Elsevier

Biochemical Pharmacology

Volume 68, Issue 12, 15 December 2004, Pages 2443-2450
Biochemical Pharmacology

The interactions between the N-terminal and C-terminal domains of the human UDP-glucuronosyltransferases are partly isoform-specific, and may involve both monomers

https://doi.org/10.1016/j.bcp.2004.08.019Get rights and content

Abstract

The pathological mutation Y486D was previously shown to reduce the activities of the UDP-glucuronosyltransferases (UGTs) 1A1 and 1A6 by about 88% and 99%, respectively. Surprisingly, the corresponding mutation in UGT1A9 (Y483D) doubled the Vmax of scopoletin glucuronidation, whereas the entacapone glucuronidation rate was decreased by about 50%. Due to the primary structure identity of the C-terminal half of all the human UGTs of the 1A subfamily, the sharp differences between them in the effect of a mutation deep inside the C-terminal half suggested that there are isoform-specific interactions between the variable N- and the conserved C-terminal halves. In dimeric enzymes, like the UGTs, such interactions might either occur within the same polypeptide, or between opposite monomers. The latter implies functional monomer–monomer interactions, and this was investigated using hetero-dimeric UGTs. Insect cells were co-infected with mixtures containing different combinations of recombinant baculoviruses encoding either UGT1A4 or 1A9Sol. The UGT1A4 was selected because it glucuronidates neither entacapone nor scopoletin at significant rates. The active enzyme in these hetero-dimers was 1A9Sol, a truncation mutant of UGT1A9 that exhibited a very low ratio of entacapone to scopoletin glucuronidation rates. Interestingly, the ratio of entacapone to scopoletin glucuronidation rates in the co-infected cells was dependent on, and markedly increased with, the probability that 1A9Sol forms hetero-dimers with UGT1A4. In addition, the apparent Km for entacapone in the hetero-dimers was much lower than in 1A9Sol, and resembled the corresponding value in full-length UGT1A9. The results, thus, revealed important monomer–monomer interactions within the UGTs.

Introduction

The UDP-glucuronosyltransferases (UGTs) are membrane-bound proteins of the endoplasmic reticulum that play important roles in the metabolism of xenobiotics and endobiotics. The UGTs catalyse glucuronic acid transfer from UDP-glucuronic acid (UDP-GA) to aglycones that are mostly small lipophilic compounds, including endogenous molecules like bilirubin and steroids, carcinogens from the environment, and many drugs or drug metabolites [1], [2], [3], [4]. The human genome contains about 16 functional UGT genes that are divided into two main subfamilies, UGT1A and UGT2B [3]. At the protein level, the UGTs seem to be composed of two major domains of rather similar size, the relatively variable N-terminal half, and the highly conserved C-terminal half. Furthermore, due to exon sharing in the UGT1A gene locus, the C-terminal halves of all the members of the UGT1A subfamily are identical [3]. The aglycone specificity of individual UGTs appears, therefore, to be determined by the N-terminal domain, while the binding site of the sugar donor, UDP-glucuronic acid, is probably located primarily within the C-terminal domain. Nevertheless, this division of labour may be superficial, since the UGTs are dimeric [5], [6], [7], [8], [9], and monomer–monomer interactions may affect substrate binding. Little is presently known, however, on the interactions between the monomers within the dimeric UGTs.

The UGTs are bound to the endoplasmic reticulum membrane so that most of their mass is located on the lumenal side of the membrane. A short trans-membrane segment is present close to the C-terminus of these 50–60 kDa proteins, and the last 20–26 amino acids are exposed on the cytoplasmic side of the membrane [1]. We have recently demonstrated that the UGT1A9 is an exception among the human UGTs in its resistance to inhibition by Triton X-100 solubilization [9]. The identity of the C-terminal half among all the human UGTs of the 1A subfamily implies that the differences in detergent sensitivity between the UGT1A9 and the other human UGTs arise from residues within the N-terminal half. On the other hand, the likely binding of detergent micelle(s) to the single trans-membrane helix, as well as the removal of the trans-membrane helix, together with the cytoplasmic tail, affect the activity of the truncation mutant [10], suggesting that the fully identical C-terminal half of the enzyme is involved in the variable detergent sensitivity of the UGTs. This apparent contradiction may be explained by protein–protein interactions between the two major domains of the UGTs. It thus appears that the interactions between the N- and C-terminal domains may play an important role in the structure and perhaps also in the function of the UGTs.

The pathological Y486D mutation in UGT1A1 causes Crigler–Najjar syndrome type II (CN-II) [11], [12], and it severely lowers the activity of both UGTs 1A1 and 1A6 [13], [14]. This mutation changes the Tyr residue in the highly conserved QYHSLDV segment, and the last Val in this sequence is the first in the stretch of 17 hydrophobic amino acids that form the trans-membrane helix. Interestingly, the DV in this segment is the point where previous attempts to generate water-soluble UGTs, by truncating the human UGT1A6, or the rat UGT2B1, failed [15], [16]. We have, nevertheless, truncated UGT1A9 at exactly the same point and in this case the mutant, 1A9Sol, was active and water-soluble [10]. The latter results raised the possibility that a corresponding Y to D mutation in UGT1A9 might not be detrimental to enzymatic activity, thereby revealing isoform-specific differences in the sensitivity to a mutation within the C-terminal half. We have, therefore, generated the equivalent of the 1A1/Y486D mutant in UGT1A9, namely 1A9/Y483D, and analysed its scopoletin and entacapone glucuronidation activities. In addition, we have examined hetero-dimers containing the truncation mutant of UGT1A9, 1A9Sol, together with the full-length UGT1A4. The results reveal extensive protein–protein interactions within the UGTs.

Section snippets

Materials and methods

Scopoletin, saccharolactone, and UDP-GA were purchased from Sigma. Entacapone was kindly provided by Orion Pharma (Espoo, Finland), scopoletin and entacapone glucuronides were synthesised in our laboratory [17]. Restriction enzymes were purchased from New England Biolabs and from MBI Fermentas.

Results

The Y486D mutation in UGT1A1 affects the highly conserved Tyr residue that is located in the vicinity of the trans-membrane helix near the C-terminus of the protein. The mutation causes CN-II, and inhibits the activity of UGTs 1A1 and 1A6 [14]. The mode of exon sharing that governs the transcription of all the UGTs of the 1A subfamily implies that these CN-II patients carry analogous mutants of the other UGT1A enzymes. We have now generated the corresponding mutation in UGT1A9, namely

Discussion

Each of the UGTs can be divided into two large and almost equal size sections, namely the N-terminal half that is likely to play a major role in the aglycone binding, and the highly conserved C-terminal half that probably harbours the UDP-GA binding site. Interactions between these domains may be necessary for the structure and the function of these enzymes. For example, they might bring the glucuronic acid moiety of the bound UDP-GA into the vicinity of the sugar-accepting group on the

Acknowledgements

We thank Johanna Mosorin, Saila Mörsky and Sanna Sistonen for skilful technical assistance. This research was supported by the Academy of Finland (Project No. 207535) and the National Technology Agency, Finland.

References (19)

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