Expression of a functionally active human hepatic UDP-glucuronosyltransferase (UGT1A6) lacking the N-terminal signal sequence in the endoplasmic reticulum

• Published in: FEBS letters Vol. 454; no. 3; pp. 187 - 191
• Main Authors: Ouzzine, M., Magdalou, J., Burchell, B., Fournel-Gigleux, S.
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 09-07-1999
• Subjects: 4-MU, 4-methylumbelliferone; Animals; Biological Transport; Cell Line; Cricetinae; DMEM, Dulbecco's modified Eagle's medium; Endoplasmic Reticulum - metabolism; Endoplasmic reticulum targeting; ER, endoplasmic reticulum; Glucuronosyltransferase - genetics; Glucuronosyltransferase - metabolism; Human; Humans; Liver - enzymology; Liver - ultrastructure; Membrane translocation; PBS, phosphate buffer saline; PCR, polymerase chain reaction; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis; Sequence Deletion; Signal peptide; UDP-glucuronosyltransferase; UGT, UDP-glucuronosyltransferase; Human; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis; Endoplasmic reticulum targeting; PBS, phosphate buffer saline; UDP-glucuronosyltransferase; DMEM, Dulbecco's modified Eagle's medium; Signal peptide; PCR, polymerase chain reaction; 4-MU, 4-methylumbelliferone; UGT, UDP-glucuronosyltransferase; Membrane translocation; ER, endoplasmic reticulum
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(99)00797-8

UDP-glucuronosyltransferase 1A6 (UGT1A6) is a membrane glycoprotein of the endoplasmic reticulum playing a key role in drug metabolism. It is synthesized as a precursor with an N-terminal cleavable signal peptide. We demonstrate that deletion of the signal peptide sequence does not prevent membrane targeting and integration of this human isoform when expressed in an in vitro transcription-translation system, as shown by
N-glycosylation, resistance to alkaline treatment and protease protection. Furthermore, UGT1A6 lacking the signal peptide (UGT1A6Δsp) was targeted to the endoplasmic reticulum in mammalian cells as shown by immunofluorescence microscopy and was catalytically active with kinetic constants for 4-methylumbelliferone glucuronidation similar to that of the wild-type. These results provide evidence that the signal peptide is not essential for the membrane assembly and activity of UGT1A6 suggesting that additional topogenic element(s) mediate(s) this process.