Functional properties of Notch specific xylosyltransferases and effects of xylosylation on cellular Notch signaling: A comparative study of human and Drosophila enzymes

Notch 特异性木糖基转移酶的功能特性以及木糖基化对细胞 Notch 信号传导的影响：人类和果蝇酶的比较研究

• 批准号: 243069142
• 负责人: Dr. Hendrikus Hans Bakker
• 金额: --
• 依托单位: Institut für Klinische Biochemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2014-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/243069142?language=en
• 关键词: Functional; properties; Notch; specific; xylosyltransferases

It has been clearly established that O-fucosylation of EGF like repeats of the Notch receptor has a strong impact on the signaling function of the molecule. Extension of fucose by the glycosyltransferase Fringe influences the signaling strength of different ligands. In contrast, the importance of O-glucosylation, which provides the second type of abundant glycosylation on Notch, and the extension to the xylose-xylose-glucose-O-Ser glycotope is largely unknown because the genes encoding the enzymes involved in its biosynthesis were only recently cloned. We identified the human genes encoding xylosyltransferases involved in the building of the xylose-xylose-glucose trisaccharide. Two genes named GXYLT1 and GXYLT2 encode enzymes catalyzing the first xylose transfer, while a single gene (XXYLT1) is responsible for the addition of the second xylose residue. Pioneering experiments in Drosophila revealed that increased xylosylation attenuates Notch signaling strength and that the mammalian enzymes are more potent than the Drosophila enzymes. This study is directed at understanding the mode of action of the different Notch xylosyltransferases and their influence on Notch signaling at cellular level, with a particular focus on the difference between the human and Drosophila enzymes transferring the second xylose. This will be done by determination of the acceptor requirements of the enzymes, resolving subcellular localization and establishing of the minimal catalytic domain, which is directed at a collaborative effort to resolve the crystal structure. At cellular level, influence of xylosylation will be studied using reporter assays in Drosophila S2 cells.

已经清楚地确定，Notch受体的EGF样重复序列的0-岩藻糖基化对分子的信号传导功能具有强烈的影响。通过糖基转移酶Fringe的岩藻糖延伸影响不同配体的信号强度。相比之下，O-葡萄糖基化的重要性，它提供了第二种类型的丰富的糖基化的Notch，并延伸到木糖-木糖-葡萄糖-O-Ser糖表位在很大程度上是未知的，因为编码的酶参与其生物合成的基因只是最近克隆。我们鉴定了编码参与木糖-木糖-葡萄糖三糖构建的木糖基转移酶的人类基因。命名为GXYLT 1和GXYLT 2的两个基因编码催化第一次木糖转移的酶，而单个基因（XXYLT 1）负责添加第二个木糖残基。在果蝇中进行的开创性实验表明，木糖基化增加会减弱Notch信号强度，并且哺乳动物酶比果蝇酶更有效。本研究旨在了解不同Notch木糖基转移酶的作用模式及其在细胞水平上对Notch信号传导的影响，特别关注人类和果蝇转移第二木糖的酶之间的差异。这将通过确定酶的受体要求、解析亚细胞定位和建立最小催化结构域来完成，这是针对解析晶体结构的协作努力。在细胞水平上，将使用果蝇S2细胞中的报告基因测定来研究木糖基化的影响。