Interactions of substrate with calreticulin, an endoplasmic reticulum chaperone
Interactions of substrate with calreticulin, an endoplasmic reticulum chaperone
复制标题
DOI：
10.1074/jbc.m209132200
复制
发表时间：
2003-02-21
影响因子：
4.8
通讯作者：
Surolia, A
中科院分区：
文献类型：
--
作者：
Kapoor, M;Srinivas, H;Surolia, A
钙网蛋白是真核生物内质网中发现的分子伴侣，其与N-糖基化多肽的相互作用由靶糖蛋白上存在的聚糖Glc（1）Man（7-9）GlcNAc（2）介导。在这里，我们报告的热力学参数的相互作用与二糖，三糖，四糖，这是截短版本的葡萄糖基化臂的葡萄糖（1）曼（7-9）GlcNAc（2），确定了定量技术的等温滴定量热法。该方法提供了结合常数（K-B）和结合焓变化（Δ H（B）度）以及反应的化学计量的直接估计。与过去的推测不同，这些研究明确表明，钙网蛋白每个分子只有一个位点用于结合其互补的葡萄糖基化配体。虽然葡萄糖本身的结合是不可检测的，但当葡萄糖以α-1，3键与ManalphaMe结合时，如Glcalpha 1 - 3 ManalphaMe中那样，在279 K下获得4.19 × 10（4）m（-1）的结合常数。从二糖到三糖的结合常数增加25倍，从三糖到四糖的结合常数增加一倍，表明寡糖的整个Glcalpha 1 - 3 Manalpha 1 - 2 Manalpha 1-ManalphaMe结构被钙网蛋白识别。由此获得的热力学参数支持的建模研究，这表明，增加的氢键和货车德瓦尔斯相互作用的数量发生的寡糖的大小增加。此外，一些新的发现有关的糖配体的钙网蛋白维斯6维斯豆类凝集素，具有相同的倍，这种伴侣，进行了讨论。
Calreticulin is a molecular chaperone found in the endoplasmic reticulum in eukaryotes, and its interaction with N-glycosylated polypeptides is mediated by the glycan Glc(1)Man(7-9)GlcNAc(2) present on the target glycoproteins. Here, we report the thermodynamic parameters of its interaction with di-, tri-, and tetrasaccharide, which are truncated versions of the glucosylated arm of Glc(1)Man(7-9)GlcNAc(2), determined by the quantitative technique of isothermal titration calorimetry. This method provides a direct estimate of the binding constants (K-b) and changes in enthalpy of binding (DeltaH(b)degrees) as well as the stoichiometry of the reaction. Unlike past speculations, these studies demonstrate unambiguously that calreticulin has only one site per molecule for binding its complementary glucosylated ligands. Although the binding of glucose by itself is not detectable, a binding constant of 4.19 X 10(4) m(-1) at 279 K is obtained when glucose occurs in alpha-1,3 linkage to ManalphaMe as in Glcalpha1-3ManalphaMe. The binding constant increases by 25-fold from di- to trisaccharide and doubles from tri- to tetrasaccharide, demonstrating that the entire Glcalpha1-3Manalpha1-2Manalpha1-ManalphaMe structure of the oligosaccharide is recognized by calreticulin. The thermodynamic parameters thus obtained were supported by modeling studies, which showed that increased number of hydrogen bonds and van der Waals interactions occur as the size of the oligosaccharide is increased. Also, several novel findings about the recognition of saccharide ligands by calreticulin vis 6 vis legume lectins, which have the same fold as this chaperone, are discussed.