Cloning, expression and characterization of Bombyx mori α1,6-fucosyltransferase
Cloning, expression and characterization of Bombyx mori α1,6-fucosyltransferase
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DOI：
10.1016/j.bbrc.2014.06.087
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发表时间：
2014-07-25
影响因子：
3.1
通讯作者：
Ikeda, Yoshitaka
中科院分区：
文献类型：
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作者：
Ihara, Hideyuki;Okada, Takahiro;Ikeda, Yoshitaka
尽管核心 α 1,6-岩藻糖基化在脊椎动物和无脊椎动物的 N-聚糖中都很常见，但与脊椎动物相比，无脊椎动物中负责的酶 α 1,6-岩藻糖基转移酶的特征要少得多。为了研究α 1,6-岩藻糖基转移酶在昆虫中的功能，我们克隆了来自家蚕的α 1,6-岩藻糖基转移酶（Bm α 1,6FucT）的cDNA，并表征了使用昆虫细胞系制备的重组酶。 Bm α 1,6FucT 的编码区由 1737 bp 组成，编码推导的氨基酸序列中的 578 个氨基酸，与其他 α 1,6-岩藻糖基转移酶显示出显着的相似性。酶活性测定表明，Bm α 1,6FucT 具有酶活性，尽管与人类酶相比活性较低。研究结果还表明，与人类酶不同，Bm α 1,6FucT 是 N-糖基化的，并形成二硫键同型二聚体。这些发现有助于更好地了解 α1,6-岩藻糖基化在无脊椎动物中的作用，也有助于开发昆虫细胞中重组糖蛋白的更有效的 N-糖基化工程。 (C) 2014 Elsevier Inc. 保留所有权利。
Although core alpha 1,6-fucosylation is commonly observed in N-glycans of both vertebrates and invertebrates, the responsible enzyme, alpha 1,6-fucosyltransferase, has been much less characterized in invertebrates compared to vertebrates. To investigate the functions of alpha 1,6-fucosyltransferase in insects, we cloned the cDNA for the alpha 1,6-fucosyltransferase from Bombyx mori (Bm alpha 1,6FucT) and characterized the recombinant enzyme prepared using insect cell lines. The coding region of Bm alpha 1,6FucT consists of 1737 bp that code for 578 amino acids of the deduced amino acid sequence, showing significant similarity to other alpha 1,6-fucosyltransferases. Enzyme activity assays demonstrated that Bm alpha 1,6FucT is enzymatically active in spite of being less active compared to the human enzyme. The findings also indicate that Bm alpha 1,6FucT, unlike human enzyme, is N-glycosylated and forms a disulfide-bonded homodimer. These findings contribute to a better understanding of roles of alpha 1,6-fucosylation in invertebrates and also to the development of the more efficient engineering of N-glycosylation of recombinant glycoproteins in insect cells. (C) 2014 Elsevier Inc. All rights reserved.