Expression of chimeric cDNAs in cell culture defines a region of UDP glucuronosyltransferase involved in substrate selection.
The cDNAs encoding two forms of UDP glucuronosyltransferase have been expressed in cultured cells to demonstrate that one form, UDPGTr-3, glucuronidates testosterone, whereas the second form, UDPGTr-4, is mainly active toward etiocholanolone (Mackenzie, P. I. (1986) J. Biol. Chem. 261, 14112-14117; Mackenzie, P. I. (1987) J. Biol. Chem. 262, 9744-9749). In order to localize areas of the polypeptide chain involved in substrate selection, the 5' regions of UDPGTr-3 and -4 cDNAs were exchanged to form two chimeric cDNAs. A 53-kDa protein was synthesized in COS cells transfected with the chimeric UDPGTr-3.4 cDNA, which encodes the amino-terminal 298 residues of UDPGTr-3 and the carboxyl-terminal 232 residues of UDPGTr-4. This protein glucuronidated testosterone rather than etiocholanolone and had a faster electrophoretic mobility when transfected COS cells were cultured in the presence of tunicamycin, an inhibitor of N-linked glycosylation. The unglycosylated variant produced by this treatment also glucuronidated testosterone. In contrast, a 50-kDa protein that was more active toward etiocholanolone as substrate was synthesized in COS cells transfected with UDPGTr-4.3, a chimeric cDNA that encodes the amino-terminal region of UDPGTr-4 joined to the carboxyl-terminal region of UDPGTr-3. The electrophoretic mobility of this chimeric protein was unaffected by tunicamycin treatment. These results demonstrate that amino acid sequences that specify substrate specificity are localized in the amino-terminal half of the UDP glucuronosyltransferase polypeptide chain and that the presence of N-linked oligosaccharide chains on the protein does not affect the choice of substrate.
Mackenzie
P I
PI
Department of Clinical Pharmacology, Flinders Medical Centre, Bedford Park, South Australia.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002678:Chimera; D003062:Codon; D004247:DNA / Q000235:genetics*; D015870:Gene Expression; D014453:Glucuronosyltransferase / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D014415:Tunicamycin / Q000494:pharmacology
D003062:Codon; D015345:Oligonucleotide Probes; D014415:Tunicamycin; D004247:DNA; D014453:Glucuronosyltransferase