Modification of Epidermal Growth Factor-like Repeats withO-Fucose
Modification of Epidermal Growth Factor-like Repeats withO-Fucose
复制标题
用 O-岩藻糖修饰表皮生长因子样重复序列
DOI：
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发表时间：
2001
影响因子：
4.8
通讯作者：
R. Haltiwanger
中科院分区：
文献类型：
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作者：
Yang Wang;L. Shao;S. Shi;Reed J. Harris;M. Spellman;P. Stanley;R. Haltiwanger
O-岩藻糖修饰存在于许多细胞表面和分泌蛋白的表皮生长因子样重复序列上。 O-岩藻糖聚糖在配体诱导的受体信号传导中发挥重要作用。例如，β1,3-N-乙酰葡糖胺基转移酶 Fringe 对 Notch 上的 O-岩藻糖进行延伸，从而调节 Notch 对其配体作出反应的能力。将 O-岩藻糖添加到表皮生长因子样重复序列中的酶 GDP-岩藻糖蛋白 O-岩藻糖基转移酶 (O-FucT-1) 先前从中国仓鼠卵巢 (CHO) 细胞中纯化。在这里，我们报告了编码人 O-FucT-1 的 cDNA 的分离。从纯化的 CHOO-FucT-1 的 N 端序列分析推导出的探针用于筛选人心脏 cDNA 文库和表达序列标签和基因组数据库。该 cDNA 包含一个开放阅读框，编码 388 个氨基酸的蛋白质，具有典型的 II 型膜方向的预测 N 端跨膜序列。同样，从小鼠肝脏cDNA文库的表达序列标签和5'-快速扩增的cDNA末端获得的小鼠同源物编码393个氨基酸的II型跨膜蛋白，与人O-FucT-1具有90.4%的同一性。在果蝇和秀丽隐杆线虫中也发现了同源物，与人 O-FucT-1 的同源性分别为 41.2% 和 29.4%。人类基因 (POFUT1) 位于 20 号染色体上的 PLAGL2 和 KIF3B 之间，靠近 20p11 的着丝粒。小鼠基因 (Pofut1) 位于小鼠 2 号染色体同源区域的 Plagl2 附近。POFUT1 基因转录物在所有检查的组织中都有表达，这与修饰的广泛定位一致。可溶形式的人 O-FucT-1 在昆虫细胞中的表达产生了预测分子量的蛋白质，其 O-FucT-1 动力学和酶学特性与从 CHO 细胞中纯化的 O-FucT-1 相似。编码蛋白 O-岩藻糖基转移酶 I 的基因的鉴定现在使突变策略成为可能，以检查不寻常的 O-岩藻糖翻译后修饰的功能。
The O-fucose modification is found on epidermal growth factor-like repeats of a number of cell surface and secreted proteins. O-Fucose glycans play important roles in ligand-induced receptor signaling. For example, elongation of O-fucose on Notch by the β1,3-N-acetylglucosaminyltransferase Fringe modulates the ability of Notch to respond to its ligands. The enzyme that addsO-fucose to epidermal growth factor-like repeats, GDP-fucose protein O-fucosyltransferase (O-FucT-1), was purified previously from Chinese hamster ovary (CHO) cells. Here we report the isolation of a cDNA that encodes human O-FucT-1. A probe deduced from N-terminal sequence analysis of purified CHOO-FucT-1 was used to screen a human heart cDNA library and expressed sequence tag and genomic data bases. The cDNA contains an open reading frame encoding a protein of 388 amino acids with a predicted N-terminal transmembrane sequence typical of a type II membrane orientation. Likewise, the mouse homolog obtained from an expressed sequence tag and 5′-rapid amplification of cDNA ends of a mouse liver cDNA library encodes a type II transmembrane protein of 393 amino acids with 90.4% identity to humanO-FucT-1. Homologs were also found inDrosophila and Caenorhabditis elegans with 41.2 and 29.4% identity to human O-FucT-1, respectively. The human gene (POFUT1) is on chromosome 20 betweenPLAGL2 and KIF3B, near the centromere at 20p11. The mouse gene (Pofut1) maps near Plagl2 on a homologous region of mouse chromosome 2. POFUT1 gene transcripts were expressed in all tissues examined, consistent with the widespread localization of the modification. Expression of a soluble form of human O-FucT-1 in insect cells yielded a protein of the predicted molecular weight with O-FucT-1 kinetic and enzymatic properties similar to those of O-FucT-1 purified from CHO cells. The identification of the gene encoding proteinO-fucosyltransferase I now makes possible mutational strategies to examine the functions of the unusual O-fucose post-translational modification.