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Uridine diphosphate glucuronosyltransferases (UGTs) are key enzymes responsible for the body's ability to process a variety of endogenous and exogenous compounds. Significant gains in understanding UGT function have come from the analysis of variants seen in patients. We cared for a Sudanese child who showed clinical features of type 1 Crigler-Najjar syndrome (CN-1), namely severe unconjugated hyperbilirubinemia leading to liver transplantation. CN-1 is an autosomal recessive disorder caused by damaging mutations in the gene for UGT1A1, the hepatic enzyme responsible for bilirubin conjugation in humans. Clinical genetic testing was unable to identify a known pathogenic UGT1A1 mutation in this child. Instead, a novel homozygous variant resulting in an in-frame deletion, p.Val275del, was noted. Sanger sequencing demonstrated that this variant segregated with the disease phenotype in this family. We further performed functional testing using recombinantly expressed UGT1A1 with and without the patient variant, demonstrating that p.Val275del results in a complete lack of glucuronidation activity, a hallmark of CN-1. Sequence analysis of this region shows a high degree of conservation across all known catalytically active human UGTs, further suggesting that it plays a key role in the enzymatic function of UGTs. Finally, we note that the patient's ethnicity likely played a role in his variant being previously undescribed and advocate for greater diversity and inclusion in genomic medicine., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

The aim of this work was to highlight a considerable and broad problem in UGT1A10 activity assessment that has led to underestimation of its role in intestinal glucuronidation of drugs and other xenobiotics. The reason appears to be poor activity of the commercial UGT1A10 that is used by many laboratories, and here we have tested it by comparison with our recombinant His-tagged UGT1A10 (designated as UGT1A10-H), both expressed in insect cells. The glucuronidation rates of morphine, estradiol, estrone, SN-38, diclofenac, 4-methylumbelliferone, 7-amino-4-methylcoumarin, N-(3-carboxypropyl)-4-hydroxy-1,8-naphthalimide, and bavachinin were assayed. The results revealed that the activity of commercial UGT1A10 was low, very low, and in the cases of morphine, estrone, 7-methyl-4-aminocoumarin, and bavachinin it was below the detection limit. On the other hand, under the same conditions, UGT1A10-H exhibited high glucuronidation rates toward all these compounds. Moreover, using estradiol, morphine, and estrone, in the presence and absence of suitable inhibitors, nilotinib or atractylenolide I, it was demonstrated that UGT1A10-H, but not the commercial UGT1A10, provides a good tool to study the role of native UGT1A10 in the human intestine. The results also suggest that much of the data in the literature on UGT1A10 activity may have to be re-evaluated.

The glucuronidation of estrone and 16α-hydroxyestrone by recombinant human UDP-glucuronosyltransferase enzymes (UGTs) of subfamilies 1A, 2A and 2B was studied. Microsomes from human liver and small intestine were also tested for the glucuronidation of these two estrogens. The results revealed that UGT1A10 is by far the most active enzyme in estrone glucuronidation. UGT1A10 also exhibited high rate of 16α-hydroxyestrone conjugation at the 3-OH, whereas UGT2B7 catalyzed its glucuronidation at high rates at the 16-OH. Human liver microsomes exhibited high rates of 16α-hydroxyestrone-16-glucuronide formation, but very low formation rates of either 16α-hydroxyestrone-3-glucuronide or estrone glucuronide. On the other hand, human intestine microsomes catalyzed the formation of all these 3 different glucuronides at high rates. Kinetic analyses revealed very low Km value for 16α-hydroxyestrone glucuronidation by UGT2B7, below 4 μM, suggesting higher affinity than commonly found among UGTs and their substrates. In further studies with UGT1A10, mutant F93G exhibited increased glucuronidation rates of 16α-hydroxyestrone, but not estrone, whereas mutations in F90 did not reveal any activity with either estrogen. Taken together, the results of this study significantly expand our understanding on the metabolism of estrogens and their interactions with the human UGTs., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Understanding drug glucuronidation in the dog, a preclinical animal, is important but currently poorly characterized at the level of individual enzymes. We have constructed cDNAs for the 10 dog UDP-glucuronosyltransferases of subfamily 1A (dUGT1As), expressed them in insect cells, and assayed their activity as well as the activity of the nine human UGT1As, toward 14 compounds. The goal was to find out whether individual dUGT1As and individual human UGT1As have similar substrate specificities. The results revealed similarities but also many differences. For example, similarly to the human UGT1A10, dUGT1A11 exhibited high glucuronidation activity toward the 3-OH of 17-β-estradiol, 17-α-estradiol, and ethinylestradiol, and also conjugated the drug entacapone. Unlike the human UGT1A10, however, it failed to catalyze considerable rates of R-propranolol, diclofenac, and indomethacin glucuronidation. The estrogen glucuronidation assays revealed that dUGT1A8 and dUGT1A10 have a capacity to catalyze the formation of (linked) diglucuronides, an activity no human UGT1A exhibited. dUGT1A2-dUGT1A4 are homologs of the human UGT1A4, but none of them catalyzed N-glucuronidation of dexmedetomidine. Contrary to the human UGT1A4, however, dUGT1A2-dUGT1A4 catalyzed indomethacin and diclofenac glucuronidation. It may be concluded that, perhaps with the exception of UGT1A6, high similarities in substrate specificity between individual dog and human UGTs of subfamily 1A are rare or partial. Activity assays with liver and intestine microsomes of both dog and human further revealed interspecies differences, particularly in glucuronidation rates. In the dog, the microsomes assays also strongly suggested important roles for dUGTs of other subfamilies, mainly in the liver., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)