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

Abstract

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 (UGT1A6Asp) was targeted to the endoplasmic reticulum in mammalian ceils as shown by immunofluorescence microscopy and was catalytically active with kinetic constants for 4-methylumbelliferone glucuronidation similar to that of the wildtype. These results provide evidence that the signal peptide is not essential for the membrane assembly and activity of UGTIA6 suggesting that additional topogenic element(s) mediate(s) this process.

Characterization of a membrane-bound C-glucosyltransferase responsible for carminic acid biosynthesis in Dactylopius coccus Costa

“…If the truncated DcUGT2 protein is indeed targeted to the ER without the putative signal peptide, another unknown ER-targeting signal must be present in the DcUGT2 protein and clearly not contained within the transmembrane domain/cytoplasmic tail. Such a signal has, in fact, been demonstrated to occur within an amino acid stretch, encompassing residues 140–240 of the human UGT1A6, although an exact motif was not defined 33 , 34 . Expression of UGT1A6 without the sequence encoding the N-terminal signal peptide showed that the ΔSP-UGT1A6 protein was translocated into and retained in the ER via this 100 amino acid stretch in mammalian cells 33 , 34 .…”

“…Such a signal has, in fact, been demonstrated to occur within an amino acid stretch, encompassing residues 140–240 of the human UGT1A6, although an exact motif was not defined 33 , 34 . Expression of UGT1A6 without the sequence encoding the N-terminal signal peptide showed that the ΔSP-UGT1A6 protein was translocated into and retained in the ER via this 100 amino acid stretch in mammalian cells 33 , 34 . The amino acid alignment of DcUGT2 to UGT1A6 indeed identified regions of homology between the two proteins in this 100 amino acid stretch, but whether the ER-targeting signal is contained within these regions remains to be established (Supplementary Fig.…”

“…4 ). In addition to being N-glycosylated in an in vitro transcription–translation system with pancreatic microsomal membranes and in vivo when expressed in the Pichia pastoris yeast, the ΔSP-UGT1A6 protein was also functionally active and had similar kinetic parameters to UGT1A6 33 , 34 . It was therefore concluded that the signal peptide was not essential for membrane assembly and functional activity of UGT1A6.…”

The crystal structure of human UDP-glucuronosyltransferase 2B7 C-terminal end is the first mammalian UGT target to be revealed: the significance for human UGTs from both the 1A and 2B families

“…UGTs are synthesized as precursors of ~530 residues containing an N-terminal signal peptide that mediates the integration of the polypeptide chain into the ER (Mackenzie and Owens, 1984; Ouzzine et al, 1999a, 1999b). The signal peptide is subsequently cleaved and the protein is N-glycosylated.…”

Revisiting the Latency of Uridine Diphosphate-Glucuronosyltransferases (UGTs)—How Does the Endoplasmic Reticulum Membrane Influence Their Function?

“…Following membrane integration, the N-terminal signal peptide is cleaved and the enzyme is N -glycosylated in the ER lumen. Notably, other internal topological elements within the UGTs might be required to regulate the integration process, as demonstrated for UGT1A6 [ 49 , 50 ]. In this way, the UGTs become mature ER enzymes of just over 500 residues [ 1 ].…”