Cloning of two human liver bilirubin UDP-glucuronosyltransferase cDNAs with expression in COS-1 cells.
We report the isolation and characterization of two human liver cDNA clones, HUG-Br1 and HUG-Br2; each encodes a UDP-glucuronosyltransferase enzyme which glucuronidates bilirubin IX alpha to form both the IX alpha C8 and IX alpha C12 monoconjugates and a diconjugate. HUG-Br1 cDNA (2351 base pairs) and HUG-Br2 cDNA (2368 base pairs) encode proteins with 533 and 534 amino acid residues, respectively, with a typical membrane-insertion signal peptide, membrane-spanning domain, and 3 or 5 potential asparagine-linked glycosylation sites. At the nucleic acid and deduced amino acid sequence levels the two clones are 82% similar overall, 66% similar in the amino termini, and identical after codon 287, thus encoding proteins with the same carboxyl terminus. The mRNA encoding HUG-Br1 is of high abundance, and the one encoding HUG-Br2 is of low abundance; both are 2.6 kilobases in length. Both messages (2.6 kilobases) were present in the explanted liver of a Type I Crigler-Najjar patient, although the level for that of HUG-Br1 was reduced 4.5-fold. Northern blot analysis of poly(A)+ RNA isolated from the liver of an untreated and a phenobarbital-treated Erythrocebus patas monkey with 5'-specific probes for each clone indicated that the HUG-Br2-encoded message is induced two fold, but that for HUG-Br1 is not. These data indicate that bilirubin is glucuronidated by at least two different proteins, most likely present in very different amounts. These cDNAs which encode functional bilirubin UDP-glucuronosyltransferases will allow the isolation of an appropriate gene to develop a gene therapy model for patients which have the totally deficient trait.
Ritter
J K
JK
Section on Genetic Disorders of Drug Metabolism, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Crawford
J M
JM
Owens
I S
IS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001663:Bilirubin / Q000235:genetics* / Q000378:metabolism; D015152:Blotting, Northern; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004901:Erythrocebus patas; D015870:Gene Expression; D014453:Glucuronosyltransferase / Q000235:genetics*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D008099:Liver / Q000201:enzymology*; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection
D007527:Isoenzymes; D012333:RNA, Messenger; D004247:DNA; D014453:Glucuronosyltransferase; D001663:Bilirubin