Cloning, expression, and chromosomal localization of mouse liver bile acid CoA:amino acid N-acyltransferase

Abstract

A mouse liver lambda Zap XR cDNA library was screened using the coding region of human bile acid CoA:amino acid N-acyltransferase (BAT) cDNA as a probe. Ten positive clones were isolated and purified, two of which apparently possessed complete open reading frames for BAT based on sequence analysis of the ends of the cDNAs. One clone (mBAT#9) was selected for sequence analysis and characterization. mBAT#9 is 1869 basepairs in length and the full-length cDNA possesses a 189 basepair 5‘-nontranslated region, an open-reading frame of 1260 basepairs, and a 404 basepair 3‘-nontranslated region followed by a poly(A) tail. The open-reading frame codes for a 420 amino acid protein with a calculated molecular mass of 46,525 daltons. The structural gene for mBAT was mapped to mouse Chromosome 4. The amino acid sequence of mBAT is 69% identical and 84% similar to that of hBAT, and 86% identical and 95% similar to that of kan-1, a putative rat liver BAT. Enzymatically active mBAT was expressed in E. coli using the bacterial expression vector pKK233-2. Immunoblot analysis of expressed mBAT with rabbit anti-human BAT polyclonal antibodies detected a single protein with a molecular mass of approximately 45,000 daltons. Cytosol from cells transformed with mBAT#9/pKK233-2 possessed significant amounts of BAT-catalyzed conjugating activity with taurine as substrate but the expressed enzyme did not use glycine or fluoro-beta-alanine as substrates. The K(m) value for taurine was 1.9 mM +/- 0.1 mM in reactions with cholyl CoA as a cosubstrate. The specificity of mBAT for taurine as a substrate was confirmed by the demonstration, using HPLC-electrospray ionization mass spectrometry, that mouse gallbladder bile contained only taurine conjugates of bile acids. The identification of the types of amino acid conjugates of bile acids present in mouse bile had not been previously reported. These results indicate that a taurine-specific form of BAT has been cloned and expressed from mouse liver.