BACKGROUND Previous structural analyses showed that human α1,6-fucosyltransferase, FUT8 contains a catalytic domain along with two additional domains, N-terminal α-helical domain and C-terminal Src homology 3 domain, but these domains are unique to FUT8 among glycosyltransferases. The role that these domains play in formation of the active form of FUT8 has not been investigated. This study reports on attempts to determine the involvement of these domains in the functions of FUT8. METHODS Based on molecular modeling, the domain mutants were constructed by truncation and site-directed mutagenesis, and were heterologously expressed in Sf21 or COS-1 cells. The mutants were analyzed by SDS-PAGE and assayed for enzymatic activity. In vivo cross-linking experiments by introducing disulfide bonds were also carried out to examine the orientation of the domains in the molecular assembly. RESULTS Mutagenesis and molecular modeling findings suggest that human FUT8 potentially forms homodimer in vivo via intermolecular hydrophobic interactions involving α-helical domains. Truncation or site-directed mutagenesis findings indicated that α-helical and SH3 domains are all required for enzymatic activity. In addition, in vivo cross-linking experiments clearly indicated that the SH3 domain located in close proximity to the α-helical domain in an intermolecular manner. CONCLUSIONS α-Helical and SH3 domains are required for a fully active enzyme, and are also involved in homophilic dimerization, which probably results in the formation of the active form of human FUT8. GENERAL SIGNIFICANCE α-Helical and SH3 domains, which are not commonly found in glycosyltransferases, play roles in the formation of the functional quaternary structure of human FUT8.

中文翻译：

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α-螺旋和 Src 同源性 3 结构域参与人 α1,6-岩藻糖基转移酶 FUT8 的分子组装和酶活性。

背景 先前的结构分析表明，人 α1,6-岩藻糖基转移酶 FUT8 包含一个催化结构域以及两个额外的结构域，即 N 端 α 螺旋结构域和 C 端 Src 同源 3 结构域，但这些结构域在糖基转移酶中是 FUT8 独有的。这些结构域在 FUT8 活性形式的形成中所起的作用尚未得到研究。本研究报告了试图确定这些域参与 FUT8 功能的尝试。方法基于分子模型，通过截短和定点诱变构建结构域突变体，并在Sf21或COS-1细胞中异源表达。通过SDS-PAGE分析突变体并测定酶活性。还进行了通过引入二硫键的体内交联实验来检查分子组装中结构域的方向。结果 诱变和分子建模结果表明，人类 FUT8 可能通过涉及 α-螺旋结构域的分子间疏水相互作用在体内形成同型二聚体。截短或定点诱变结果表明，α-螺旋结构域和 SH3 结构域都是酶活性所必需的。此外，体内交联实验清楚地表明，SH3结构域以分子间方式紧邻α-螺旋结构域。结论 α-Helical 和 SH3 结构域是完全活性酶所必需的，并且也参与同源性二聚化，这可能导致人 FUT8 活性形式的形成。