DISCRIMINATION BETWEEN CRIGLER-NAJJAR TYPE-I AND TYPE-II BY EXPRESSION OF MUTANT BILIRUBIN URIDINE DIPHOSPHATE-GLUCURONOSYLTRANSFERASE
DISCRIMINATION BETWEEN CRIGLER-NAJJAR TYPE-I AND TYPE-II BY EXPRESSION OF MUTANT BILIRUBIN URIDINE DIPHOSPHATE-GLUCURONOSYLTRANSFERASE
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DOI：
10.1172/jci117604
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发表时间：
1994-12-01
影响因子：
15.9
通讯作者：
ELFERINK, RPJO
中科院分区：
文献类型：
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作者：
SEPPEN, J;BOSMA, PJ;ELFERINK, RPJO
Crigler-Najjar（CN）病分为两种亚型，I型和II型。这些类型之间的差异的分子基础还没有很好地理解。胆红素UDP-葡萄糖醛酸基转移酶（B-UGT）基因的6个CN I型和CN II型患者的几个突变被确定。在COS细胞中表达含有这些突变的重组cDNA。使用HPLC测量B-UGT活性，并使用夹心ELISA定量表达的蛋白质的量。这使我们能够确定所表达的酶的比活性。检查的所有I型患者都有B-UGT 1基因突变，导致酶完全失活。II型CN患者的B-UGT 1基因突变仅部分灭活了该酶。在饱和胆红素浓度（75 μ M）时，CN II型患者A有4.4+/-2%的残余活性，CN II型患者B有38+/-2%的残余活性。COS细胞中表达的B-UGT 1和人肝微粒体中表达的B-UGT对胆红素的亲和力相似，Km分别为5.1+/-0.9 mu M和7.9+/-5.3 mu M。来自患者B的B-UGT 1对胆红素的亲和力降低了10倍，Km = 56+/-23 μ M。在生理浓度的胆红素下，两种II型患者的结合能力都大大降低，而I型患者没有B-UGT活性。我们得出结论，CN I型是由完全缺乏功能性B-UGT引起的，CN II型B-UGT活性降低。
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 mu M) 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 mu M and 7.9+/-5.3 mu M, respectively. B-UGT1 from patient B had a tenfold decreased affinity for bilirubin, K-m = 56+/-23 mu M.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.