Publication

Comparative characterization of a wild type and transmembrane domain-deleted fatty acid amide hydrolase: identification of the transmembrane domain as a site for oligomerization

Hilal Lashuel
1998
Article
Résumé

Fatty acid amide hydrolase (FAAH) is an integral membrane protein responsible for the hydrolysis of a number of primary and secondary fatty acid amides, including the neuromodulatory compounds anandamide and oleamide. Analysis of FAAH's primary sequence reveals the presence of a single predicted transmembrane domain at the extreme N-terminus of the enzyme. A mutant form of the rat FAAH protein lacking this N-terminal transmembrane domain (DeltaTM-FAAH) was generated and, like wild type FAAH (WT-FAAH), was found to be tightly associated with membranes when expressed in COS-7 cells. Recombinant forms of WT- and DeltaTM-FAAH expressed and purified from Escherichia coli exhibited essentially identical enzymatic properties which were also similar to those of the native enzyme from rat liver. Analysis of the oligomerization states of WT- and DeltaTM-FAAH by chemical cross-linking, sedimentation velocity analytical ultracentrifugation, and size exclusion chromatography indicated that both enzymes were oligomeric when membrane-bound and after solubilization. However, WT-FAAH consistently behaved as a larger oligomer than DeltaTM-FAAH. Additionally, SDS-PAGE analysis of the recombinant proteins identified the presence of SDS-resistant oligomers for WT-FAAH, but not for DeltaTM-FAAH. Self-association through FAAH's transmembrane domain was further demonstrated by a FAAH transmembrane domain-GST fusion protein which formed SDS-resistant dimers and large oligomeric assemblies in solution.

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