Homodimerization of human bilirubin-uridine-diphosphoglucuronate glucuronosyltransferase-1 (UGT1A1) and its functional implications.
Genetic lesions of bilirubin-uridine-diphosphoglucuronate glucuronosyltransferase-1 (UGT1A1) completely or partially abolish hepatic bilirubin glucuronidation, causing Crigler-Najjar syndrome type 1 or 2, respectively. Clinical observations indicate that some mutant forms of human UGT1A1 (hUGT1A1) may be dominant-negative, suggesting their interaction with the wild-type enzyme. To evaluate intermolecular interaction of hUGT1A1, Gunn rat fibroblasts were stably transduced with hUGT1A1 cDNA. Gel permeation chromatography of solubilized microsomes suggested dimerization of hUGT1A1 in solution. Nearest-neighbor cross-linking analysis indicated that, within microsomal membranes, hUGT1A1 dimerized more efficiently at pH 7.4 than at pH 9. Two-hybrid analysis in yeast and mammalian systems demonstrated positive interaction of hUGT1A1 with itself, but not with another UGT isoform, human UGT1A6, which differs only in the N-terminal domain. Dimerization was abolished by deletion of the membrane-embedded helix from the N-terminal domain of hUGT1A1, but not by substitution of several individual amino acid residues or partial deletion of the C-terminal domain. A C127Y substitution abolished UGT1A1 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 may explain the dominant-negative effect of some mutant forms of the enzyme.
Ghosh
S S
SS
Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Sappal
B S
BS
Kalpana
G V
GV
Lee
S W
SW
Chowdhury
J R
JR
Chowdhury
N R
NR
D000818:Animals; D019556:COS Cells; D002850:Chromatography, Gel; D019281:Dimerization; D014453:Glucuronosyltransferase / Q000737:chemistry* / Q000502:physiology; D006801:Humans; D011994:Recombinant Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship; D000099148:UGT1A1 Enzyme
D014453:Glucuronosyltransferase; D011994:Recombinant Proteins; D000099148:UGT1A1 Enzyme