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ER-60 Domains Responsible for Interaction with Calnexin and Calreticulin

机译：ER-60负责与Calnexin和Caltretitulin相互作用的域

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ER-60 is a thiol oxidoreductase family protein of the endoplasmic reticulum that facilitates the oxidative folding of glycoproteins via interaction with calnexin (CNX) and calreticulin (CRT).In this study,we tried to identify the site of interaction with CNX and CRT in the ER-60 molecule.ER-60 was shown to be composed of at least four domains,named a,b,b',and a',by limited proteolysis.Recombinant fragments of ER-60,a,b',and a'c,were each expressed in Escherichia coli as an individual soluble folded protein that underwent a cooperative unfolding transition along a urea gradient.These fragments each gave the circular dichroism (CD) spectrum of the folded protein.On the other hand,fragment b,which did not undergo the cooperative unfolding transition along a urea gradient gel,did not show any sign of the folded structure on the CD measurement.However,subtraction of the spectra showed that the b domain was folded in wild-type ER-60 or abb'.Both a and a'c,which have a catalytic center CGHC motif,showed activity almost equivalent to half of that of wild-type ER-60.Extension from a or a'c to ab and abb' or b'a'c had little effect on their isomerase activity,suggesting that the b and b' domains hardly contribute to the catalytic activity of ER-60.The contribution of both the b and b' domains to the binding with CNX and CRT was revealed by surface plasmon resonance analysis and oxidative-refolding experiments of monoglucosylated RNase B with addition of the luminal domain of CNX.
机译：ER-60是内质网的硫醇氧化还原酶蛋白质，促进糖蛋白的氧化折叠通过与Calnexin（CNX）和Calretitulin（CRT）的相互作用。在本研究中，我们尝试识别与CNX和CRT的互动部位ER-60分子。显示-60，其由至少四个结构域组成，由有限的蛋白水解。ER-60，A，B'和A的特征碎片。 'C，各自在大肠杆菌中表达，作为一种单独可溶的折叠蛋白，沿尿素梯度进行合作展开过渡。这些片段各自给出了折叠蛋白的圆形二色性（CD）光谱。另一方面，碎片B，其中没有经历沿尿素梯度凝胶的合作展开过渡，没有显示CD测量上的折叠结构的任何迹象。然而，光谱的减法表明B域在野生型ER-60或ABB中折叠'，A和A'C，具有催化中心CGHC M OTIF，显示了几乎相当于野生型ER-60的一半的活动。从A或A'C到AB和ABB'或B'a'C对其异构酶活性影响，表明B和B '结构域几乎没有导致ER-60的催化活性。通过加入单葡糖基化的RNase B的表面等离子体共振分析和氧化 - 重折叠实验，揭示了B和B'结构域对与CNX和CRT的结合的贡献。 CNX的腔域。

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  • 来源
    《Biochemistry》 |2004年第27期|共11页
  • 作者

    Reiko Urade; Hirokazu Okudo; Hiroyuki Kato; Tatsuya Moriyama; Yukino Arakaki;

  • 作者单位

    Graduate School of Agriculture Kyoto University Uji Kyoto 611-0011 Japan;

    Graduate School of Agriculture Kyoto University Uji Kyoto 611-0011 Japan;

    Graduate School of Agriculture Kyoto University Uji Kyoto 611-0011 Japan;

    Graduate School of Agriculture Kyoto University Uji Kyoto 611-0011 Japan;

    Graduate School of Agriculture Kyoto University Uji Kyoto 611-0011 Japan;

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  • 正文语种 eng
  • 中图分类 生物化学;
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