Mutagenesis of cellulase EGZ for studying the general protein secretory pathway in Erwinia chrysanthemi
Mutagenesis of cellulase EGZ for studying the general protein secretory pathway in Erwinia chrysanthemi
复制标题
纤维素酶EGZ诱变研究菊欧文氏菌一般蛋白分泌途径
作者：
Béatrice Py;M. Chippaux;F. Barras
来自欧文氏菌的内切葡聚糖酶Z（EGZ）的细胞外分泌由所谓的Out一般分泌途径介导，并且可能涉及通过一种或多种Out蛋白识别EGZ携带的结构信息。本研究的目的是探讨EGZ的结构与分泌性之间的关系。EGZ由两个独立的结构域组成，位于N和C近端侧，由富含Ser/Thr的区域分开，分别负责催化和纤维素结合。通过研究修饰的EGZ衍生物的可分泌性来研究分泌区域（“靶向信号”）的存在。这些由缺失或肽插入产生，并且通过使用上文引用的结构域组织作为指导来设计。催化和/或纤维素结合试验表明，所有蛋白质都至少表现出一个功能性EGZ结构域，而免疫印迹分析证实，插入或缺失都不会导致严重错误折叠的蛋白质。相反，在E.查里岛这表明至少存在两个分泌基序，一个位于每个功能域中。随后测试了富含Ser/Thr的接头区的作用。因此，研究了两种含有接头区的蛋白质和一种缺乏接头区的蛋白质，所述接头区的长度通过添加8个和18个额外的残基而增加。所有三种都表现出内切葡聚糖酶活性和纤维素结合能力，证实了EGZ/多糖相互作用背景下结构域的独立性。而E.查里岛总的来说，我们对EGZ的研究结果（i）表明存在多个分泌相关位点，这些位点依次起作用或形成单个三维分泌信号，（ii）表明分泌能力不是由两个功能结构域中的任何一个单独决定的，（iii）揭示接头区在分泌中起作用。我们建议，所有EGZ衍生物受损的识别步骤，其性质进行了讨论。
Extracellular secretion of endoglucanase Z (EGZ) from Erwinia chrysanthemi is mediated by the so‐called Out general secretion pathway and, presumably, involves recognition of EGZ‐carried structural information by one or more of the Out proteins. Investigating the relationships between structure and secretability of EGZ was the purpose of the present work. EGZ is made of two independent domains, located at the N‐and C‐proximal sides, separated by a Ser/Thr‐rich region, which are responsible for catalysis and cellulose‐binding, respectively. The existence of a secretion region (‘targeting signal’) was investigated by studying the secretability of modified EGZ derivatives. These resulted from deletion or peptide insertion and were designed by using the domain organization cited above as a guide. Catalytic and/or cellulose‐binding tests showed that all proteins exhibited at least a functional EGZ domain while immunoblot analyses confirmed that neither the insertions nor the deletions led to grossly misfolded proteins. In contrast, all of the proteins lost their secretability in E. chrysanthemi. This suggested that at least two secretion motifs existed, one lying within each functional domain. The role of the Ser/Thr‐rich linker region was subsequently tested. Accordingly, two proteins containing a linker region whose length was increased by the addition of 8 and 18 additional residues and one protein lacking the linker region were studied. All three exhibited endoglucanase activity and cellulose‐binding ability, confirming the independence of the domains within the context of EGZ/polysaccharide interaction. In contrast, none was secreted by E. chrysanthemi. Collectively, our results with EGZ (i) suggest the occurrence of multiple secretion‐related sites either acting sequentially or forming a single three‐dimensional secretion signal, (ii) show that secretability is not determined by either one of the two functional domains alone, and (iii) reveal that the linker region plays a role in secretion. We propose that all EGZ derivatives were impaired in the recognition step, the nature of which is discussed.
大肠杆菌 K12 中蛋白质输出的遗传分析。
影响因子：
16.6
作者：
Benson,SA;Hall,MN;Silhavy,TJ
通讯作者：
Silhavy,TJ