Identification and recombinant expression of anandamide hydrolyzing enzyme from Dictyostelium discoideum

Dhamodharan Neelamegan,Ian C Schoenhofen,James C Richards,Andrew D Cox

doi:10.1186/1471-2180-12-124

Dhamodharan Neelamegan, Ian C Schoenhofen + Show 2 more

Open Access

https://doi.org/10.1186/1471-2180-12-124

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Journal: BMC microbiology	Publication Date: Jun 25, 2012
Citations: 34	License type: cc-by

Affiliation: Institute for Biological Sciences

Abstract

BackgroundAnandamide (Arachidonoyl ethanolamide) is a potent bioactive lipid studied extensively in humans, which regulates several neurobehavioral processes including pain, feeding and memory. Bioactivity is terminated when hydrolyzed into free arachidonic acid and ethanolamine by the enzyme fatty acid amide hydrolase (FAAH). In this study we report the identification of a FAAH homolog from Dictyostelium discoideum and its function to hydrolyze anandamide.ResultsA putative FAAH DNA sequence coding for a conserved amidase signature motif was identified in the Dictyostelium genome database and the corresponding cDNA was isolated and expressed as an epitope tagged fusion protein in either E.coli or Dictyostelium. Wild type Dictyostelium cells express FAAH throughout their development life cycle and the protein was found to be predominantly membrane associated. Production of recombinant HIS tagged FAAH protein was not supported in E.coli host, but homologous Dictyostelium host was able to produce the same successfully. Recombinant FAAH protein isolated from Dictyostelium was shown to hydrolyze anandamide and related synthetic fatty acid amide substrates.ConclusionsThis study describes the first identification and characterisation of an anandamide hydrolyzing enzyme from Dictyostelium discoideum, suggesting the potential of Dictyostelium as a simple eukaryotic model system for studying mechanisms of action of any FAAH inhibitors as drug targets.

Highlights

Anandamide (Arachidonoyl ethanolamide) is a potent bioactive lipid studied extensively in humans, which regulates several neurobehavioral processes including pain, feeding and memory
The consensus amidase signature sequence has a conserved GSS(G/A/S)G motif shared among many proteins in the amidase class including glutamyl-t-RNA amidotransferase subunit A of Methanococcus jannaschii and fatty acid amide hydrolase (FAAH) from human, porcine, rat, Arabidopsis and Dictyostelium
FAAH full length protein amino acid sequence from Dictyostelium lacks significant identity when compared to FAAH from human (20%), porcine (20%), rat (20%), and Arabidopsis (32%) (Figure 1), but identity across the amidase signature sequence increased to 40%, 38%, 38%, and 50%, for the human, procine, rat, and Arabidopsis FAAH homologs

Summary

Introduction

Anandamide (Arachidonoyl ethanolamide) is a potent bioactive lipid studied extensively in humans, which regulates several neurobehavioral processes including pain, feeding and memory. Bioactivity is terminated when hydrolyzed into free arachidonic acid and ethanolamine by the enzyme fatty acid amide hydrolase (FAAH). In this study we report the identification of a FAAH homolog from Dictyostelium discoideum and its function to hydrolyze anandamide. Action of anandamide is terminated by the enzyme fatty acid amide hydrolase (FAAH) [3]. Identification of FAAH suggests that regulation of NAE signalling could occur in Dictyostelium and Dictyostelium could be utilised as a simple eukaryotic model to study NAE functions in parallel with mammalian systems. The identification of any new drug target enzyme such as FAAH or any drug processing mechanisms in Dictyostelium suggests further potential for the use of Dictyostelium in human biomedical research. Dictyostelium offers an attractive system to study such processes by gene manipulation studies because of its small 34 Mbp haploid genome harbouring many homologous genes found in higher eukaryotes [18]

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