Co- and/or post-translational modifications are critical for TCH4 XET activity
Co- and/or post-translational modifications are critical for TCH4 XET activity
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DOI：
10.1046/j.1365-313x.1998.00239.x
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发表时间：
1998-08-01
期刊：
影响因子：
7.2
通讯作者：
Braam, J
中科院分区：
文献类型：
--
作者：
Campbell, P;Braam, J
TCH4编码拟南芥的木葡聚糖内糖基转移酶(XET)。XETs内切裂解木葡聚糖聚合物，木聚糖是植物细胞壁的主要结构成分之一。因此，XET功能可能影响细胞形态和植物形态发生。为了深入了解TCH4的生化功能，我们定义了最佳XET活性的结构要求。重组杆状病毒被设计成产生不同形式的TCH4。TCH4蛋白被设计成在胞浆中合成，因此缺乏正常的协同和翻译后修饰，实际上是不活跃的。TCH4蛋白，有或没有多组氨酸标签，含有完整的H端，被定向到分泌途径。因此，正如预测的那样，TCH4的高端区起到信号肽的作用。在二硫苏糖醇(DTT)存在的情况下，通过SDS-PAGE的迁移率变化监测到TCH4至少有一个二硫键。这种二硫键(S)对于XET的全部活性是必不可少的。TCH4在体内是糖基化的；去除N-连接糖基化的糖苷酶消除了98%的XET活性。因此，共翻译和/或翻译后修饰对于最佳的TCH4 XET活性至关重要。此外，利用定点突变，我们证明了保守的DEIDFEFL蛾(E97)的第一个谷氨酸残基是活性所必需的。谷氨酰胺在这个位置的变化导致蛋白质失活；天冬氨酸的变化导致蛋白质的错误定位。这些数据支持这样一种假设，即与芽孢杆菌β-葡聚糖酶类似，该区域可能是XET酶的活性部位。
TCH4 encodes a xyloglucan endotransglycosylase (XET) of Arabidopsis thaliana. XETs endolytically cleave and religate xyloglucan polymers; xyloglucan is one of the primary structural components of the plant cell wall. Therefore, XET function may affect cell shape and plant morphogenesis. To gain insight into the biochemical function of TCH4, we defined structural requirements for optimal XET activity. Recombinant baculoviruses were designed to produce distinct forms of TCH4. TCH4 protein engineered to be synthesized in the cytosol and thus lack normal co-and post-translational modifications is virtually inactive. TCH4 proteins, with and without a polyhistidine tag, that harbor an intact hi-terminus are directed to the secretory pathway. Thus, as predicted, the hi-terminal region of TCH4 functions as a signal peptide. TCH4 is shown to have at least one disulfide bond as monitored by a mobility shift in SDS-PAGE in the presence of dithiothreitol (DTT). This disulfide bond(s) is essential for full XET activity. TCH4 is glycosylated in vivo; glycosidases that remove N-linked glycosylation eliminated 98% of the XET activity. Thus, co- and/or post-translational modifications are critical for optimal TCH4 XET activity. Furthermore, using site specific mutagenesis, we demonstrated that the first glutamate residue of the conserved DEIDFEFL moth (E97) is essential for activity. A change to glutamine at this position resulted in an inactive protein; a change to aspartic acid caused protein mislocalization. These data support the hypothesis that, in analogy to Bacillus beta-glucanases, this region may be the active site of XET enzymes.