UDP-glucuronosyltransferases: a family of detoxifying enzymes

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276

UDP-glucuronosyltransferases: a family of detoxifying enzymes Thomas R. Tephly and Brian Burchell Glucuronidation is an important process in the metabolism of xenobiotic and endogenous substances leading to enhancement of excretion of these compounds from the body. A multigene family encodes a number of UDPglucuronosyltransferase enzymes which catalyse this route of metabolism. Recent advances in biochemical and molecular biological approaches, reviewed here by Thomas Tephly and Brian Burchell, have given new insight into the function and structure of UDP-glucuronosyltransferases. These proteins have surprisinn similarities and wet az7t7ear to be capable of conjugating a remarkablk num”ber of different chemicals. .I

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Excretion of xenobiotics from the animal organism generally requires conversion of the xenobiotic to a more water-soluble metabolite. Glucuronidation represents a major means of generating water-soluble substances from xenobiotics or endobiotics, which leads to the ultimate excretion of the substances into the urine or the bile. Whereas enzymatic oxidation and reduction reactions have been extensively studied, only recently have we begun to understand the enzymatic processes responsible for the conversion of chemicals to glucuronide conjugates. The conventional view has it that conjugation reactions proceed in the second of a two-phase sequence: oxidation (phase l), where a substance might be hydroxylated, followed by glucuronidation (phase 2). In fact, this is probably the exception rather than the rule. Many drugs and environmental chemicals (e.g. morphine, naphthols and phenols) already exist in the hydroxylated state when the animal is exand carboxylic posed; amines acids, other classes of substrate for glucuronidation, need not be converted by other metabolic reactions in order to serve as subT. R. Tephly is Professor

at the Department of

Pharmacology, Bowen Science Building, University of Iowa, Iowa City, IA 52242, USA, and B. Burchell is Professor and Head at the Department of Biochemical Medicine, Ninewells Hospital and Medical School, Dundee DDI 9SY, UK. 0

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strates for glucuronide formation. Thus, glucuronidation, as one of many types of conjugation reactions available to the organism, is a first-line reaction in the metabolism of xenobiotic agents. The reaction that proceeds to a glucuronide is:

R-OH + UDPGA 1 R-OGA

+ UDP

where R-OH is a xenobiotic or endobiotic and UDPGA is UDPglucuronic acid. This ancient reaction is catalysed by a family of enzymes, the UDP-glucuronosyltransferases (UDPGTs), which have evolved discrete specificities in response to the need to eliminate potentially toxic endobiotic and environmentally produced xenobiotic compounds. The remarkable efficiency’ of the glucuronidation systems in detoxification has largely endured the rigours of the modern chemical environment. However, recently glucuronidation has been implicated in adverse drug reactions of certain carboxylic acid drugsl. The chemical revolution has inevitably outpaced biological evolution and UDPGTs randomly handle modern chemicals and may be ‘duped’ into production of toxic glucuronides. 00

Multiplicity and heterogeneity The UDP-glucuronosyltransferases are located in the endoplasmic reticulum of cells from a number of tissues but are usually found in highest activity in the liver. Multiple forms of UDPGT have been observed in most species studied, on the basis of purification of these proteins to apparent homogeneity, expression of cDNAs or other strong evidence (Table I). These proteins are extremely labile after membrane perturbation and are dependent upon phospholipid for activity. They possess molecular weights between 50 kDa and 60 kDa, and are inducible by a number of chemicals. From studies on substrate specificity carried out with either purified, homogeneous proteins or cDNAs expressed in COS cells it is clear that many UDPGTs are capable of reacting with more than one xenobiotic and with different classes of xenobiotic. In addition, numerous xenobiotics react with more than one isofonn of UDPGT (Table II). Thus it is generally not possible to use a single xenobiotic substrate in microsomal preparations to evaluate the activity of a UDPGT since multiple forms may react with that substrate. For example, 4-nitrophenol glucuronidation has often been used to assess ‘UDPGT activity’. Evidence suggests that this sub-

TABLE I. Hepatic UDPGTs

Rat 4-nitrophenol UDPGT (two forms) 17fi-hydroxy steroid UDPGT (two forms) Sor-hydroxy steroid UDPGT morphine UDPGT (two forms) digitoxigenin UDPGT bilirubin UDPGT estrone UDPGT 4-hydroxybiphenyl UDPGT Rabbit . 4-nitrophenol UDPGT (two forms) estrone UDPGT morphine UDPGT Mouse ~18.5 UDPGT ~16.7 UDPGT Pig 4-nitrophenol UDPGT (two forms) Human pi 7.4 UDPGT (estriol) ~16.2 UDPGT bilirubin UDPGT morphine UDPGT (two forms) 6-hydroxy bile acid UDPGT tertiary amine UDPGT phenol UDPGT For details, see Refs 1, 19 and 24-26.

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1990 [Vol. 211

TABLE II. Substrates reacting with multiple forms of UDPGT in rat liver 4-Nitrophenol

(four forms)

4-Methylumbelliferone

(four forms)

1-Naphthol (three forms) a-Naphthylamine

(three forms)

f3-Naphthylamine

(three forms)

Testosterone

(two forms)

Estradiol (two forms) 4-Hydroxybiphenyl

(two forms)

For details, see Refs 1,24 and 26. reacts primarily (but not exclusively) with the 17P-hydroxy steroid UDPGT in microsomal preparations from untreated rat liver. However, in hepatic microsomes from 3-methylcholanthrenetreated rats, one or two highly inducible forms of 4-nitrophenol UDPGT(s) predominate for 4-nitrophenol glucuronidation. This situation is further complicated in hepatic microsomes from phenobarbital-treated rats: a 4-hydroxybiphenyl UDPGT is induced which may be the major isoform reacting with 4-nitrophenol. These results indicate that different UDPGTs have overlapping specificity, and that 4-nitrophenol is not a good substrate for the study of an individual UDPGT. Furthermore, at least three different isoforms react with 4-nitrophenol in human liver microsomal preparations, each of which has different physical properties and abundance. The UDPGTs exhibit higher subspecificity for endobiotic strates. Current knowledge suggests that many endogenous substrates such as androgens, estrogens and bilirubin react primarily with one form. Thus, the 17fi-hydroxy steroid, 3a-hydroxy steroid, 6-hydroxy bile acid, estrone, bilirubin and p1 7.4 (estriol 16~0H) human UDPGTs are examples of enzymes named for their relatively selective action on endogenous Where endogenous substrates. substrates have not been identified, isoforms have been termed on the basis of their reaction with selective xenobiotics. As information continues to accumulate on the structures of these proteins it will soon be possible to classify them according to family and subfamily. strate

Topological location microsomal The assay of UDPGTs always gives cause for

277 concern and is responsible for many of the variations in results obtained by different laboratories studying drug glucuronidation in vitro. The major reason for this problem is the latencv of membrane-bound UDPGT (Refs 1 and 2), and it has further implications for variation in drug glucuronidation. Parallels can be drawn between the hepatic microsomal glucose-6-phosphatase system and glucuronidation. The molecular characterization of the hepatic microsomal glucase-6-phosphatase insystem volved in the hydrolysis of mannose 6-phosphate, which has many similar membrane properties to UDPGTs, has recently been reported. Lubrol PX and other detergents have been demonstrated effectively to release latent UDPGT activity towards l-naphthol in a manner that exactly correlates with the release of latent mannose-6-phosphatasez4, indicating that the detergent disrupts the membrane structure and that under optimal conditions this does not disrupt protein structure. A model to explain latency has been proposed, which suggests that UDP-glucuronic acid transport is rate limiting for glucuronidation by intact microsomes5 and that disruption of the membrane barrier by detergents allows free access of the donor substrate and reveals the full catalytic potential of the transferases. The model

CYTOPLASM

Fig. 1. Model of the

topology of UDPglucuronosyltransferases. a: UDP-glucuronic acid binding domain, conserved sequence. b: Xenobiotic or endobiotic substrate-binding region, variable sequence. c: N-terminal region, conserved sequence. Lys-Lys, stoptransfer sequence.

could be extended to implicate transporters for the extrusion of UDP, glucuronide and possibly phosphate from the lumen of the endoplasmic reticulum6. Polymorphic variations in individual transporter components of this system could be implicated in drug side-effects or might represent targets for pharmacological intervention. The transmembrane topology of UDPGTs has been examined using proteases and proteases with antibodies7, complemented by computer-based DNA sequence analysis&lo. The picture that emerges is that a small C-terminal domain exposed on the cytoplasmic surface of the membrane is joined by a transmembrane region to the majority of the protein, including the active site, on the lumen of the endoplasmic reticulum (Fig. 1). A more detailed examination of the sequence data has enabled predictions of certain structural features of UDPGT proteins, which are now being confirmed by biochemical analysis. For example, all the cDNAs isolated so far, with the exception of that encoding 17P-hydroxy steroid UDPGT, possess at least one putative asparagine-linked glycosylation consensus sequence (AsnX-Ser/Thr) in the translated protein. Similarly, although there are marked differences between the derived amino acid sequences of

TiPS -July

278 different UDPGTs, they appear to be very closely related structurally. Hydropathy profiles indicate that all UDPGTs possess hydrophobic (cleaved) signal sequences and a highly hydrophobic sequence in the C-terminal region, located between two highly charged areas. This is characteristic of the stop-transfer signals of transmembrane proteinsll. Indeed, lysine residues at positions -3, -4 and -5 from the C terminus are important in retention of UDPGTs in this orientation in the endoplasmic reticulum12. The C-terminal half of the sequence in general contains the majority of the sequence homology between the different UDPGTs, which is suggestive of some conserved function - perhaps UDPGA binding or some form of conformational requirement (Fig. 1). Further support for this concept has recently been provided by Mackenzie13. Indeed, analysis of other UDP-sugar binding protein sequences such as baculovirus UDP-glucosyltransferase, UDP-glucose pyrophosphorylase, glycogenin and galactosyltransferases may reveal the salient features of the UDP binding portion of these homologous proteins. The major region of dissimilarity, even between enzymes showing more than 80% identity, is concentrated in the N-terminal part of the protein (between residues 60 and 120), which presumably provides the specificity of substrate binding. Specificity of drug binding to UDPGTs Although there is striking similarity in sequence particularly between steroid-reactive the UDPGTs, function studies (substrate specificity), subunit molecular weights, p1 values and Nterminal amino acid analysis have shown that each protein possesses unique properties and that each animal species has a unique pattern of UDPGTs. Thus, the non-conserved moieties in the amino acid sequences are critical and worthy of attention. One strategy that has been adopted to investigate active site properties involves experiments carried out with morphine UDPGT. The glucuronidation of morphine is a major metabolic

route for this substance in most animals, especially humans. Purified rat hepatic morphine UDPGT has a narrow substrate specificity in that it reacts only with morphine and morphine analoguesZ4. Early studies performed with rabbit hepatic microsomes showed that the N-alkyl position of the morphine molecule was important in determining the reactivity of these substances with morphine UDPGT15. Cyproheptadine, a compound possessing an N-methyl group, was a potent competitive inhibitor (Ki 80~~) of morphine glucuronidation although it was not itself a substrate for glucuronidation in rabbit liver microsomes16. Desmethyl cyproheptadine was found to be a rather poor inhibitor (Ki 400~~). Thus the presence of an N-alkyl group on morphine analogues or on potential inhibitors enhances reactivity with the morphine UDPGT. More recently, it has been demonstrated that certain benzodiazepines that have N-methyl moieties but are not glucuronidated are also potent competitive inhibitors of morphine glucuronidation in rat and human liver microsomes17J8. Of particular interest is the surprising specificity of benzodiazepines as inhibitors of morphine glucuronidation; no other glucuronidation reaction studied thus far appears to be inhibited by benzodiazepines except for tertiary amine glucuronidation in human liver microsomes19. Of the benzodiazepines studied, flunitrazepam is one of the most potent competitive inhibitors of morphine UDPGT (Ref. 20). It has been employed as a photoaffinity ligand for the benzodiazepine receptor and, recently, for morphine UDPGT. Photoactivation carried out with either solubilized microsomes or partially purified morphine UDPGT has shown that flunitrazepam is rapidly converted to an irreversible and a more potent inhibitor of morphine UDPGT activity21. The rapid inactivation of morphine UDPGT by flunitrazepam is protected by morphine and no other UDPGT activities appear to be inhibited by flunitrazepam after photoirradiation. Fluorography of solubilized rat hepatic microsomes treated with

2990 [Vol. 111

photoirradiation and [3H]flunitrazepam showed the presence on SDS-PAGE gels of a single band of 54-58 kDa (Ref. 21). The molecular weight of rat liver microsomal morphine UDPGT is -56 kDa (Ref. 14). Thus, it is possible that flunitrazepam will be useful in probing the active site of morphine UDPGT and yielding information on the unique portion of the morphine UDPGT or on other characteristics of this protein that are required for its activity and specificity. Variation of drug glucuronidation in humans A major challenge is to determine the extent of the UDPGT gene family in humans and factors underlying the specificity of drug glucuronidation. A description of the variations within the family of rat UDPGTs during development, in response to drug and xenobiotic administration in different tissues and due to genetic defects, has been recently presentedl. Experiments with inbred strains of rat provide readily available sources of tissue and can be easily controlled. However, a ten-fold difference in the rates of drug glucuronidation is not uncommon in a healthy human population. Whether this variation in UDPGT activities is related to age, disease state, exposure to xenobiotics or genetic background has yet to be determined. Recently, some human liver UDPGTs have been purified, cloned and/or expressed in cell cultures, providing the first glimpses of specificities and heterogeneity of human UDPGTs (Ref. 1). An intriguing question is whether the genetic basis for variation of drug glucuronidation could &ad to adverse drug effects in humans. There is no doubt that genetic variation occurs within the multigene family encoding enzymes responsible for glucuronidation in humans; this has already been observed for bilirubin glucuronidation in Crigler-Najjar and Gilbert’s syndromesl. Although no obvious case of adverse drug response has led to an identification of genetic variation in drug glucuronidation, a search is under way for polymorphisms of drug glucuronidation. The accumulated evidence from studies

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279

[Vol. 111

of Crigler-Najjar patients suggests that levels of menthol glucuronidation vary independently from those of bilirubin glucuronidation**. Kinetic analysis of the inhibition of bilirubin glucuronidation by menthol in human liver homogenates suggests that two different isoenzymes catalyse these two reactions (K. Robertson and B. Burchell, unpublished) and it is likely that these two different UDPGTs are encoded by different genes. It may be difficult to demonpolymorphisms in the strate human population by administering drugs and searching for different levels of glucuronides in the urine especially in cases where there is no apparent drug toxicity. Firstly, glucuronidation of a particular drug may be carried out by more than one UDPGT. Secondly, large numbers of healthy individuals will be required for screening in order to find rare genetic variations. Lastly, compounds should be selected for analysis that are not metabolized extensively by oxidation or sulfate conjugation. This is a formidable task since most drugs are metabolized by a number of different systems in

vivo.

UDPGTs are members of a class of proteins involved in detoxification of endobiotics and xenobiotics encoded by a complex multigene family. These enzymes are located and retained mainly in the endoplasmic reticulum, so that drug substrates are transported into and glucuronides transported out of the lumen of this organelle. The membrane location and the variations in the profiles of these enzymes have significant implications for the efficient elimination of potentially toxic compounds. Many aspects of the function of this gene family are unresolved. How many genes are there and how many functional enzymes are present in humans? The specificity of drug binding to UDPGTs and the effectiveness of glucuronidation still need to be determined to facilitate prediction of xenobiotic elimination in humans. Other intriguing possibilities are that UDPGTs may be involved in regulating neurotransmitter function in brain and that they have a

significant role in olfactory sory perception23.

sen12

Acknowledgements We thank agencies such as the National Institute of General Medical Sciences, the Wellcome Trust and the Medical Research Council for grants supporting our work. In addition we acknowledge the work of our students and colleagues who have participated in the research presented here.

13 14 15 16 17

18

References Burchell, B. and Coughtrie, M. W. H. (1989) Pharmacol. Ther. 43, 261-289 Dutton, G. J. (1980) Glucuronidatioti of Drugs and Other Compounds, CRC Press Scragg, I. M., Arion, W. J. and Burchell, 8. (1985) in Advances in Glucuromde Conjugation (Matern, S., Bock, K. W. and Gerok, W., eds), pp. 390-391, MTP Press Vanstapel, F. and Blanckaert, N. (1988) Arch. Biochem. Biophys. 263, 216-225 Hallinan, T. and DeBrito, A. E. R. (1981) in Hormones and Cell Regulation (Vol. 5) (Dumont, J. E. and Nunez, J., eds), pp. 7%95, Elsevier Burchell, B. and Burchell, A. (1989) Curr. Opin. Cell Biol. 1, 712-717 Shepherd, S. R. I’., Baird, S. J., Hallinan, T. and Burchell, B. (1989) Biochem. 1.259, 617-620 Jackson, M. R. and Burchell, B. (1986) Nucleic Acids Res. 14, 779-795 9 Mackenzie, P. (1986) 1. Biol. Chem.261, 6119-6125 i0 Iyanagi, T. rt al. (1987) 1. Biol. Chem. 261, 15607-15614 11 Sabatini, D. D., Kreibich, G., Morimoto,

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T. and Adesnik, M. (1982) I. Cell Biol. 92, l-22 Nilsson, T., Jackson, M. and Peterson, I’. A. (1989) Cell 58, 707-718 Mackenzie, I’. (1990) 1. Bzol. Chem. 256, 3432-3435 Puig, J. and Tephly, T. R. (1986) Mol. Pharmacol. 33, 97-101 Sanchez, E., de1 Villar, E. and Tephly, T. R. (1978) Biochem. 1. 169, 17%177 de1 Villar, E., Sanchez, E. and Tephly, T. R. (1977) Life SCI. 21, 1801-1806 de1 Villar, E., Sanchez, E., Letelier, M. E. and Vega, I’. (1984) Res. Commun. Chem. Pathol. Pharmacol. 33, 43-47 Rane, A., Sawe, J., Pacifici, G. M., Svendson, J. 0. and Kager, L. (1986) Adv. Pain Res. Ther. 8, 57-64 Styczyski, I’. B., Coffman, 8. L., Green, M. D. and Tephly, T. R. (1989) Pharmacologist 31, 131 Vega, P., Carrasco, M., Sanchez, E. and de1 Villar, E. (1984) Res. Commun. Chem. Pathol. Pharmacol.‘44, 179-198 Thomassin, J. and Tephly, T. R. Mol. Pharmacol. (in press) Burchell, B. and Coughtrie, M. (1990) in European Consensus Conference on Phnrmacogenetics (Alvan, G. et al., eds), pp. 153-160, Commission of European Communities, Cost Bl Medicine, Luxembourg Lazard, D., Tal, N., Rubinstein, M., Khen, M., Lancet, D. and Zupko, K. Biochemistry (in press) Tephly, T., Green, M., Puig, J. and Irshaid, Y. (1988) Xenobiotica 18, 1201-1210 Mackenzie, I’. I., Joffe, M. M., Munson, P. J. and Owens, I. S. (1985) Blochem. Pharmacol. 34, 737-746 Tephly, T. R., Townsend, M. and Green, M. D. (1989) Dru,? Metub. Rev. 20, 689-695

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