Barbier O, Girard C, Breton R, Bélanger A, Hum D W
Oncology and Molecular Endocrinology Research Center, Laval University Medical Center (CHUL) and Laval University, Quebec, Quebec, G1V 4G2, Canada.
Biochemistry. 2000 Sep 26;39(38):11540-52. doi: 10.1021/bi000779p.
The recent cloning of several human and monkey UDP-glucuronosyltransferase (UGT) 2B proteins has allowed the characterization of these steroid metabolic enzymes. However, relatively little is known about the structure-function relationship, and the potential post-translational modifications of these proteins. The mammalian UGT2B proteins contain at least one consensus asparagine-linked glycosylation site NX(S/T). Endoglycosidase H digestion of the human and monkey UGT2B proteins demonstrates that only UGT2B7, UGT2B15, UGT2B17, and UGT2B20 are glycosylated. Although UGT2B15 and UGT2B20 contain three and four potential glycosylation sites, respectively, site-directed mutagenesis revealed that both proteins are glycosylated at the same first site. In both proteins, abolishing glycosylation decreased glucuronidation activity; however, the K(m) values and the substrate specificities were not affected. Despite the similarities between UGT2B15 and UGT2B20, UGT2B20 is largely more labile than UGT2B15. Treating HK293 cells stably expressing UGT2B20 with cycloheximide for 2 h decreased the enzyme activity by more than 50%, whereas the activity of UGT2B15 remained unchanged after 24 h. The UGT2B20 protein is unique in having an isoleucine at position 96 instead of an arginine as found in all the other UGT2B enzymes. Changing the isoleucine in UGT2B20 to an arginine stabilized enzyme activity, while the reciprocal mutation in UGT2B15 R96I produced a more labile enzyme. Secondary structure predictions of UGT2B proteins revealed a putative alpha-helix in this region in all the human and monkey proteins. This alpha-helix is shortest in UGT2B20; however, the helix is lengthened in UGT2B20 I96R. Thus, it is apparent that the length of the putative alpha-helix between residues 84 and 100 is a determining factor in the stability of UGT2B enzyme activity. This study reveals the extent and importance of protein glycosylation on UGT2B enzyme activity and that the effect of residue 96 on UGT2B enzyme stability is correlated to the length of a putative alpha-helix.
最近几种人类和猴子的尿苷二磷酸葡萄糖醛酸基转移酶（UGT）2B蛋白的克隆，使得对这些类固醇代谢酶的特性研究成为可能。然而，对于这些蛋白的结构-功能关系以及潜在的翻译后修饰，人们了解得还相对较少。哺乳动物的UGT2B蛋白至少含有一个共有天冬酰胺连接的糖基化位点NX(S/T)。对人类和猴子的UGT2B蛋白进行内切糖苷酶H消化表明，只有UGT2B7、UGT2B15、UGT2B17和UGT2B20被糖基化。尽管UGT2B15和UGT2B20分别含有三个和四个潜在的糖基化位点，但定点诱变显示这两种蛋白都在同一个第一位点被糖基化。在这两种蛋白中，去除糖基化会降低葡萄糖醛酸化活性；然而，米氏常数（K(m)）值和底物特异性不受影响。尽管UGT2B15和UGT2B20之间存在相似性，但UGT2B20在很大程度上比UGT2B15更不稳定。用环己酰亚胺处理稳定表达UGT2B20的HK293细胞2小时，酶活性降低超过50%，而UGT2B15的活性在24小时后保持不变。UGT2B20蛋白的独特之处在于其第96位是异亮氨酸，而在所有其他UGT2B酶中该位点是精氨酸。将UGT2B20中的异亮氨酸替换为精氨酸可稳定酶活性，而在UGT2B15中进行R96I的反向突变则产生了一种更不稳定的酶。UGT2B蛋白的二级结构预测显示，在所有人类和猴子的蛋白中，该区域都有一个假定的α螺旋。这个α螺旋在UGT2B20中最短；然而，在UGT2B20 I96R中该螺旋变长。因此，很明显，84至100位残基之间假定的α螺旋长度是UGT2B酶活性稳定性的一个决定因素。这项研究揭示了蛋白糖基化对UGT2B酶活性的影响程度和重要性，以及第96位残基对UGT2B酶稳定性的影响与假定的α螺旋长度相关。
