Molecular characterization of human and mouse fatty acid amide hydrolases

Abstract

Recently, we reported the isolation, cloning, and expression of a rat enzyme, fatty acid amide hydrolase (FAAH), that degrades bioactive fatty acid amides like oleamide and anandamide to their corresponding acids, thereby serving to terminate the signaling functions of these molecules. Here, we report the molecular characterization of both a mouse and a human FAAH and compare these enzymes to the rat FAAH. The enzymes are well conserved in primary structure, with the mouse and rat FAAHs sharing 91% amino acid identity and the human FAAH sharing 82% and 84% identity with the rat FAAH and mouse FAAH, respectively. In addition, the expressed human and rat FAAHs behave biochemically as membrane proteins of comparable molecular size and show similar, but distinguishable, enzymological properties. The identification of highly homologous FAAH proteins in rat, mouse, and human supports a general role for the fatty acid amides in mammalian biology.

Figure 1

Comparison of deduced amino acid sequences from human, mouse, and rat FAAH cDNAs. Sequence identity shared by at least two of the three FAAH proteins is shaded.

Figure 2

Expression and comparison of human and rat FAAHs in COS-7 cells. (A) FAAH activity as measured by oleamide hydrolysis was present in COS-7 cells transiently transfected with either rat FAAH or human FAAH (lanes 2 and 3, respectively) but not in untransfected COS-7 cells (lane 1). (B) Western blot analysis of COS-7 cell extracts with polyclonal anti-FAAH antibodies (see text) identified a single immunoreactive 60- to 65-kDa protein present in COS-7 cells transfected with either rat or human FAAH (lanes 2 and 3, respectively) but not in untransfected COS-7 cells (lane 1). (C and D) Association of FAAH activity and immunoreactivity (lanes 1 and 2, rat FAAH; lanes 3 and 4, human FAAH) with COS-7 cell membranes upon cellular fractionation (supernatant, soluble protein fraction; pellet, membrane protein fraction).

Figure 3

Immunofluorescence microscopy of COS-7 cells transfected with either human FAAH (A and B, cell phase; A′ and B′, anti-FAAH antibody staining) or rat FAAH (C and D, cell phase; C′ and D′, anti-FAAH antibody staining). Note the fibrous, rod-like staining pattern of the human FAAH (arrow) and, in contrast, the more diffuse staining pattern of the rat FAAH. No significant immunostaining was observed in untransfected COS-7 cells, and both the rat and the human FAAH immunoreactivities were effectively competed away by excess peptide antigen.

Figure 4

Southern (A) and Northern (B) blot analyses of human FAAH. (A) Southern blot of human genomic DNA digested with several restriction enzymes (4 μg of digested DNA per lane); restriction enzymes used are indicated at the top of each lane. (B) Northern blot of human mRNA from different human tissues (CLONTECH; 2 μg of mRNA per lane).