Biochemical and Proteomic Characterization of Recombinant Human α/β Hydrolase Domain 6

Sci Rep. 2019 Jan 29;9(1):890. doi: 10.1038/s41598-018-36633-4.


Human alpha/beta hydrolase domain 6 (hABHD6) is an enzyme that hydrolyzes 2-arachidonoylglycerol (2-AG), a potent agonist at both cannabinoid CB1 and CB2 receptors. In vivo modulation of ABHD6 expression has been shown to have potential therapeutic applications, making the enzyme a promising drug target. However, the lack of structural information on hABHD6 limits the discovery and design of selective inhibitors. We have performed E. coli expression, purification and activity profiling screening of different hABHD6 constructs and identified a truncated variant without N-terminal transmembrane (TM) domain, hΔ29-3-ABHD6, as the most promising protein for further characterization. The elimination of the TM domain did not affect 2-AG or fluorogenic arachidonoyl, 7-hydroxy-6-methoxy-4-methylcoumarin ester (AHMMCE) substrates hydrolysis, suggesting that the TM is not essential for enzyme catalytic activity. The hΔ29-3-ABHD6 variant was purified in a single step using Immobilized Metal Affinity Chromatography (IMAC), in-solution trypsin digested, and proteomically characterized by Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). The N-terminal peptide without methionine was identified indicating on a post-translational modification of the recombinant protein. The mechanism of inhibition of hABHD6 with AM6701 and WWL70 covalent probes was elucidated based on MS analysis of trypsin digested hABHD6 following the Ligand Assisted Protein Structure (LAPS) approach. We identified the carbamylated peptides containing catalytic serine (Ser148) suggesting a selective carbamylation of the enzyme by AM6701 or WWL70 and confirming an essential role of this residue in catalysis. The ability to produce substantial quantities of functional, pure hABHD6 will aid in the downstream structural characterization, and development of potent, selective inhibitors.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Chromatography, Affinity
  • Enzyme Activation
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Expression
  • Humans
  • Monoacylglycerol Lipases / chemistry*
  • Monoacylglycerol Lipases / genetics
  • Monoacylglycerol Lipases / isolation & purification
  • Monoacylglycerol Lipases / metabolism*
  • Recombinant Fusion Proteins
  • Recombinant Proteins*
  • Solubility
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Substrate Specificity


  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • ABHD6 protein, human
  • Monoacylglycerol Lipases