Stable expression of two human UDP-glucuronosyltransferase cDNAs in V79 cell cultures.
Two human liver UDP-glucuronosyltransferase cDNA clones (HLUGP1 and HLUG25) were individually inserted into the eukaryotic expression vector pKCRH2. Each recombinant plasmid was cotransfected with a SFVneo vector, thereby allowing establishment of several V79 cell lines retaining the exogenous UDP-glucuronosyltransferase cDNA after selection with G418 (Geneticin). Southern blot analysis suggested that the cDNAs were integrated into the host cell genome. Northern blot and immunoblot analyses indicated that the cDNAs were correctly transcribed and translated for the production of functional enzymes. The established recombinant V79 cell lines stably expressed the UDP-glucuronosyltransferase activities towards 1-naphthol (HLUGP1) and hyodeoxycholic acid (HLUG25) at levels 10-20-fold higher than with transient expression, and in the range found in human liver. These high levels of expression of UDP-glucuronosyltransferase activity allowed the determination of apparent kinetic constants and substrate specificities of glucuronidation in the genetically engineered cell lines. HLUG25 cDNA encoded an isoform with restricted specificity towards the 6-OH group of the bile acid hyodeoxycholic acid. The other steroids, bile acids, endobiotics, and xenobiotics tested as substrates were glucuronidated in various samples of human liver microsomes, but not by this isoenzyme. This study, allowing the expression of individual UDP-glucuronosyltransferases in heterologous cells with no endogenous transferases, offered a unique solution for the characterization of UDP-glucuronosyltransferase functional heterogeneity.
Fournel-Gigleux
S
S
Centre du Médicament, URA CNRS 597, Nancy, France.
Sutherland
L
L
Sabolovic
N
N
Burchell
B
B
Siest
G
G
D000818:Animals; D002472:Cell Transformation, Viral / Q000235:genetics; D002478:Cells, Cultured; D002999:Clone Cells / Q000201:enzymology; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics*; D003840:Deoxycholic Acid / Q000378:metabolism; D005347:Fibroblasts / Q000502:physiology; D015870:Gene Expression; D015698:Genomic Library; D014453:Glucuronosyltransferase / Q000235:genetics*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D019868:Kanamycin Kinase; D010770:Phosphotransferases / Q000235:genetics; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D013539:Simian virus 40 / Q000235:genetics; D013379:Substrate Specificity; D014162:Transfection
D007527:Isoenzymes; D012333:RNA, Messenger; D003840:Deoxycholic Acid; C010471:hyodeoxycholic acid; D004247:DNA; D014453:Glucuronosyltransferase; D010770:Phosphotransferases; D019868:Kanamycin Kinase