Homodimerization of human bilirubin-uridine-diphosphoglucuronate glucuronosyltransferase-1 (UGT1A1) and its functional implications

Genetic lesions of bilirubin-uridine-diphosphoglucuronate glucuronosyltrans ferase-1 (UGT1A1) completely or partially abolish hepatic bilirubin glucuro nidation, causing Crigler-Najjar syndrome type I or 2, respectively. Clinic al observations indicate that some mutant forms of human UGT1A1 (hUGT1A1) m ay be dominant-negative, suggesting their interaction with the wildtype enz yme. To evaluate intermolecular interaction of hUGT1A1, Gunn rat fibroblast s were stably transduced with hUGT1A1 cDNA. Gel permeation chromatography o f solubilized microsomes suggested dimerization of hUGT1A1 in solution. Nea rest-neighbor cross-linking analysis indicated that, within microsomal memb ranes, hUGT1A1 dimerized more efficiently at pH 7.4 than at pH 9. Two-hybri d analysis in yeast and mammalian systems demonstrated positive interaction of hUGT1A1 with itself, but not with another UGT isoform, human UGT1A6, wh ich differs only in the N-terminal domain. Dimerization was abolished by de letion of the membrane-embedded helix from the N-terminal domain of hUGT1A1 , but not by substitution of several individual amino acid residues or part ial deletion of the C-terminal domain. A C127Y substitution abolished UGT1A 1 activity, but not its dimerization. Coexpression of mutagenized and wild- type hUGT1A1 in COS-7 cells showed that the mutant form markedly suppressed the catalytic activity of wild-type hUGT1A1. Homodimerization of hUGT1A1 m ay explain the dominant-negative effect of some mutant forms of the enzyme.