Role of cysteine residues in the function of human UDP glucuronosyltransferase isoform 1A1 (UGT1A1)
Role of cysteine residues in the function of human UDP glucuronosyltransferase isoform 1A1 (UGT1A1)
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DOI：
10.1042/bj20050381
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发表时间：
2005-12-15
影响因子：
4.1
通讯作者：
Roy-Chowdhury, N
中科院分区：
文献类型：
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作者：
Ghosh, SS;Lu, Y;Roy-Chowdhury, N
由UGT 1A 1 [UGT（UDP葡萄糖醛酸转移酶）亚型1A 1，EC 2.4.1.17]催化的胆红素葡萄糖醛酸化对于胆红素的胆汁消除至关重要。UGT 1A 1缺乏会导致CN-1（Crigler-Najjar综合征1型），其特征是潜在致命的未结合高胆红素血症。两名CN-1患者的UGT 1A 1核苷酸序列分析显示，患者A为nt 530 G -> A纯合子（其中nt 530 G -> A表示核苷酸530处鸟嘌呤到腺嘌呤的转换）突变，预测C177 Y置换，并且患者B在一个等位基因上具有nt 466 T -> C突变，在另一个等位基因上具有nt 1070 A -> G突变，分别预测C156 R和Q357 R取代。成熟人UGT 1A 1的所有II半胱氨酸残基在其他人UGT同种型以及大鼠、小鼠和恒河猴UGT 1A 1中高度保守，表明其功能重要性。突变UGT 1A 1质粒的表达表明，位于内质网池内的7个半胱氨酸残基（包括患者A和13中突变的那些）中的任何一个被取代都会消除UGT 1A 1活性或显著增加其表观K值。胆红素C-末端胞质尾部内的三个半胱氨酸残基的取代对基础UGT 1A 1活性的影响最小，但阻止UDP-G1 cNAc激活UGT 1A 1。N-乙基马来酰亚胺未抑制天然微粒体中的UGT 1A 1活性，但阻止UGT 1A 1被UDP-GlcNAc激活，并抑制毛地黄皂苷透化微粒体中的活性。二硫苏糖醇不影响人肝微粒体中的UGT 1A 1活性。总之，结果表明，人UGT 1A 1脑池内区域内7个半胱氨酸残基的游离巯基（而非二硫键）对其催化活性很重要，而胞质结构域中的半胱氨酸残基可能参与UDP-GlcNAc对其的生理激活。
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 I I cysteine residues of mature human UGT1A1 are highly conserved in other human UGT isoforms, and in rat, mouse and Rhesus monkey UGT I A I, 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 13) abolished UGT1A1 activity or markedly increased its apparent K. for bilirubin. Substitution of the three cysteine residues within the C-terminal cytosolic tail had minimal effect on basal UGT I A I activity, but prevented UGT1A1 activation by UDP-G1cNAc. N-Ethylmaleimide did not inhibit UGT1A1 activity in native microsomes, but prevented UGT1A1 activation by UDP-GIcNAc 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-GIcNAc.