Douglas J. Clarke

Genetic defects of the UDP-glucuronosyltransferase-1 (UGT1) gene that cause familial non-haemolytic unconjugated hyperbilirubinaemias.

Congenital familial non-haemolytic hyperbilirubinaemias are potentially lethal syndromes caused by genetic lesions that reduce or abolish hepatic bilirubin UDP-glucuronosyltransferase activity. Here we describe genetic defects that occur in the UGT1 gene complex that cause three non-haemolytic unconjugated hyperbilirubinaemia syndromes. The most severe syndrome, termed Crigler-Najjar syndrome type I, is mainly associated with mutations in exons 2 to 5 that affect all UGT1 enzymes and many of the mutations result in termination codons and frameshifts. Crigler-Najjar type II syndrome which is treatable with phenobarbital therapy is associated with less dramatic missense mutations or heterozygous expression of mutant and normal alleles. Gilberts syndrome, the most prevalent (2-19% in population studies) and mildest of the three syndromes is principally caused by a TA insertion at the TATA promoter region upstream of the UGT1A1 exon. Current methods used for the diagnosis and treatment of these diseases are discussed.

Differential expression and induction of UDP-glucuronosyltransferase isoforms in hepatic and extrahepatic tissues of a fish, Pleuronectes platessa: immunochemical and functional characterization.

Glucuronidation of three substrates prototypical for different UDP-glucuronosyltransferase (UDPGT) isoforms in hepatic, renal, intestinal, and branchial microsomes of corn oil, 3-methylcholanthrene, Aroclor 1254, and clofibrate-pretreated plaice was investigated. The differential expression of UDPGT in the four tissues clearly demonstrated for the first time that multiple isoforms with differing substrate specificities were present in fish. The liver was quantitatively the most important site for the glucuronidation of all three compounds studied. Phenol UDPGT activity was ubiquitous to all tissues and was induced by 3-methylcholanthrene and Aroclor 1254 in hepatic tissue and by Aroclor 1254 in renal tissue. The glucuronidation of testosterone was restricted to liver and intestinal tissue, while conjugation of bilirubin was expressed solely in hepatic tissue. The biotransformation of the endogenous compounds was not induced in the xenobiotic-treated animals. The presence of immunoreactive UDPGTs in the four tissues was demonstrated by immunoblot analysis using sheep anti-plaice UDPGT antibodies. Hepatic tissue displayed a range of immunoreactive polypeptides of 52 to 57 kDa, while a 55-kDa polypeptide was detected in extrahepatic tissues. An increased intensity of the latter polypeptide species was demonstrated in liver and kidney microsomes in which there was a concomitant induction of phenol UDPGT activity in xenobiotic-treated fish. The results indicate that the 55-kDa polypeptide was the major polyaromatic hydrocarbon-inducible UDPGT isoenzyme in hepatic and renal microsomes.

Characterisation of a human bilirubin UDP-glucuronosyltransferase stably expressed in hamster lung fibroblast cell cultures

A cDNA encoding a human bilirubin UDP‐glucuronosyltransferase has been isolated and stably expressed in Chinese hamster V79 lung fibroblast cell line. Western blotting of cell homogenates with anti‐UGT antibody revealed a highly expressed protein of approx. 55.5 kDa in size. The expressed enzyme specifically catalysed the formation of bilirubin mono‐ and diglucuronides, and also catalysed the glucuronidation of two phenolic compounds, which are good substrates for other human UGT isoenzymes, at low rates.

Isolation and characterisation of a new hepatic bilirubin UDP‐glucuronosyltransferase Absence from Gunn rat liver

A novel UDP‐glucuronosyltransferase that conjugates bilirubin IXα, bilirubin monoglucuronide and an arylalkanoic acid was purified to homogeneity from clofibrate treated Wistar rats. The enzyme displayed a subunit molecular mass of 54 kDa, a pI of 7.6 and was demonstrated to be N‐glycosylated. Sequence analysis of peptides derived by endoproteinase Glu‐C cleavage of the purified enzyme indicated that it was a new member of the recently identified UGT1 subfamily. Immunoblot analysis demonstrated that this enzyme was absent from Gunn rat liver. The molecular derivation of this enzyme and the lack of it in Gunn rats is discussed.

Functional and immunochemical comparison of hepatic UDP-glucuronosyltransferases in a piscine and a mammalian species

1. The aglycone specificity of hepatic microsomal glucuronidation was compared under uniform conditions in a fish, Pleuronectes platessa and a mammal, Rattus norvegicus, representative of the most primitive and advanced vertebrate classes. 2. Both species exhibited comparable UDP-glucuronosyltransferase (UDPGT) activity towards planar phenolic substrates (1-naphthol, 4-nitrophenol); however, plaice activity towards bulky non-planar substrates such as (-)-morphine was either 200-fold lower, or for an arylacetic acid (RS-2-phenylpropionic acid) and an aryloxyacetic acid (clofibric acid) non-detectable. 3. Conjugation of the endogenous substrates, bilirubin and steroids were 4- to 40-fold lower in the plaice than in the rat. Whilst both species formed diglucuronides of the asymmetrical bilirubin IX alpha, they displayed a reciprocal preference for the initial esterification, conjugation of the C-8 side chain predominating in the rat and of C-12 in the fish. 4. Immunoblot analysis using two polyclonal antisera preparations raised against rat UDPGTs demonstrated the presence of multiple weakly cross-reacting polypeptides in fish microsomes indicative of multiple isoforms and conservation of common structural motifs over more than 350 million years since evolutionary divergence of the mammals.

Characterization and molecular analysis of hepatic microsomal UDP-glucuronosyltransferases in the marine fish, Pleuronectes platessa

Abstract Here we report the results of the first molecular enzymological study of fish UDP-glucuronosyltransferase (UDPGT). UDPGT activities of plaice liver microsomes were greatest for planar phenols and there was low but measurable conjugation of bilirubin, testosterone and androsterone. A highly purified preparation was isolated which possessed activities towards 1-naphthol, bilirubin and testosterone, containing several molecular weight species of Mr 52–56 kDa. On immunoblot analysis these proteins cross-reacted with a polyspecific anti-rat UDPGT antibody, suggesting that a number of plaice UDPGT isoforms with epitopes in common with the corresponding rat enzymes had been purified.

The UDP-glucuronosyltransferase gene family: Function and expression of human UGTs

The UDP-glucuronosyltransferase UGTs are a group of isoenzymes of 50-60 kDa localised primarily in hepatic endoplasmic reticulum and nuclear envelope (Tephly and Burchell, 1990). The UGTs are encoded by a large multigene family, which has evolved to produce protein catalysts with different, but sometimes apparently overlapping substrate specificities. The expression of UGTs is differentially regulated by chemical and hormonal inducers and during development, thereby producing different tissue specific profiles of activities (Burchell and Coughtrie, 1989).