Role of cysteine residues in the function of human UDP glucuronosyltransferase isoform 1A1 (UGT1A1)

Siddhartha S. Ghosh, Yang Lu, Sung W. Lee, Xia Wang, Chandan Guha, Jayanta Roy-Chowdhury, Namita Roy-Chowdhury

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Bilirubin glucuronidation, catalysed by UGT1A1 [UGT (UDP glucuronosyltransferase) isoform 1A1, EC 2.4.1.17], is critical for biliary elimination of bilirubin. UGT1A1 deficiency causes CN-1 (Crigler-Najjar syndrome type 1), which is characterized by potentially lethal unconjugated hyperbilirubinaemia. Nucleotide sequence analysis of UGT1A1 in two CN-1 patients revealed that patient A was homozygous for a nt 530 G → A (where nt 530 G → A means guanine to adenine transition at nucleotide 530) mutation, predicting a C177Y substitution, and patient B had a nt 466 T → C mutation on one allele and a nt 1070 A → G mutation on the other, predicting a C156R and a Q357R substitution respectively. All 11 cysteine residues of mature human UGT1A1 are highly conserved in other human UGT isoforms and in rat, mouse and Rhesus monkey UGT1A1, suggesting their functional importance. Expression of mutagenized UGT1A1 plasmids showed that substitution of any of the seven cysteine residues located within the endoplasmic reticulum cisternae (including those mutated in patients A and B) abolished UGT1A1 activity or markedly increased its apparent Km for bilirubin. Substitution of the three cysteine residues within the C-terminal cytosolic tail had minimal effect on basal UGT1A1 activity, but prevented UGT1A1 activation by UDP-GlcNAc. N-Ethylmaleimide did not inhibit UGT1A1 activity in native microsomes, but prevented UGT1A1 activation by UDP-GlcNAc and inhibited the activity in digitonin-permeabilized microsomes. Dithiothreitol did not affect UGT1A1 activity in human liver microsomes. Together, the results suggested that free thiol groups, but not disulphide bonding, of seven cysteine residues within the intracisternal region of human UGT1A1 are important for its catalytic activity, while cysteine residues in the cytosolic domain may be involved in its physiological activation by UDP-GlcNAc.

Original languageEnglish (US)
Pages (from-to)685-692
Number of pages8
JournalBiochemical Journal
Volume392
Issue number3
DOIs
StatePublished - Dec 15 2005

Fingerprint

Glucuronosyltransferase
Cysteine
Protein Isoforms
Uridine Diphosphate
Crigler-Najjar Syndrome
Substitution reactions
Bilirubin
Chemical activation
UGT1A1 enzyme
Microsomes
Mutation
Nucleotides
Digitonin
Ethylmaleimide
Hyperbilirubinemia
Dithiothreitol
Guanine
Liver Microsomes
Adenine
Macaca mulatta

Keywords

  • Alkylation
  • Bilirubin
  • Crigler-Najjar syndrome type 1
  • Cysteine
  • UDP glucuronosyltransferase isoform 1A1 (UGT1A1)
  • UDP-GlcNac

ASJC Scopus subject areas

  • Biochemistry

Cite this

Role of cysteine residues in the function of human UDP glucuronosyltransferase isoform 1A1 (UGT1A1). / Ghosh, Siddhartha S.; Lu, Yang; Lee, Sung W.; Wang, Xia; Guha, Chandan; Roy-Chowdhury, Jayanta; Roy-Chowdhury, Namita.

In: Biochemical Journal, Vol. 392, No. 3, 15.12.2005, p. 685-692.

Research output: Contribution to journalArticle

Ghosh, Siddhartha S. ; Lu, Yang ; Lee, Sung W. ; Wang, Xia ; Guha, Chandan ; Roy-Chowdhury, Jayanta ; Roy-Chowdhury, Namita. / Role of cysteine residues in the function of human UDP glucuronosyltransferase isoform 1A1 (UGT1A1). In: Biochemical Journal. 2005 ; Vol. 392, No. 3. pp. 685-692.
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N2 - Bilirubin glucuronidation, catalysed by UGT1A1 [UGT (UDP glucuronosyltransferase) isoform 1A1, EC 2.4.1.17], is critical for biliary elimination of bilirubin. UGT1A1 deficiency causes CN-1 (Crigler-Najjar syndrome type 1), which is characterized by potentially lethal unconjugated hyperbilirubinaemia. Nucleotide sequence analysis of UGT1A1 in two CN-1 patients revealed that patient A was homozygous for a nt 530 G → A (where nt 530 G → A means guanine to adenine transition at nucleotide 530) mutation, predicting a C177Y substitution, and patient B had a nt 466 T → C mutation on one allele and a nt 1070 A → G mutation on the other, predicting a C156R and a Q357R substitution respectively. All 11 cysteine residues of mature human UGT1A1 are highly conserved in other human UGT isoforms and in rat, mouse and Rhesus monkey UGT1A1, suggesting their functional importance. Expression of mutagenized UGT1A1 plasmids showed that substitution of any of the seven cysteine residues located within the endoplasmic reticulum cisternae (including those mutated in patients A and B) abolished UGT1A1 activity or markedly increased its apparent Km for bilirubin. Substitution of the three cysteine residues within the C-terminal cytosolic tail had minimal effect on basal UGT1A1 activity, but prevented UGT1A1 activation by UDP-GlcNAc. N-Ethylmaleimide did not inhibit UGT1A1 activity in native microsomes, but prevented UGT1A1 activation by UDP-GlcNAc and inhibited the activity in digitonin-permeabilized microsomes. Dithiothreitol did not affect UGT1A1 activity in human liver microsomes. Together, the results suggested that free thiol groups, but not disulphide bonding, of seven cysteine residues within the intracisternal region of human UGT1A1 are important for its catalytic activity, while cysteine residues in the cytosolic domain may be involved in its physiological activation by UDP-GlcNAc.

AB - Bilirubin glucuronidation, catalysed by UGT1A1 [UGT (UDP glucuronosyltransferase) isoform 1A1, EC 2.4.1.17], is critical for biliary elimination of bilirubin. UGT1A1 deficiency causes CN-1 (Crigler-Najjar syndrome type 1), which is characterized by potentially lethal unconjugated hyperbilirubinaemia. Nucleotide sequence analysis of UGT1A1 in two CN-1 patients revealed that patient A was homozygous for a nt 530 G → A (where nt 530 G → A means guanine to adenine transition at nucleotide 530) mutation, predicting a C177Y substitution, and patient B had a nt 466 T → C mutation on one allele and a nt 1070 A → G mutation on the other, predicting a C156R and a Q357R substitution respectively. All 11 cysteine residues of mature human UGT1A1 are highly conserved in other human UGT isoforms and in rat, mouse and Rhesus monkey UGT1A1, suggesting their functional importance. Expression of mutagenized UGT1A1 plasmids showed that substitution of any of the seven cysteine residues located within the endoplasmic reticulum cisternae (including those mutated in patients A and B) abolished UGT1A1 activity or markedly increased its apparent Km for bilirubin. Substitution of the three cysteine residues within the C-terminal cytosolic tail had minimal effect on basal UGT1A1 activity, but prevented UGT1A1 activation by UDP-GlcNAc. N-Ethylmaleimide did not inhibit UGT1A1 activity in native microsomes, but prevented UGT1A1 activation by UDP-GlcNAc and inhibited the activity in digitonin-permeabilized microsomes. Dithiothreitol did not affect UGT1A1 activity in human liver microsomes. Together, the results suggested that free thiol groups, but not disulphide bonding, of seven cysteine residues within the intracisternal region of human UGT1A1 are important for its catalytic activity, while cysteine residues in the cytosolic domain may be involved in its physiological activation by UDP-GlcNAc.

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KW - Crigler-Najjar syndrome type 1

KW - Cysteine

KW - UDP glucuronosyltransferase isoform 1A1 (UGT1A1)

KW - UDP-GlcNac

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