cDNA cloning and expression of the human hepatic bile acid-binding protein. A member of the monomeric reductase gene family.
In human liver, we previously identified one isoform of dihydrodiol dehydrogenase activity that expresses high affinity bile acid binding (HBAB) with minimal 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) activity for bile acids. This protein may assist in the rapid intracellular transport of bile acids from the sinusoidal to the canalicular pole of the cell. We now report the cDNA cloning and bacterial expression of this novel, multifunctional protein. A 1252-base pair HBAB cDNA was cloned from a HepG2 lambda GT11 library using a rat hepatic bile acid binder cDNA probe. Bacterial expressed recombinant HBAB oxidized racemic trans dihydrodiol benzene (0.455 mumol NADPH/mg/min) with minimal 3 alpha-HSD activity for bile acids (< 0.003 mumol NADPH/mg/min). Lithocholic acid and chenodeoxycholic acid dissociation constants as determined by displacement of the fluorescent probe, bis-1-anilino-8 sulfonate, were higher than those previously reported for the native protein (1 microM versus 10 nM). Significant amino acid sequence homology was found with the human chlordecone reductase, bovine prostaglandin F synthetase, and rat hepatic-3 alpha-HSD suggesting, that HBAB is also a member of the recently identified, monomeric oxidoreductase gene family. Future studies will define the physiologic significance of this novel, multifunctional protein in bile acid transport and xenobiotic metabolism.
Stolz
A
A
Division of Gastrointestinal and Liver Diseases, University of Southern California School of Medicine, Los Angeles 90033.
Hammond
L
L
Lou
H
H
Takikawa
H
H
Ronk
M
M
Shively
J E
JE
D015096:3-Hydroxysteroid Dehydrogenases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001647:Bile Acids and Salts / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D015342:DNA Probes; D004273:DNA, Neoplasm / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D006801:Humans; D006913:Hydroxysteroid Dehydrogenases; D007700:Kinetics; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D005810:Multigene Family; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods; D014176:Protein Biosynthesis; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)
D015096:3-Hydroxysteroid Dehydrogenases; D001647:Bile Acids and Salts; D002352:Carrier Proteins; D004247:DNA; D015342:DNA Probes; D004273:DNA, Neoplasm; D006913:Hydroxysteroid Dehydrogenases; D008562:Membrane Glycoproteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C037643:bile acid binding proteins; C435496:AKR1C2 protein, human; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)