Full mass spectrometric characterization of human monoacylglycerol lipase generated by large-scale expression and single-step purification.
The serine hydrolase monoacylglycerol lipase (MGL) modulates endocannabinoid signaling in vivo by inactivating 2-arachidonoylglycerol (2-AG), the main endogenous agonist for central CB1 and peripheral CB2 cannabinoid receptors. To characterize this key endocannabinoid enzyme by mass spectrometry-based proteomics, we first overexpressed recombinant hexa-histidine-tagged human MGL (hMGL) in Escherichia coli and purified it in a single chromatographic step with high yield (approximately 30 mg/L). With 2-AG as substrate, hMGL displayed an apparent V max of 25 micromol/(microg min) and K m of 19.7 microM, an affinity for 2-AG similar to that of native rat-brain MGL (rMGL) (Km=33.6 microM). hMGL also demonstrated a comparable affinity (Km approximately 8-9 microM) for the novel fluorogenic substrate, arachidonoyl, 7-hydroxy-6-methoxy-4-methylcoumarin ester (AHMMCE), in a sensitive, high-throughput fluorometric MGL assay. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) unequivocably demonstrated the mass (34,126 Da) and purity of this hMGL preparation. After in-solution tryptic digestion, hMGL full proteomic characterization was carried out, which showed (1) an absence of intramolecular disulfide bridges in the functional, recombinant enzyme and (2) the post-translational removal of the enzyme's N-terminal methionine. Availability of sufficient quantities of pure, well-characterized hMGL will enable further molecular and structural profiling of this key endocannabinoid-system enzyme.
Zvonok
Nikolai
N
Center for Drug Discovery, Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, USA.
Williams
John
J
Johnston
Meghan
M
Pandarinathan
Lakshmipathi
L
Janero
David R
DR
Li
Jing
J
Krishnan
Srinivasan C
SC
Makriyannis
Alexandros
A
D000595:Amino Acid Sequence; D000818:Animals; D063385:Cannabinoid Receptor Modulators / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D008994:Monoacylglycerol Lipases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D063385:Cannabinoid Receptor Modulators; D008994:Monoacylglycerol Lipases