Bilirubin and many other non-polar metabolites or exogenous toxins and drugs are converted to polar conjugates, primarily in the liver, by UDP-glucuronyl transferase (UDPGT)-catalyzed glucuronidation prior to excretion in bile or urine. UDPGT is a principal enzyme in the conjugatory detoxication mechanism of the body. Inherited diseases with defective UDPGT function are associated with uncojungated hyperbilirubinemia; in severe cases (Crigler-Najjar syndrome, Type 1) patients usually die from bilirubin-induced brain damage in infancy. Gunn rats are an animal model of Crigler-Najjar syndrome, Type 1. Our studies indicate that in rat liver, UDPGT exists as a family of distinct but related proteins; two of these isoforms have activity for bilirubin, others catalyze the glucuronidation of phenolic, steroidal, N- and S-substrates. Bilirubin-UDPGT appears to exist in Gunn rat in a biological defective form. UDPGT activities develop in perinatal life and the individual isoforms are specifically inducible. The molecular mechanisms of multiplicity, development, induction and inherited deficiency of UDPGTs are not known.
Aims of this proposal are to determine (a) whether the UDPGT isoforms are derived from a family of multiple genes, or by differential processing of a single gene-product; (b) the molecular mechanism of induction and perinatal development of UDPGT activities; and c) the molecular basis of bilirubin-UDPGT deficiency in Gunn rats. To these ends, we have purified and characterized multiple UDPGT-isoforms, have developed antisera which specific for groups of UDPGT isoforms and have developed immunological methods for identification and quantitation of UDPGTs. We have partially purified UDPGT-specific mRNAs from rat liver and have characterized these by cell-free translation. We plan to develop cDNA and isoform-specific restriction fragment clones, and to use these probes for quantitation of UDPGT isoform-specific mRNAs during development and induction of UDPGT. We also wish to study the structure of UDPGT-genes (including the intervening sequences) and to investigate a possible error in the nucleotide sequence of the bilirubin-UDPGT specific mRNA in Gunn rats. This system represents the application of recently developed technology in the investigation of this biologically important protein which has multiple isoforms, is differentially inducible, has defective function in mutants, and is of low abundance indicating tight control.
