The fuc1 gene product (20 kDa FUC1) of Pisum sativum has no α-L-fucosidase activity
The fuc1 gene product (20 kDa FUC1) of Pisum sativum has no α-L-fucosidase activity
复制标题
豌豆的fuc1基因产物（20 kDa FUC1）没有α-L-岩藻糖苷酶活性
作者：
T. Tarragó;I. Martínez;M. Torrent;A. Codina;E. Giralt;P. Puigdomènech;D. Ludevid
从豌豆（Pisum sativum L. cv Alaska）上胚轴纯化的 α-L-岩藻糖苷酶先前被描述为 20 kDa 的细胞壁酶，可水解木葡聚糖寡糖片段的末端 α-L-岩藻糖苷键。进一步分离 cDNA 和基因组拷贝并进行测序。 cDNA 和基因组克隆 (fuc1) 的预测产物是含有信号肽和 5 个半胱氨酸的 20 kDa 蛋白质。这是第一个在植物中克隆的α-L-岩藻糖苷酶基因，但其岩藻糖苷酶活性尚未得到证实。在这里，我们的生化和免疫分析表明，fuc1 不编码 α-L-岩藻糖苷酶。在大肠杆菌、昆虫细胞和拟南芥中表达的豌豆fuc1产生不具有α-L-岩藻糖苷酶活性的重组蛋白。豌豆植物具有内源 α-L-岩藻糖苷酶活性，但该酶不能被针对大肠杆菌中表达的重组 FUC1 蛋白产生的抗体识别。相比之下，抗体免疫沉淀了无活性的 20 kDa 蛋白质。通过对豌豆蛋白提取物进行色谱分析，我们从 20 kDa 蛋白组分中分离出了 α-L-岩藻糖苷酶活性组分。我们得出的结论是，α-L-岩藻糖苷酶活性并非归因于 20 kDa FUC1 蛋白。提出了fuc1基因产物的新功能，现命名为PIP20（来自豌豆的蛋白酶抑制剂）。
An α-L-fucosidase purified from pea (Pisum sativum L. cv Alaska) epicotyl was previously described as a cell wall enzyme of 20 kDa that hydrolyses terminal α-L-fucosidic linkages from oligosaccharide fragments of xyloglucan. cDNA and genomic copies were further isolated and sequenced. The predicted product of the cDNA and the genomic clone (fuc1), was a 20 kDa protein containing a signal peptide and five cysteines. This was the first α-L-fucosidase gene to be cloned in plants but its fucosidase activity has not been demonstrated. Here, our biochemical and immuno analyses suggest that fuc1 does not encode an α-L-fucosidase. Pea fuc1 expressed in Escherichia coli, insect cells and Arabidopsis thaliana produced recombinant proteins without α-L-fucosidase activity. Pea plants had endogenous α-L-fucosidase activity, but the enzyme was not recognised by an antibody produced against recombinant FUC1 protein expressed in E. coli. In contrast, the antibody immunoprecipitated a 20 kDa protein which was inactive. By chromatographic analysis of pea protein extracts, we separated α-L-fucosidase-active fractions from the 20 kDa protein fractions. We conclude that the α-L-fucosidase activity is not attributable to the 20 kDa FUC1 protein. A new function for fuc1 gene product, now named PIP20 (for protease inhibitor from pea) is proposed.