The interactions between the N-terminal and C-terminal domains of the human UDP-glucuronosyltransferases are partly isoform-specific, and may involve both monomers
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.
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2020, Biochemical and Biophysical Research CommunicationsCitation Excerpt :In this study we show that Ugt2b1 acquires catalytic capacity for morphine glucuronidation by forming hetero-oligomers with another mouse Ugt isoform, Ugt1a1 (Fig. 2). It has been reported that oligomerization may alter UGT activity, as noted with enhanced 17β-estradiol glucuronidation of the oligomers of Ugt1a1 and Ugt2b1 (Fig. 4) in this study, in addition to affecting UGT substrate-specificity [24–26]. Our previous study with the guinea pig UGTs, UGT2B21 and UGT2B22, showed that their co-expression markedly increased M-6-G generation catalyzed by UGT2B21.
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2013, Archives of Biochemistry and BiophysicsCitation Excerpt :Interestingly, while the effects of UGT2B7 expression on UGT1A1, UGT1A4, UGT1A6 and UGT1A9 activities have been reported [23], only UGT1A9 activity was altered when UGT2B7 expression was down regulated in human hepatocytes transfected with UGT2B7 siRNA. While the detailed mechanism of UGT protein interactions in human hepatocytes remains to be elucidated, our findings specific to UGT1A9 and UGT2B7 do not discount the physiological relevance of protein interactions between UGT2B7 and UGT1A1, UGT1A4 or UGT1A6, but may further support the effects that lipid and membrane composition of the individual expression systems may have on UGT protein interactions in general [23,24,40]. In summary, the data presented in this manuscript supports the utility of siRNA down regulation as an important process for evaluating UGT enzymology and suggests that UGT protein interactions are a physiologically relevant phenomena whose effects can be observed in human hepatocytes.
Homodimerization of UDP-glucuronosyltransferase 2B7 (UGT2B7) and identification of a putative dimerization domain by protein homology modeling
2011, Biochemical PharmacologyCitation Excerpt :FRET and Co-IP data indicated that UGT1A1 may heterodimerize with UGT 1A3, 1A4, 1A6, 1A7, 1A8, 1A9 and 1A10 [8], although two-hybrid analysis did not identify an association between UGT1A1 and UGT1A6 [7]. Consistent with protein–protein interactions, altered enzyme activity and thermal stability have been demonstrated following co-expression of various human UGT1A proteins [11–14] and co-expression of UGT2B7 and UGT1A proteins [15]. Co-expression of guinea pig UGT2B21 and UGT2B22 similarly resulted in the altered glucuronidation of chloramphenicol and morphine [16,17], while immunopurification and chemical cross-linking studies are suggestive of interactions between rat UGT2B1 and UGT1A proteins [18].
Phenylalanine 93 of the human UGT1A10 plays a major role in the interactions of the enzyme with estrogens
2011, SteroidsCitation Excerpt :In this respect, it may be noted here that addition of 0.2% of the detergent Triton X-100 to the full-length membrane-bound UGT1A9, or the preparation of a water-soluble mutant of UGT1A9 by truncating the enzyme before the predicted start of the trans-membrane helix and the cytoplasmic tail affected the scopoletin glucuronidation activity of the enzyme differently than glucuronidation of other aglycone substrates [9,10], respectively). It is thus assumed that the differences in glucuronidation rates of the F90 and F93 mutants toward scopoletin on one hand, and toward 4-MU on the other hand (Fig. 2) are linked in some way to the previous observations with UGT1A9 and these substrates [9,10]. Nevertheless, it remains to be studied how these findings are connected.
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