Bilirubin and many other non-polar endogenous metabolites or exogenous carcinogens, toxins and drugs are converted to polar conjugates, primarily in the liver, by UDP-glucuronosyltransferase (UDPGT)-mediated glucuronidation prior to excretion in the bile or urine. UDPGT is a principal enzyme-system in conjugative detoxification. Inherited disorders with defective UDPGT function are associated with unconjugated hyperbilirubinemia; in severe cases (e.g. Crigler-Najjar syndrome, Type I), patients die in infancy or around the age of puberty from bilirubin induced brain damage. Gunn rats are an animal model of Crigler-Najjar syndrome, Type I. Our studies indicate that in rat liver, UDPGT exists as multiple distinct but structurally related isoforms. Two of these isoforms catalyze the glucuronidation of bilirubin, others mediate the conjugation of phenolic, steroidal, N- and S- substrates. In Gunn rats, two UDPGT isoforms, Isoform V (normally active toward bilirubin), and Isoform I (normally active toward 4-nitrophenol), are present in immunoreactive but functionally defective forms. UDPGT activities toward various substrates develop at different periods of perinatal life, are specifically inducible and are expressed differentially during hepatocellular proliferation. Molecular mechanisms of multiplicity, ontogenic development, induction, and inherited functional defects of UDPGT are not known. As a part of this project during the last two years we have purified normal and functionally defective UDPGT isoforms from Gunn and normal rats and developed a series of UDPGT-specific cDNA clones from rat liver.
Specific aims of this continuation application are to use these clones for development of UDPGT isoform-specific cDNA probes, which will be used for (a) the study the molecular mechanism of development and induction of UDPGTs and (b) determination of whether individual UDPGT isoforms are products of distinct genes; and (b) full-length cDNA clones for UDPGT Isoforms I and V, which will be used to define the structural basis for defective UDPGT function in Gunn rats by nucleotide sequence determination. We also wish to introduce the normal cDNA sequences for Isoforms I and V into isolated Gunn rat hepatocytes for correction of the inherited disorder of bilirubin glucuronidation; these cells will be subsequently transplanted into syngeneic Gunn rat recipients by a method which was also developed in our laboratory during execution of this project. This system represents the application of recently developed technology in the investigation of these biologically important protein that have multiple isoforms, are differentially regulated, have defective function in mutants and are of low abundance indicating tight control.
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