
et al., 1995). In this study, we successfully used HPLC coupled the 14,15-EET-EA appears to proceed at a significantly faster
to a triple quadrupole detector utilizing one of the most sen- rate when compared with the production of 14,15-EET from
sitive commercially available ion traps (QTRAP 5500), which AA despite sharing a similar qualitative product profile and
enabled a highly sensitive, accurate, and reproducible method binding characteristics, as discussed later.
for detection of all the EET regioisomers in MRM mode. The
CYP2J2-Mediated 2-AG Ester Cleavage and Produc-
limit of quantitation with a 10:1 signal to noise ratio for 5(6)-EET tion of Free AA. The LC-ESI-MS product analysis of 2-AG
was 5.0 ng/ml, whereas 8,9-, 11,12-, and 14,15-EET were cal- incubations revealed significant amounts of free AA in solu-
culated at 2.5 ng/ml. Prior to each run, a six-point standard tion (Fig. 3). Control experiments showed that the conversion
curve was generated for each set of samples using authentic of 2-AG to AA required the presence of both NADPH and CPR
standards. Extraction efficiency was calculated for each of the for catalytic turnover, indicating that the reaction proceeded
EET regioisomers using the acidification and the three-step through an oxidative mechanism (controls not shown). As seen
ethyl acetate extraction. The 14,15-, 11,12-, 8,9-, and 5,6-EETs in Fig. 4D, analysis of 2-AG incubations followed Michaelis-
were extracted from the reaction mixture with a 95% or greater Menten kinetics, indicating that the reaction was dependent
Fig. 3. Metabolism of 2-AG by incubations with
CYP2J2-ND-CPR revealed the following metabolic pro-
file as represented in the chromatogram that consists
of an overlay of two EET-G regioisomers, 14,15-EET-G
and 11,12-EET-G (m/z 393); three detectable EET
regioisomers, 14,15-EET, 11,12-EET, and 8,9-EET (m/z
319); and arachidonic acid (m/z 303).

622
McDougle et al.
Fig. 4. Kinetics of CYP2J2-mediated metabolism of 2-AG. (A) Schematic of the proposed routes of 2-AG metabolism by CYP2J2 that produces AA, EET-G,
and EET. The kinetic rates of the three products were quantified, as described in Materials and Methods. (B) Kinetics of the formation of the four
regioisomers, 14,15-, 11,12-, 8,9-, and 5,6-EET, were detected by LC-MS/MS. These correspond to the free EETs in solution that were generated either from
epoxidation of AA or oxidative ester cleavage of EET-G. (C) Kinetics of the formation of the two EET-G regioisomers, 14,15-EET-G and 11,12-EET-G,
analyzed as described in Materials and Methods. (D) The kinetics of CYP2J2-ND–mediated 2-AG ester cleavage and arachidonic acid production were
determined by monitoring AA formation. The data were fitted to Michaelis-Menten kinetics in Origin Pro 8.6 for calculation of Vmax and Km. The kinetic
rates of the epoxidation reactions and side reactions are summarized in Table 1.
on substrate binding to the active site. The Km of the ester porcine species because they both contain CYP2J2 with high
cleavage reaction was calculated at 3.25 mM with a Vmax of 2.2 6 sequence homology to humans (Supplemental Fig. 3). CYP2J2
0.2 nmol/min per nanomole CYP2J2. Samples lacking NADPH immunoblotting of the bovine and porcine microsomes was
and CYP2J2 were run in parallel as controls. AA was not performed using a polyclonal rabbit IgG antibody for human
detected in the NADPH-free sample. Additionally, 2-AG was CYP2J2. The microsomal preparations cross-reacted with the
stable in buffer at 37°C with negligible hydrolysis. The ability CYP2J2 antibody demonstrating the presence of CYP2J2 in
of P450 to mediate ester cleavage reactions has long been bovine and porcine hearts (Supplemental Fig. 4). First, the
reported, and rates determined in this work show similar functionality was assessed by measuring AA metabolism by
catalytic properties of other P450 isozymes (Guengerich, bovine and porcine microsomes, which produced 1.8 6 0.3 and
1987; Peng et al., 1995). However, this is the first account of 2.08 6 0.02 pmol min21 mg21 microsomal protein of EETs (all
the P450-mediated cleavage of an endogenous fatty acid ester. regioisomers), respectively. Next, we quantified 2-AG metab-
The same reactions were run with CYP2C8 (supersomes), which olism by directly measuring the production of the 14,15-EET-G
also produced AA in a NADPH-dependent reaction (Supplemental regioisomer by bovine and porcine microsomes that was
Fig. 1). However, CYP2C8 epoxygenase did not demonstrate the calculated at 1.17 6 0.57 and 1.1 6 0.55 pmol min21 mg21
ability to oxygenate the 2-AG olefin bonds.
microsomal protein, respectively. The 14,15-EET-EA isomer
Bovine and Porcine Heart Microsome Metabolism of was produced with a higher turnover in both bovine and
AA and 2-AG. To examine whether the novel endocannabi- porcine microsomes with values of 1.96 6 0.05 and 2.25 6 0.07
noid epoxygenation reactions would occur within a complex pmol min21 mg21 microsomal protein, respectively.
environment in the presence of other proteins, we prepared
CYP2J2-Mediated Lipase Activity. CYP2J2-mediated
heart microsomes from the tissue of bovine and porcine left oxidative cleavage of 2-AG into AA raised questions as to
ventricular myocardium. The rate of metabolism of AA, AEA, whether this enzyme could act as a lipase and use AA-containing
and 2-AG is summarized in Table 2. We chose bovine and phospholipids as direct substrates. PIP2 is present in the plasma
membrane, where it is cleaved by phospholipase C to produce
AA for subsequent entry into the eicosanoid cascade (Rouzer and
Marnett, 2011). Thus, we incubated PIP2 with the CYP2J2-ND-
CPR-NADPH system utilizing PIP2 concentrations below its
critical micelle formation. In these experiments, we did not ob-
TABLE 1
Summary of 2-arachidonoyl glycerol epoxidation kinetics
The product formation kinetics for CYP2J2 nanodisc incubations with 2-arachidonoyl
glycerol.
serve formation of AA or diacylglycerol (Huang et al., 2011).
Km
Vmax
Vmax/Km
Spectral Binding Studies of CYP2J2 with AA, AEA,
and 2-AG. In an effort to understand the binding interactions
of AA and its derivatives (AEA and 2-AG) with the active site of
CYP2J2, we performed spectroscopic binding studies. First, the
interactions of AA, AEA, 2-AG, ebastine, and MS-PPOH with
the CYP2J2-ND active site were investigated using UV-visible
spectral binding titrations (Fig. 5). As controls, the spectral
mM
pmol/min per nmol
14,15-EET-G
11,12-EET-G
14,15-EET
11,12-EET
8,9-EET
32.6 6 1.7
13.6 6 1.3
8.3 6 2.0
6.7 6 1.3
6.2 6 1.8
12.7 6 3.8
265.4 6 12.3
125.5 6 5.2
53.3 6 2.9
34.8 6 1.5
8.9 6 1.1
8.1 6 0.3
9.2 6 0.2
6.4 6 1.25
5.1 6 0.4
1.4 6 0.3
0.2 6 .05
5,6-EET
3.1 6 0.3

CYP2J2-Mediated Metabolism of Endocannabinoids
623
TABLE 2
Metabolism of arachidonic acid, 2-arachidonoyl glycerol, and anandamide by bovine and porcine heart microsomes
Substrate
Metabolite(s) Detected
Bovine Microsomes
Porcine Microsomes
pmol min21 mg21
AA
2-AG
AEA
Total EET (all regioisomers)
14,15-EET-G
14,15-EET-EA
1.8 6 0.3
1.1 6 0.57
1.96 6 0.05
2.08 6 0.02
1.18 6 0.55
2.25 6 0.07
binding properties of the potent epoxygenase inhibitor Soret. The apparent Ks of binding for 2-AG was calculated at 18
MS-PPOH (Falck et al., 1997) and ebastine (Fig. 5D) were in- 6 2.7 mM. Taken together, these data demonstrate that the
vestigated, and both induced type-I shifts with apparent spectral modification of the carboxylic acid of the AA with the glycerol
binding constants (Ks) of 10.8 6 1.5 mM and 5.6 6 0.7, moiety affects binding in CYP2J2’s large, yet narrow binding
respectively.
cavity (Lafite et al., 2007). Note that the observed Soret shifts
The fatty acids presented with smaller changes in spin- are not as prominent as xenobiotic P450 substrates, yet are
state and minor shifts in Soret band. Specifically, the Soret consistent with the few published spectra of other fatty acids
band at 417 nm red-shifted by 0.5 nm with a slight decrease in binding to membrane-bound P450s (Loughran et al., 2001).
amplitude for both AA and AEA. The difference spectra Moreover, the lack of significant Soret perturbations corresponds
revealed prominent troughs for both ligands at 414 nm and to the relatively low turnover rates of the AA-metabolizing
broad peaks at 434 and 435 nm for AA and AEA, respectively epoxygenases, such as CYP2J2 (Westphal et al., 2011). Ad-
(Fig. 5A). Both substrates were fitted to single binding ditionally, these experiments were further validated using
isotherm with apparent Ks of 11.3 6 1.3 mM for AA and 13.1 6 steady-state cyanide-binding experiments in the presence of
1.4 mM for AEA (Fig. 5D). The similarities between AA and AA, AEA, and 2-AG to probe the access and binding of cyanide
AEA spectral shifts and binding equilibrium suggest that the to the heme (Supplemental Fig. 2).
presence of the ethanolamide (AEA) motif does not significantly
Kinetics of Cyanide Binding to CYP2J2-ND in the
alter substrate distances and positioning in relation to the Presence of Substrates. The cyanide-binding kinetics of
heme when compared with AA. Conversely, 2-AG spectral CYP2J2 was investigated in the absence or presence of hy-
titrations resulted in a blue shift while slightly decreasing the drophobic substrates using stopped-flow spectroscopy. For
Fig. 5. Equilibrium binding using absorption spectra for interactions of substrate with CYP2J2 nanodiscs. (A) AEA titration Ks was determined to be
13.1 6 1.4 mM (inset: difference spectrum of AEA-bound CYP2J2-NDs). (B) 2-AG titration Ks was determined to be 18 6 2.7 mM (inset: difference
spectrum of 2-AG–bound CYP2J2 nanodiscs). (C) MS-PPOH titration Ks was determined to be 10.8 6 1.5 mM (inset: difference spectrum of
MS-PPOH–bound CYP2J2-NDs showing a type I binding mode). (D) Summary of the lmax (nm) of peak/trough, Ks, and DΑmax for binding of arachidonic
acid, anandamide, 2-arachidonoyl glycerol, ebastine, and MS-PPOH to CYP2J2 nanodiscs.

624
McDougle et al.
each substrate tested, rapid mixing and formation of the slow phase ∼10%. To rule out nonspecific fatty acid binding,
heme-cyanide complex red-shifted the Soret (Fig. 6A). As we examined the rates of cyanide binding in the presence of
shown in Fig. 6B, the difference spectra D (A444–414nm) were saturating concentrations of palmitate as a nonbinding sub-
analyzed as a function of time and yielded an exponential strate. As shown in Fig. 6H, the presence of palmitate does not
biphasic curve that was comprised of a fast and slow phase noticeably alter the ratio of the two phases. Although not
(Bidwai et al., 2013). The fast phase corresponds to the rate of dramatic, the reported results provide important characteriza-
cyanide binding to the ferric heme active site. Specifically, it tion of the nature of fatty acid binding.
can be used as a direct measure of substrate egress from the
CYP2J2 Homology Modeling with AA, AEA, and
immediate vicinity of the heme active site to accommodate 2-AG. Molecular Operating Environment (Chemical Com-
formation of the cyanide complex. In the fast phase, the pres- puting Group, Montreal, QC, Canada) software was used to
ence of increasing concentrations of ebastine significantly de- identify substrate-binding characteristics within the active site
creased cyanide-binding rates in a concentration-dependent of a CYP2J2 homology model. A hybrid structure model was
manner. Conversely, the fatty acid ligands only slightly de- constructed from the crystal Protein Data Bank coordinates of
creased cyanide binding (Fig. 6C). The ratio of the fast- and similar P450 isozymes (class-dependent sequence alignment
slow-phase amplitudes was compared to determine how ligand strategy), as described in Materials and Methods (Baudry et al.,
concentrations affect the presence of the ratios. As seen in 2006). This approach takes advantage of the conserved se-
Fig. 6, D–G, saturating concentrations of ebastine significantly quences that maintain the secondary and tertiary P450 folds to
increase the ratio of the slow phase (17.4 to 66.4%), whereas the generate the core structures surrounding the buried catalytic
arachidonate-containing ligands only slightly increased the sites. Given the fatty acids’ relatively low turnover, there are
likely many unproductive conformations within the active site.
As shown in Fig. 7A, AA was docked in the most preferred
configuration for epoxidation of the 14,15-olefin when closest to
the heme (5.1 Å), followed by the 11,12-, 8,9-, and 5,6-olefins
with calculated heme distances of 8.6, 11.3, and 12.9 Å, re-
spectively. As shown in Fig. 7B, the distances of the AEA
14,15-, 11,12-, 8,9-, and 5,6-olefins were calculated at 6, 8.5,
12.1, and 13.5 Å, respectively. Interestingly, 2-AG (Fig. 7C)
yielded significantly different active site interactions albeit
with similar heme distances of the 14,15-, 11,12-, 8,9-, and
5,6-olefin estimated at 5.9, 7.6, 9, and 11.5 Å, respectively. The
interaction potential energies predict feasibility of binding and
were similar for each polyunsaturated fatty acid with calcu-
lations of 268.3 kcal/mol, 269.4 kcal/mol, and 273.4 kcal/mol
for AA, 2-AG, and AEA, respectively. Thus, the olefin distances
of the Molecular Operating Environment model were in agree-
ment with experimental differences observed in AA, AEA,
and 2-AG metabolism. Additionally, the model predicted sig-
nificantly different binding characteristics based on the pres-
ence of the carboxylate/ethanolamide or glycerol moiety. For
example, a patch of hydrophobic residues appeared to me-
diate AA and AEA positioning within the CYP2J2 active site
with Pro112, Ile487, and Ile376 common for both substrates.
Interactions of polar residues were fewer with only Gln228 in-
teracting with the polar carboxylate/ethanolamide groups.
Conversely, positioning of the 2-AG used both hydrophobic
residues (Ile127, Val380, and Ile487) as well as interactions
of the hydrophilic glycerol moiety with polar CYP2J2 resi-
dues (Asn231, Ser224, Gln228, and Ser64). The relatively fewer
polar interactions of AA and AEA might enable sampling of
different conformations of these substrates in the active site
leading to the epoxidation of the 8,9- and 5,6-olefin groups.
Fig. 6. Stopped-flow cyanide-binding spectroscopy with CYP2J2-ND and
AA, 2-AG, AEA, and ebastine. (A) Stopped-flow spectroscopy was used to
monitor kinetics of cyanide binding to CYP2J2-ND by recording the
formation of the cyanide-heme complex at 444 nm as a function of time in
the presence of saturating [CN-]. (B) The difference spectra (DAbs 444–414
nm) was plotted against time and fitted to a double exponential equation
for determination of the rate of the (C) fast phase for each substrate
concentration tested and determination of statistical significance relative
to the substrate-free condition when *P , 0.05 and ***P , 0.001. The ratio
of the amplitudes of the fast phase (black) to slow phase (gray) was
dramatically altered in the presence of (D) ebastine, whereas the presence
of the polyunsaturated fatty acids such as (E) AEA, (F) 2-AG, and (G) AA
was modestly changed. (H) The saturated fatty acid palmitate did not alter
cyanide-binding characteristics.
Conversely, the glycerol group appears tethered and cannot
sample orientations for epoxidation of the 8,9- and 5,6-olefins. In
absence of a high-resolution CYP2J2 crystal structure, this
model provides a useful tool to predict the interactions of AA,
AEA, and 2-AG within the active site that provides CYP2J2’s
regioselectivity.
Discussion
Extrahepatic CYP2J2 epoxygenase metabolizes AA to form
EETs that are essential for regulation of blood pressure and

CYP2J2-Mediated Metabolism of Endocannabinoids
625
Fig. 7. Substrate-bound CYP2J2 homology model with
arachidonic acid, anandamide, and 2-arachidonoyl glyc-
erol. Proposed docking of the polyunsaturated fatty
acids into the active site of CYP2J2. (Left) The substrate
(A) AA, (B) AEA, and (C) 2-AG are represented as stick
figures (green) surrounded by space-filling dots. The
CYP2J2 ribbon structure is colored blue, and the heme-
oxygen motif is in red. The interaction potential energies
predict feasibility of binding and were similar for
each polyunsaturated fatty acid with calculations of
269.4 kcal/mol and 273.4 kcal/mol for 2-AG and AEA,
respectively. (Right) Predicted ligand-residue interactions
within the CYP2J2 active site for (A) AA, (B) AEA, and
(C) 2-AG.
cardiovascular function (Spector, 2009). AA-derived endocan- that is highly expressed in the myocardium and endocardium.
nabinoids play an important role in the regulation of the Given the increasing reports of an endocannabinoid-mediated
cardiovascular system. Therefore, we explored the metabo- regulation of blood pressure and the cardiovascular system, the
lism of functionalized arachidonate-containing lipids such as role of the oxygenated AEA metabolites may provide an in-
the endocannabionoids with the cytochrome P450 most highly teresting avenue for future studies. For instance, AEA is a
expressed in the human heart. The two most well characterized, partial agonist of both CB1 and CB2 receptors, whereas the 5,
AA-derived endocannabinoids are AEA and 2-AG, whose dys- 6-EET-EA metabolite selectively binds CB2 with 1000-fold greater
regulation has been implicated in a wide range of pathophys- affinity (Snider et al., 2009). Additionally, there is mounting
iological states (Pacher et al., 2006; Kogan and Mechoulam, evidence of CB2 cardioprotection versus CB1 cardiotoxicity,
2007; Pacher and Steffens, 2009). In this study, we demonstrate and the ability to selectively target these pathways may provide
CYP2J2-mediated metabolism of AEA and 2-AG using recombi- a useful therapeutic target for the treatment of heart disease
nant human CYP2J2 and heart microsomes derived from bovine (Mukhopadhyay et al., 2008; Pacher and Steffens, 2009).
and porcine tissues. The results provided in this work show the Moreover, less is known about the physiologic effects of the
endocannanbinoids 2-AG and AEA are both substrates of human other EET-EA metabolites and whether they also exhibit dif-
CYP2J2 and that the predominant products, 14,15-EET-EA and ferential receptor-binding profiles and in vivo effects.
14,15-EET-G, are produced in heart microsomal systems of two
different mammalian species, lending credibility to the proposed 11,12-EET-G were found endogenously in rat kidney and
pathway in tissues. spleen tissues. These metabolites bound both CB1 and CB2
Previously, the epoxide metabolites of 2-AG, 14,15-, and
The qualitative product analysis of CYP2J2-AEA incuba- with greater affinity than 2-AG (Chen et al., 2008). Incubations
tions by LC-MS revealed four epoxide regioisomers at the 5,6-, with family 2 P450s, CYP2C8, CYP2C11, and CYP2C3 failed to
8,9-, 11,12-, and 14,15-olefin positions. The quantitative anal- produce these metabolites (Chen et al., 2008). Therefore, it was
ysis of 14,15-EET-EA exhibited fourfold more product when concluded that these epoxide metabolites do not arise from
compared with the rate of formation of 14,15-EET regioisomer epoxidation of 2-AG through the P450 pathway. Our modeling
from AA. Previously, the oxygenation of AEA into a single studies show that CYP2J2 contains a relatively large active site
hydroxy product, 20-HETE ethanolamide, as well as four that can accommodate 2-AG as compared with other family
epoxide regioisomers, 5,6-, 8,9-, 11,12-, and 14,15-EET-EA, was 2 P450s. Moreover, we show it is possible to directly oxygenate
demonstrated by several hepatic P450s, including CYP2D6, 2-AG by CYP2J2 to produce relatively high levels of both
CYP3A4, and CYP4F2 (Snider et al., 2010). However, this is 14,15- and 11,12-EET-G. Additionally, our results show that
the first report of the AEA metabolism by a P450 epoxygenase CYP2J2 produces only two epoxide metabolites, 14,15-EET-G

626
McDougle et al.
and 11,12-EET-G, similar to the two 2-AG epoxide metabolites showed modest inhibition of cyanide binding as shown in Fig. 6C
identified in vivo. Thus, these results provide an alternative and indicated that they either more easily egress from the active
pathway for the biosynthesis of 2-AG epoxides through the site or are bound as to not block cyanide binding. Similarly, the
direct enzymatic oxygenation by CYP2J2.
ratio of the fast to slow phase was significantly decreased in the
Due to the demonstrated beneficial functions of the presence of increasing concentrations of ebastine and only
endocannabinoids, there are several drugs in the market that modestly decreased in the presence of increasing concentrations
target the enzymes that attenuate the in vivo levels of these of AA, AEA, and 2-AG (Fig. 6, E–G). This effect was consistent
substrates (Cravatt and Lichtman, 2003). Previous inhibition and repeatable and not due to nonspecific fatty acid binding, as
of enzymes that inactivate AEA and 2-AG has shown that the evidenced by the lack of change induced by palmitate (Fig. 6H).
inhibition is incomplete (Blankman et al., 2007), indicating
The enzymatic turnover of CYP2J2 is 1 to 2 orders of mag-
that there may be alternative pathways of attenuation of nitude lower than xenobiotic metabolizing hepatic P450s, and it
these endocannabinoids. We demonstrate in this work that might be tempting to disregard the potential biologic significance
2-AG is converted to AA by CYP2J2 and CYP2C8. Subsequent of the CYP2J2 pathway based solely on these low enzymatic
control experiments confirmed that the cleavage proceeded turnover rates. However, the extrahepatic epoxygenases, in-
via an oxidative NADPH-dependent CYP2J2-CPR mechanism. cluding CYP2J2, function to regulate complex homeostatic func-
To our knowledge, these results provide the first evidence of tions within the body and exhibit significantly lower turnover
a P450-mediated endocannabinoid attenuation pathway for of the endogenous fatty acids (Westphal et al., 2011) in a highly
production of free AA for subsequent metabolism in the classic regulated manner. Thus, the formation of these metabolites
eicosanoid pathways (COX, LOX, and epoxygenase). To date, most likely represents a far more elegant mechanism for con-
there have been no reports of an oxidative ester cleavage mecha- trol of the many delicate homeostatic processes within organs,
nism of endogenous 2-AG. However, other P450 isozymes have such as the heart and kidneys. Therefore, similar to the steroid-
long been recognized as important sources of carbonyl pro- synthesizing P450s, the enzymatic expression levels rather
ducts through nonhydrolytic ester cleavage reactions (Guengerich, than the catalytic turnover are likely the prominent mecha-
1987; Peng et al., 1995).
nism for metabolic control in each respective organ.
The potential of CYP2J2 to produce its own substrate
In summary, we have demonstrated the ability of CYP2J2
through the cleavage of AA from PIP2 was also explored. Sev- to metabolize a diverse range of AA-containing substrates into
eral different conditions were examined, and it was concluded a number of unique metabolites that have previously been shown
the PIP2 is not a CYP2J2 substrate. Considering the tissue to regulate important physiologic effects in vivo. Additionally, we
distribution of cytochrome P450s, the role of a P450-mediated provide important evidence that CYP2J2 can directly attenuate
attenuation of 2-AG may constitute further examination in 2-AG levels through a NADPH-dependent oxidative cleavage
vivo.
mechanism, thereby producing free AA for subsequent metab-
The binding interactions of AA and its derivatives with the olism within the classic eicosanoid cascade. We further show
active site of CYP2J2 revealed relatively low spin state changes. that the formation of the metabolites of 2-AG and AEA is in
Thus, the use of the nanoscale lipid bilayers of NDs was agreement with the spectral binding studies and modeling.
essential for the characterization of AA, AEA, and 2-AG
binding in spectroscopic studies with minimal spectral scatter-
ing. In general, there are very few examples of human P450
titrations with lipid substrates in the literature, most likely due
to issues with solubility, scattering, and low spin state changes.
Interestingly, the few that have been published typically pres-
ent with a similar spectra to what is presented in this work
(Loughran et al., 2001). The ebastine, MS-PPOH, and 2-AG
Soret shifts present with classic type I binding modes (Fig. 5).
The binding of AA and AEA was less clear and difficult to
conclusively assign the classic type I and type II shifts due to the
low signal to noise ratio. However, all the binding shifts were
concentration dependent and reach saturation and are present
with clear peaks/troughs, lending credibility to the calculated
binding constants. Although the spin-state changes of the AA
and the derivatives are minor when compared with ebastine,
they do correspond to the relatively low catalytic turnover of
these substrates. Importantly, the relatively slow turnover rates
of CYP2J2 resemble the functions of the steroid-synthesizing
P450s that regulate complex homeostatic processes rather than
the very fast detoxification functions of liver P450s.
Acknowledgments
The authors thank Dr. Ilia Denisov and Dr. Matthew Edin for
helpful discussions. The authors thank Mr. William Arnold for editing
the paper. The authors greatly appreciate the contributions of Dr. Zhong
Li at the Metabolomics Laboratory of Roy J. Carver Biotechnology
Center, University of Illinois at Urbana-Champaign. The authors
thank Furong Sun and Dr. Kevin Tucker for continued support at
the School of Chemical Sciences mass spectrometry facility, University
of Illinois at Urbana-Champaign. The authors thank Prof. Stephen
Sligar for providing the gene encoding MSP1D1 and MSP1E3D1;
Prof. Robert Gennis for the use of stopped-flow equipment; and
Dr. Hanlin Ouyang for technical assistance. The authors thank
Prof. Mary Schuler, Brendon Colón, and Eryk Radziszewski for
training and assistance with the Molecular Operating Environment
modeling software. The authors thank Prof. Fan and Holly Pondenis
for assistance with CYP2J2 immunoblotting. The authors thank Prof.
Bunick for providing the homogenizer.
Authorship Contributions
Participated in research design: McDougle, Das.
Conducted experiments: McDougle, Kambalyal.
Performed data analysis: McDougle, Meling, Kambalyal, Das.
Wrote or contributed to the writing of the manuscript: McDougle,
Das.
The results for the CYP2J2-ND substrate-free and substrate-
bound stopped-flow cyanide-binding experiments are summa-
rized in Fig. 6 and Supplemental Table 1. For cyanide-binding
studies, we employed ebastine and showed that the rates of the
fast phase were significantly decreased with increasing ebas-
tine concentrations similar to other P450-substrate systems
(Denisov et al., 2007). Conversely, AA, AEA, and 2-AG only
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