Cloning and characterisation of the first drug-metabolising canine UDP-glucuronosyltransferase of the 2B subfamily.
Glucuronidation is a major route of clearance for a diverse set of both drug and endogenous substrates. The present study was undertaken to redress the lack of molecular information currently available on drug glucuronidation by the dog, a species widely used in metabolism studies by the pharmaceutical industry. A novel dog uridine diphosphate glucuronosyltransferase (UGT), designated UGT2B31 (GenBank Accession Number: AY135176), has been isolated from a dog cDNA library, expressed in V79 cells and characterised using various methods: (i) UGT2B31 sequence has been compared with mammalian UGT sequences using both sequence alignments and phylogenetic analysis; and (ii) the substrate specificity of UGT2B31 has been determined using functional analysis and compared with that obtained using UGT2B7 and dog liver microsomes. The following results were obtained: (i) sequence alignments between UGT2B31 and UGT2B15 gave the greatest degree of identity (76%); however, human UGT2B4, human UGT2B7, monkey UGT2B9 (all 75%), and rat UGT2B1 (73%) also gave a high degree of identity; (ii) phylogenetic analysis determined UGT2B31 to be most closely related to rat UGT2B1; (iii) UGT2B31 displayed a substrate specificity similar to human UGT2B7 and rat UGT2B1, catalysing the glucuronidation of phenols, opioids, and carboxylic acid-containing drugs; and (iv) UGT2B31 only formed morphine-3-glucuronide; however, kinetic analysis determined the K(m) of this reaction to be similar to that observed with UGT2B7 (both approximately 1300 microM). The results suggest that UGT2B31 plays a crucial role in drug detoxification by the dog and may be the canine equivalent of human UGT2B7.
Soars
Matthew G
MG
Department of Molecular and Cellular Pathology, Ninewells Hospital and Medical School, Dundee, DD1 9SY, Scotland, UK. mattsoars100@hotmail.com
Fettes
Michelle
M
O'Sullivan
Audrey C
AC
Riley
Robert J
RJ
Ethell
Brian T
BT
Burchell
Brian
B
D000595:Amino Acid Sequence; D000818:Animals; D001711:Biotransformation; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D004285:Dogs; D015723:Gene Library; D014453:Glucuronosyltransferase / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008168:Lung; D008322:Mammals; D008862:Microsomes, Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014162:Transfection
D011994:Recombinant Proteins; D014453:Glucuronosyltransferase