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

已明确证实Notch受体的EGF样重复序列的O-岩藻糖基化对该分子的信号功能有很大的影响。岩藻糖被糖基转移酶边缘延伸会影响不同配体的信号强度。相比之下，O-糖基化在Notch上提供了第二种类型的丰富的糖基化，以及木糖-木糖-葡萄糖-O-丝氨酸糖位的延伸，其重要性在很大程度上是未知的，因为编码参与其生物合成的酶的基因最近才被克隆。我们鉴定了编码木糖转移酶的人类基因，这些基因参与了木糖-木糖-葡萄糖三糖的构建。两个基因GXYLT1和GXYLT2编码催化第一个木糖转移的酶，而一个基因(XXYLT1)负责第二个木糖残基的添加。对果蝇的开创性实验表明，增加木糖化可以减弱Notch信号强度，哺乳动物的酶比果蝇的酶更有效。本研究旨在从细胞水平上了解不同的Notch木糖转移酶的作用方式及其对Notch信号的影响，特别是人类和果蝇转移第二木糖的酶的差异。这将通过确定酶的受体要求、解决亚细胞定位和建立最小催化结构域来完成，这是针对合作努力来解析晶体结构的。在细胞水平上，将使用报告分析在果蝇S2细胞中研究木糖化的影响。

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.