Molecular cloning and expression of rat liver bile acid CoA ligase

Bile acid CoA ligase (B22222222294) is responsible for catalyzing the first step in the conjugation of bile acids with amino acids. Sequencing of putative rat liver BAL cDNAs identified a cDNA (rBAL-1) possessing a 51 nucleotide 5′-untranslated region, an open reading frame of 2,070 bases encoding a 690 aa protein with a molecular mass of 75,960 Da, and a 138 nucleotide 3′-nontranslated region followed by a poly(A) tail. Identity of the cDNA was established by: 1) the rBAL-1 open reading frame encoded peptides obtained by chemical sequencing of the purified rBAL protein; 2) expressed rBAL-1 protein comigrated with purified rBAL during SDS-polyacrylamide gel electrophoresis; and 3) rBAL-1 expressed in insect Sf9 cells had enzymatic properties that were comparable to the enzyme isolated from rat liver. Evidence for a relationship between fatty acid and bile acid metabolism is suggested by specific inhibition of rBAL-1 by cis-unsaturated fatty acids and its high homology to a human very long chain fatty acid CoA ligase.In summary, these results indicate that the cDNA for rat liver BAL has been isolated and expression of the rBAL cDNA in insect Sf9 cells results in a catalytically active enzyme capable of utilizing several different bile acids as substrates. Bile acid CoA ligase (B22222222294) is responsible for catalyzing the first step in the conjugation of bile acids with amino acids. Sequencing of putative rat liver BAL cDNAs identified a cDNA (rBAL-1) possessing a 51 nucleotide 5′-untranslated region, an open reading frame of 2,070 bases encoding a 690 aa protein with a molecular mass of 75,960 Da, and a 138 nucleotide 3′-nontranslated region followed by a poly(A) tail. Identity of the cDNA was established by: 1) the rBAL-1 open reading frame encoded peptides obtained by chemical sequencing of the purified rBAL protein; 2) expressed rBAL-1 protein comigrated with purified rBAL during SDS-polyacrylamide gel electrophoresis; and 3) rBAL-1 expressed in insect Sf9 cells had enzymatic properties that were comparable to the enzyme isolated from rat liver. Evidence for a relationship between fatty acid and bile acid metabolism is suggested by specific inhibition of rBAL-1 by cis-unsaturated fatty acids and its high homology to a human very long chain fatty acid CoA ligase. In summary, these results indicate that the cDNA for rat liver BAL has been isolated and expression of the rBAL cDNA in insect Sf9 cells results in a catalytically active enzyme capable of utilizing several different bile acids as substrates. Bile acids are one of a group of organic acids that are converted to N-acyl amino acid conjugates. They are the principal metabolites of cholesterol and have important detergent properties in the hepatobiliary system and in the lumen of the upper small intestine (1Hofmann A.F. Enterohepatic circulation of bile acids.in: Schultz S.G. Handbook of Physiology - The Gastrointestinal System III. American Physiological Society, Bethesda, MD1989: 567-596Google Scholar). The presence of the amino acid moiety lowers the pKa of the bile acid, thus preventing precipitation at the acid pH of the upper gut (2Small D.M. The physical chemistry of cholanic acids.in: Nair P.P. Kritchevsky D. In The Bile Acids. Chemistry, Physiology and Metabolism. Vol. 1. Chemistry, Plenum Press, New York1971: 249-355Google Scholar). Conjugation of bile acids with the amino acids glycine and taurine is a two-step process involving the successive action of the enzymes, bile acid CoA ligase (BAL), and bile acid CoA:amino acid N-acyltransferase (BAT). Our laboratories have previously purified BAT activity from human liver (3Johnson M. Barnes S. Kwakye J. Diasio R.B. Purification of human liver cholyl CoA: amino acid N-acyltransferase.J. Biol. Chem. 1991; 262: 10227-10233Google Scholar), and cloned a cDNA encoding hBAT from a human liver cDNA library (4Falany C.N. Johnson M. Barnes S. Diasio R.B. Molecular cloning and expression of human liver bile acid CoA: amino acid:N-acyltransferase.J. Biol. Chem. 1994; 266: 19375-19379Google Scholar). hBAT expressed in both bacterial and COS cells was demonstrated to utilize both glycine and taurine as substrates (4Falany C.N. Johnson M. Barnes S. Diasio R.B. Molecular cloning and expression of human liver bile acid CoA: amino acid:N-acyltransferase.J. Biol. Chem. 1994; 266: 19375-19379Google Scholar). Although BAL activity was identified and enzymatically characterized over 40 years ago (5Bremer J. A method for the estimation of the taurine content and its conjugation with cholic acid in rat liver.Acta Chem. Scand. 1955; 9: 683-688Google Scholar), purification of membrane-bound BAL activity has been a major challenge. Partial purification and characterization of BAL activity from guinea pig and porcine microsomes has been reported (6Vessey D.A. Benfatto A.M. Kempner E.S. Bile acid: CoASH ligases from guinea pig and porcine liver microsomes. Purification and characterization.J. Biol. Chem. 1987; 262: 5360-5365Google Scholar); however, the multiplicity and properties of the BAL proteins were not determined. Recently, our laboratory has described the purification of BAL activity from rat liver (7Wheeler J.B. Barnes S. Purification and characterization of bile acid CoA synthase from rat liver.Arch. Biochem. Biophys. 1997; 348: 15-24Google Scholar). Monoclonal anti-rBAL antibodies were generated and used to isolate rBAL so that partial amino acid sequence information could be obtained by chemical sequencing methods. The sequence of several peptides was utilized to design degenerate oligonucleotide probes for the cloning of rBAL cDNAs from a rat liver λZAP II cDNA library. The full-length rBAL-1 cDNA was expressed in insect Sf 9 cells using a baculovirus expression vector. Verification that the expressed protein was rBAL involved identification of rBAL peptide sequences in the translation of the cDNA, characterization of the enzymatic activity of expressed rBAL, and identification of the formation of bile acid CoA esters using HPLC and MALDI-TOF mass spectrometry. Sequence analysis and substrate inhibition studies indicate that rBAL is a member of the fatty acid CoA synthetase/fatty acid transport protein (FATP) gene family (8Hirsch D. Stahl A. Lodish H. A family of fatty acid transporters conserved from mycobacterium to man.Proc. Natl. Acad. Sci. USA. 1998; 95: 8625-8629Google Scholar). TRIzol reagent was purchased from Gibco BRL (Rockville, MD). Original TA Cloning and PCR optimizer kits were obtained from Invitrogen (Carlsbad, CA). The Wizard Plus Miniprep DNA purification system was from Promega (Madison, WI). The TaqStart Antibody was purchased from Clontech (Palo Alto, CA). Rat liver λZAP II cDNA library, the XL-1 MRF′ cells, and cells were from CA). reagent was obtained from and Plus kits were purchased from II gel purification was from CA). acid cholic acid acid acid and A were from acid and acid were from of was from acid was from were of reagent and were from of the rBAL amino acid sequence were obtained from and of rBAL (7Wheeler J.B. Barnes S. Purification and characterization of bile acid CoA synthase from rat liver.Arch. Biochem. Biophys. 1997; 348: 15-24Google Scholar). from rBAL were by sequencing of rBAL peptides by was by The amino acid sequence of one of the isolated peptides was of rBAL generated by a sequence was by to a and with The was from the and to peptide sequencing from the The amino acid sequence obtained from peptide was The amino acids the were at that In to a of the rBAL cDNA, rat liver was isolated using a TRIzol chain was using a A degenerate oligonucleotide was using the amino acid sequence from the was used in with to of the first was to PCR and were at and the first the was The was to for to for to for and to for The was for at major PCR and were cDNA were gel purified and the using a TA cloning The was and using for and for DNA was isolated from several using a Wizard Plus Miniprep DNA purification The purified were using an with and sequencing DNA Sequence The PCR was to the peptide The DNA was with using an to a specific activity of DNA and used as a to a λZAP II rat liver cDNA library high of the λZAP II cDNA library was as described previously C.N. H. Barnes S. Cloning and expression of liver bile acid CoA: acid N-acyltransferase.J. 1997; Scholar). XL-1 MRF′ cells were with of the λZAP library to The were to to the for at and with and in with The were at to with for The of the was with and the were The were in for and with for The were and to to the DNA to the were from from the λZAP II library were of the was with of the DNA at in and The were at for using and followed by at with The were and to at with an that were were purified by and a could be of DNA sequences were in using as described by DNA was isolated using a Wizard DNA were using the sequencing using The were the were and to were and using sequence analysis was isolated from several of an rat using of from different small intestine and were with of gel to for and in a gel in Molecular Press, New Scholar). The was to a and the was with in a as a the rBAL cDNA was isolated from the by and purified from an The rBAL-1 cDNA was with using an to a specific activity of was in at for The was and in with for at followed by in and for at The was and to at with an A was a amino acid of rBAL of with of full-length rBAL expressed in The rBAL was generated by the obtained from to a protein with an amino The was expressed in cells and purified by The protein was purified by SDS-polyacrylamide gel and using a and the of the protein by peptide using MALDI-TOF mass protein was with and at several the of a New The was at with of in the was and for by analysis and characterization of the human and Biochem. Biol. Scholar). analysis of rBAL expression in rat microsomes were from of a rat and of the proteins were by SDS-polyacrylamide gel The proteins were to a and the was with The was with anti-rBAL at a of for was used as the and of proteins was using the System active rBAL was expressed in Sf 9 insect cells using the baculovirus expression system CA). The rBAL-1 cDNA was isolated from by and and the and in the to enzyme the to the protein with a the amino of the the were cells and the isolated with a Wizard purification The were cells for of the rBAL sequence the cells were 40 and DNA were for of the DNA as described by isolate the baculovirus rBAL, Sf 9 cells in were at a of 9 in a in of II and and to at for in II was with reagent in II with and for at The Sf 9 cells were with II and with the with II The cells were at for the was and with II The cells were at for and the was and at for to cells and Sf 9 cells in in were with the and at for The Sf 9 cells were by at for were in in a and at for at The was and at at for The was in and were using reagent with as a The rBAL protein was purified by The BAL enzyme was from the of and Purification and characterization of bile amino acid: N-acyltransferase from rat Biol. Chem. Scholar). The BAL and in pH The was at for the rBAL enzyme was in a and the was for The was by the of of In the acid was the enzyme The were with of and the of in the was by spectrometry. the of expressed rBAL for the in the was from to at CoA and of and Sf 9 microsomes expressed rBAL were with to between and of in the In to the of bile acids and fatty acids to rBAL the bile acids and fatty acids were in The of bile acid fatty acid was in the and The was and at for The were and isolated as described the generated by rBAL from and were as described previously by the of the acid of the was to a HPLC with a of The of of A pH and pH The were and the of the was at A at of the The were and by a mass over the to were in both the and The partial rBAL cDNA was obtained by using rat liver as a A degenerate was to of the sequence of a peptide from of The degenerate rBAL and a were used as in the PCR major PCR and were DNA were and in encoded the amino acids the sequence of the purified peptide used to design the degenerate oligonucleotide The nucleotide PCR encoded the sequence of a peptide from purified rBAL The cDNA was used as a to a II rat liver cDNA library to isolate the full-length rBAL cDNA were isolated from the rat liver λZAP II cDNA library using high enzyme analysis and DNA sequencing that the were The nucleotide sequence and translation of the is in 1. was to rBAL-1 for at the and 9 to the in The was to rBAL-1 the The was for sequence analysis and expression The rBAL-1 cDNA 51 of sequence and 138 of 3′-nontranslated sequence with a poly(A) tail. A putative is bases from the of the poly(A) tail. rBAL-1 a protein of 690 amino acids with a molecular mass of The translation of rBAL-1 the from sequencing of peptides obtained from purified rat liver rBAL The one amino acid in the peptide sequences is encoded as a in the peptide generated by is in the translation of rBAL-1 a analysis of isolated from several of an rat was used to the and of with the rBAL that the rBAL cDNA one in in liver is in to the rBAL-1 rBAL-1 was not in isolated from however, a was for rat small intestine A was to a amino acid of the rBAL The was used in analysis of rBAL in microsomes from of a that rBAL was in liver microsomes not in microsomes from A protein with a molecular mass liver rBAL was in microsomes rBAL was in analysis of rBAL was in rat liver In to the enzymatic activity of rBAL, the rBAL-1 cDNA was expressed in Sf 9 cells using rBAL-1 was expressed in both and with an amino from Sf 9 cells with both the and of rBAL activity with as a substrate The microsomes the rBAL protein specific with the In Sf 9 insect microsomes not of the rBAL by HPLC with and the formation of of the putative by that had a molecular of in the and a molecular of in the for that expressed rBAL had generated The of the activity of the expressed of rBAL was determined. rBAL activity was at of and of enzyme generated of for rBAL expressed in Sf 9 microsomes and for (7Wheeler J.B. Barnes S. Purification and characterization of bile acid CoA synthase from rat liver.Arch. Biochem. Biophys. 1997; 348: 15-24Google have reported a of for using purified rat liver are the bile acid that from to (2Small D.M. The physical chemistry of cholanic acids.in: Nair P.P. Kritchevsky D. In The Bile Acids. Chemistry, Physiology and Metabolism. Vol. 1. Chemistry, Plenum Press, New York1971: 249-355Google Scholar). the of the of the translation of rBAL with the sequences of very and very rBAL is and to proteins cloned from rat A. Molecular cloning of cDNA encoding rat very Biol. Chem. Scholar), J. H. S. cDNA cloning and of a very 1998; Scholar), and human S. S. D. S. and to fatty acid Biophys. and of human fatty ligase 1998; Scholar). rBAL is to a protein J. H. S. A of the and fatty acid protein with a expression Biophys. 1998; Scholar). homology is with a human as (8Hirsch D. Stahl A. Lodish H. A family of fatty acid transporters conserved from mycobacterium to man.Proc. Natl. Acad. Sci. USA. 1998; 95: 8625-8629Google Scholar). has been reported to cholyl CoA activity S. S. D. The human of very is Biol. Chem. and to CoA esters of several bile acids J. D.A. of of the very family in bile acid and Biol. Chem. Scholar). The high of sequence between rBAL and these CoA proteins the of rBAL in the CoA gene family (8Hirsch D. Stahl A. Lodish H. A family of fatty acid transporters conserved from mycobacterium to man.Proc. Natl. Acad. Sci. USA. 1998; 95: 8625-8629Google Scholar). in rBAL homology with the CoA gene family (8Hirsch D. Stahl A. Lodish H. A family of fatty acid transporters conserved from mycobacterium to man.Proc. Natl. Acad. Sci. USA. 1998; 95: 8625-8629Google and at one member of family is capable of cholic acid CoA esters S. S. D. The human of very is Biol. Chem. Scholar). S. of cholic A ligase and A ligase in rat have reported that the bile acids and acid were substrates for rBAL and J. Purification and of Rat Bile CoA at has identified and as substrates of rBAL purified from rat liver. (6Vessey D.A. Benfatto A.M. Kempner E.S. Bile acid: CoASH ligases from guinea pig and porcine liver microsomes. Purification and characterization.J. Biol. Chem. 1987; 262: 5360-5365Google activity and with purified guinea pig the of several bile acids and fatty acids to the ligase activity of baculovirus expressed rBAL was that the ligase activity of rBAL was by and by and was a of rBAL activity not of rBAL activity by bile acid acid acid acid acid activity was using microsomes from insect Sf 9 cells expressed rBAL and were in the presence of of bile acids as described in and The results are as of and the of in a rBAL activity was using microsomes from insect Sf 9 cells expressed rBAL and were in the presence of of bile acids as described in and The results are as of and the of S. S. D. The human of very is Biol. Chem. have reported that human as is capable of cholyl fatty acids could and rBAL several and fatty acids were for to ligase that the fatty acid acid and acid not rBAL the fatty acid and acid and inhibition of rBAL In the of acid, acid not that the presence of the in the 9 is important for inhibition of rBAL of rBAL activity by fatty activity was using microsomes from insect Sf 9 cells expressed rBAL and were in the presence of of fatty acids as described in and The results are as of and the of that was in a rBAL activity was using microsomes from insect Sf 9 cells expressed rBAL and were in the presence of of fatty acids as described in and The results are as of and the of that was In to the of the of baculovirus expressed rBAL, used in the of proteins ligase rBAL and of were for the of enzyme activity with A of were used to that were utilized for of rBAL for its purification from rat liver (7Wheeler J.B. Barnes S. Purification and characterization of bile acid CoA synthase from rat liver.Arch. Biochem. Biophys. 1997; 348: 15-24Google Scholar). rBAL activity with inhibition of activity at protein In had rBAL activity to at of rBAL activity as with inhibition by the cloning and expression of BAL from rat the enzyme responsible for the first step in the conjugation of bile acids with amino acids. The formation of bile acid CoA the bile acids. is for conjugation with glycine Our laboratories have previously cloned and expressed bile acid CoA:amino acid N-acyltransferase the enzyme responsible for the step in the of bile acid amino acid from both human (4Falany C.N. Johnson M. Barnes S. Diasio R.B. Molecular cloning and expression of human liver bile acid CoA: amino acid:N-acyltransferase.J. Biol. Chem. 1994; 266: 19375-19379Google and liver C.N. H. Barnes S. Cloning and expression of liver bile acid CoA: acid N-acyltransferase.J. 1997; Scholar). The cloning of rBAL for the of the and of these important enzyme at the molecular The of the rBAL cDNA was by several The rBAL cDNA was isolated from a rat liver cDNA library using a cDNA generated by with rat liver as the to the partial amino acid sequence of the previously purified rBAL enzyme (7Wheeler J.B. Barnes S. Purification and characterization of bile acid CoA synthase from rat liver.Arch. Biochem. Biophys. 1997; 348: 15-24Google Scholar). peptide sequences obtained from the analysis of the purified enzyme were identified in the translation of the rBAL cDNA the relationship between the purified and cloned rBAL high of conjugation activity were in Sf 9 insect cells with a the rBAL cDNA, conjugation activity was in Sf 9 In the amino acid sequence encoded by rBAL sequence to fatty acid CoA that rBAL is a member of gene family H. M. J. D. of the analysis of amino acid sequence of and J. S. very and human characterization and relationship to fatty acid transport Acids. Scholar). rBAL was previously purified from rat liver microsomes and the of rBAL for enzymatic activity was characterized (7Wheeler J.B. Barnes S. Purification and characterization of bile acid CoA synthase from rat liver.Arch. Biochem. Biophys. 1997; 348: 15-24Google Scholar). to active full-length rBAL in in expression in to the of rBAL rBAL was expressed in Sf 9 insect cells using a vector. rBAL was expressed both in its and with an amino Sf 9 microsomes not of however, microsomes from Sf 9 cells rBAL high of conjugation The of the rBAL activity for expressed in Sf 9 microsomes was that reported with the purified enzyme The enzyme purified from rat liver was with the expressed rBAL enzyme from Sf 9 cells was as a The of different could for the in the and M. D.A. of the of for bile acid: CoA J. 1994; have reported that detergent with analysis of guinea pig and porcine BAL the in were the of acids of rBAL a region that have a in the of the protein with the The presence of the at the amino of the expressed protein the of expression of rBAL in Sf 9 by with the of the the of activity for in microsomes was that of the that the the active of the of the sequence of rBAL with and proteins from and human demonstrated a high of rBAL conserved sequence identified in the CoA gene The first is the that is conserved and chain H. M. J. D. of the analysis of amino acid sequence of and Scholar). The is a conserved in the acid transport protein gene family at amino acids J. S. very and human characterization and relationship to fatty acid transport Acids. Scholar). the that rBAL is a member of the fatty acid CoA gene rBAL high of bile acid conjugation however, the translation of the rBAL cDNA has the to a expressed in liver J. H. S. A of the and fatty acid protein with a expression Biophys. 1998; Scholar). activity and substrates for protein have not been protein with a high of to rBAL is human (8Hirsch D. Stahl A. Lodish H. A family of fatty acid transporters conserved from mycobacterium to man.Proc. Natl. Acad. Sci. USA. 1998; 95: 8625-8629Google Scholar). expressed in cells, had of activity several long chain fatty acids and was capable of cholyl CoA S. S. D. The human of very is Biol. Chem. Scholar). has been reported to CoA esters of several and bile acids as as J. D.A. of of the very family in bile acid and Biol. Chem. Scholar). J. D.A. of of the very family in bile acid and Biol. Chem. have suggested that be to as results indicate that rBAL is the rat of is expressed in the of human liver as be of a bile acid CoA ligase S. S. M. A cDNA cloning and characterization of a enzymatically active Scholar). fatty acid CoA ligase activity in microsomes isolated from rat however, cholic acid CoA ligase activity was in liver microsomes of liver cholic acid A ligase Evidence that are responsible for cholic acid and fatty acid Biol. Chem. Scholar). The that human bile acid CoA protein was in the protein with a different molecular mass during SDS-polyacrylamide gel The of protein is not and is of in human has not been in the amino acid sequences between the and rBAL are in the could sequences to for the protein J. S. very and human characterization and relationship to fatty acid transport Acids. Scholar). rBAL, and the putative identified in the and rat proteins A. Molecular cloning of cDNA encoding rat very Biol. Chem. J. H. S. cDNA cloning and of a very 1998; Scholar). S. S. D. The human of very is Biol. Chem. reported that is expressed in the and bile N-acyltransferase in rat Biol. Chem. reported that the enzyme in the bile acid is in and is not expressed in one BAT gene has been identified in human C.N. H. Barnes S. Cloning and expression of liver bile acid CoA: acid N-acyltransferase.J. 1997; and (4Falany C.N. Johnson M. Barnes S. Diasio R.B. Molecular cloning and expression of human liver bile acid CoA: amino acid:N-acyltransferase.J. Biol. Chem. 1994; 266: 19375-19379Google that the bile acid CoA esters have to for taurine glycine conjugation a bile acid CoA ligase activity is identified in The between the proteins and rBAL a in of the proteins have not been for activity bile and has been reported to bile acid CoA esters as as long chain fatty acid CoA esters S. S. D. The human of very is Biol. Chem. J. D.A. of of the very family in bile acid and Biol. Chem. Scholar). is capable of several bile acids (7Wheeler J.B. Barnes S. Purification and characterization of bile acid CoA synthase from rat liver.Arch. Biochem. Biophys. 1997; 348: 15-24Google Scholar), was by and as as by a bile acid that is to a CoA as a step in its to bile acids H. J. J. R.B. A with and a specific of J. Scholar). acid, a bile acid, not rBAL, with the that is not to a CoA in M. A. of chain bile acid and A formation of acids and by human and rat liver Scholar). results indicate that rBAL is capable of a of different bile acid CoA esters both and conjugates. was reported to both long chain fatty acid and bile acid CoA esters S. S. D. The human of very is Biol. Chem. J. D.A. of of the very family in bile acid and Biol. Chem. Scholar), fatty acids were as substrates for rBAL fatty several fatty acids were as of rBAL rBAL was not by high of to fatty acids that fatty acids are not substrates for In rBAL was by and of a in a inhibition was with acid has the in the are with the results reported by (6Vessey D.A. Benfatto A.M. Kempner E.S. Bile acid: CoASH ligases from guinea pig and porcine liver microsomes. Purification and characterization.J. Biol. Chem. 1987; 262: 5360-5365Google Scholar), that guinea pig BAL was by analysis and with acid that CoA and CoA were not and are not substrates of with the of is the for for CoA studies the of long chain A from rat liver Biol. Chem. of A from rat liver microsomes. of the of in Scholar), CoA was by analysis that acid rBAL activity in a that and acids with the active as as the of the protein not Bile acid is important for the and properties of bile acids. The and characterization of both involved in bile acid for a characterization of Recently, with have been with an to bile acid A.F. of bile acid a of bile acid Scholar). is that the of bile acid in these is to in the expression of BAL BAT The cloning and characterization of BAL for a of its in the of bile acid and fatty acid CoA esters and the between these was in by of The rBAL sequence has been to and has been acid and acid were from of The mass was purchased by from a and from of the has been in by a to the bile acid CoA ligase bile acid CoA:amino acid N-acyltransferase cholic acid acid A acid fatty acid transport protein acid acid