Discrimination between Crigler-Najjar type I and II by expression of mutant bilirubin uridine diphosphate-glucuronosyltransferase

Jurgen Seppen, Piter J. Bosma, Bart G. Goldhoorn, Conny T M Bakker, Jayanta Roy-Chowdhury, Namita Roy Chowdhury, Peter L M Jansen, Ronald P J Oude Elferink

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Abstract

Crigler-Najjar (CN) disease is classified into two subtypes, type I and II. The molecular basis for the difference between these types is not well understood. Several mutations in the bilirubin UDP-glucuronosyltransferase (B-UGT) gene of six CN type I and two CN type II patients were identified. Recombinant cDNAs containing these mutations were expressed in COS cells. B- UGT activity was measured using HPLC and the amount of expressed protein was quantitated using a sandwich ELISA. This enabled us to determine the specific activities of the expressed enzymes. All type I patients examined had mutations in the B-UGT1 gene that lead to completely inactive enzymes. The mutations in the B-UGT1 gene of patients with CN type II only partially inactivated the enzyme. At saturating concentrations of bilirubin (75 μM) CN type II patient A had 4.4±2% residual activity and CN type II patient B had 38±2% residual activity. Kinetic constants for the glucuronidation of bilirubin were determined. The affinities for bilirubin of B-UGT1 expressed in COS cells and B-UGT from human liver microsomes were similar with K(m) of 5.1±0.9 μM and 7.9±5.3 μM, respectively. B-UGT1 from patient B had a tenfold decreased affinity for bilirubin, K(m) = 56±23 μM. At physiological concentrations of bilirubin both type II patients will have a strongly reduced conjugation capacity, whereas type I patients have no B-UGT activity. We conclude that CN type I is caused by a complete absence of functional B- UGT and that in CN type II B-UGT activity is reduced.

Original languageEnglish (US)
Pages (from-to)2385-2391
Number of pages7
JournalJournal of Clinical Investigation
Volume94
Issue number6
StatePublished - Dec 1994

Fingerprint

Glucuronosyltransferase
Uridine Diphosphate
bilirubin glucuronoside glucuronosyltransferase
Bilirubin
Mutation
COS Cells
Enzymes
Genes
Liver Microsomes
Complementary DNA
Enzyme-Linked Immunosorbent Assay
High Pressure Liquid Chromatography

Keywords

  • enzyme activity
  • enzyme kinetics
  • enzyme-linked immunosorbent assay
  • hereditary diseases
  • site directed mutagenesis

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Seppen, J., Bosma, P. J., Goldhoorn, B. G., Bakker, C. T. M., Roy-Chowdhury, J., Chowdhury, N. R., ... Oude Elferink, R. P. J. (1994). Discrimination between Crigler-Najjar type I and II by expression of mutant bilirubin uridine diphosphate-glucuronosyltransferase. Journal of Clinical Investigation, 94(6), 2385-2391.

Discrimination between Crigler-Najjar type I and II by expression of mutant bilirubin uridine diphosphate-glucuronosyltransferase. / Seppen, Jurgen; Bosma, Piter J.; Goldhoorn, Bart G.; Bakker, Conny T M; Roy-Chowdhury, Jayanta; Chowdhury, Namita Roy; Jansen, Peter L M; Oude Elferink, Ronald P J.

In: Journal of Clinical Investigation, Vol. 94, No. 6, 12.1994, p. 2385-2391.

Research output: Contribution to journalArticle

Seppen, J, Bosma, PJ, Goldhoorn, BG, Bakker, CTM, Roy-Chowdhury, J, Chowdhury, NR, Jansen, PLM & Oude Elferink, RPJ 1994, 'Discrimination between Crigler-Najjar type I and II by expression of mutant bilirubin uridine diphosphate-glucuronosyltransferase', Journal of Clinical Investigation, vol. 94, no. 6, pp. 2385-2391.
Seppen, Jurgen ; Bosma, Piter J. ; Goldhoorn, Bart G. ; Bakker, Conny T M ; Roy-Chowdhury, Jayanta ; Chowdhury, Namita Roy ; Jansen, Peter L M ; Oude Elferink, Ronald P J. / Discrimination between Crigler-Najjar type I and II by expression of mutant bilirubin uridine diphosphate-glucuronosyltransferase. In: Journal of Clinical Investigation. 1994 ; Vol. 94, No. 6. pp. 2385-2391.
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abstract = "Crigler-Najjar (CN) disease is classified into two subtypes, type I and II. The molecular basis for the difference between these types is not well understood. Several mutations in the bilirubin UDP-glucuronosyltransferase (B-UGT) gene of six CN type I and two CN type II patients were identified. Recombinant cDNAs containing these mutations were expressed in COS cells. B- UGT activity was measured using HPLC and the amount of expressed protein was quantitated using a sandwich ELISA. This enabled us to determine the specific activities of the expressed enzymes. All type I patients examined had mutations in the B-UGT1 gene that lead to completely inactive enzymes. The mutations in the B-UGT1 gene of patients with CN type II only partially inactivated the enzyme. At saturating concentrations of bilirubin (75 μM) CN type II patient A had 4.4±2{\%} residual activity and CN type II patient B had 38±2{\%} residual activity. Kinetic constants for the glucuronidation of bilirubin were determined. The affinities for bilirubin of B-UGT1 expressed in COS cells and B-UGT from human liver microsomes were similar with K(m) of 5.1±0.9 μM and 7.9±5.3 μM, respectively. B-UGT1 from patient B had a tenfold decreased affinity for bilirubin, K(m) = 56±23 μM. At physiological concentrations of bilirubin both type II patients will have a strongly reduced conjugation capacity, whereas type I patients have no B-UGT activity. We conclude that CN type I is caused by a complete absence of functional B- UGT and that in CN type II B-UGT activity is reduced.",
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AU - Bosma, Piter J.

AU - Goldhoorn, Bart G.

AU - Bakker, Conny T M

AU - Roy-Chowdhury, Jayanta

AU - Chowdhury, Namita Roy

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N2 - Crigler-Najjar (CN) disease is classified into two subtypes, type I and II. The molecular basis for the difference between these types is not well understood. Several mutations in the bilirubin UDP-glucuronosyltransferase (B-UGT) gene of six CN type I and two CN type II patients were identified. Recombinant cDNAs containing these mutations were expressed in COS cells. B- UGT activity was measured using HPLC and the amount of expressed protein was quantitated using a sandwich ELISA. This enabled us to determine the specific activities of the expressed enzymes. All type I patients examined had mutations in the B-UGT1 gene that lead to completely inactive enzymes. The mutations in the B-UGT1 gene of patients with CN type II only partially inactivated the enzyme. At saturating concentrations of bilirubin (75 μM) CN type II patient A had 4.4±2% residual activity and CN type II patient B had 38±2% residual activity. Kinetic constants for the glucuronidation of bilirubin were determined. The affinities for bilirubin of B-UGT1 expressed in COS cells and B-UGT from human liver microsomes were similar with K(m) of 5.1±0.9 μM and 7.9±5.3 μM, respectively. B-UGT1 from patient B had a tenfold decreased affinity for bilirubin, K(m) = 56±23 μM. At physiological concentrations of bilirubin both type II patients will have a strongly reduced conjugation capacity, whereas type I patients have no B-UGT activity. We conclude that CN type I is caused by a complete absence of functional B- UGT and that in CN type II B-UGT activity is reduced.

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