Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA.
PLoS Genet. 2013 Jun;9(6):e1003547. doi: 10.1371/journal.pgen.1003547. Epub 2013 Jun 6.
The Notch signaling pathway controls a large number of processes during animal development and adult homeostasis. One of the conserved post-translational modifications of the Notch receptors is the addition of an O-linked glucose to epidermal growth factor-like (EGF) repeats with a C-X-S-X-(P/A)-C motif by Protein O-glucosyltransferase 1 (POGLUT1; Rumi in Drosophila). Genetic experiments in flies and mice, and in vivo structure-function analysis in flies indicate that O-glucose residues promote Notch signaling. The O-glucose residues on mammalian Notch1 and Notch2 proteins are efficiently extended by the addition of one or two xylose residues through the function of specific mammalian xylosyltransferases. However, the contribution of xylosylation to Notch signaling is not known. Here, we identify the Drosophila enzyme Shams responsible for the addition of xylose to O-glucose on EGF repeats. Surprisingly, loss- and gain-of-function experiments strongly suggest that xylose negatively regulates Notch signaling, opposite to the role played by glucose residues. Mass spectrometric analysis of Drosophila Notch indicates that addition of xylose to O-glucosylated Notch EGF repeats is limited to EGF14-20. A Notch transgene with mutations in the O-glucosylation sites of Notch EGF16-20 recapitulates the shams loss-of-function phenotypes, and suppresses the phenotypes caused by the overexpression of human xylosyltransferases. Antibody staining in animals with decreased Notch xylosylation indicates that xylose residues on EGF16-20 negatively regulate the surface expression of the Notch receptor. Our studies uncover a specific role for xylose in the regulation of the Drosophila Notch signaling, and suggest a previously unrecognized regulatory role for EGF16-20 of Notch.
Notch 信号通路在动物发育和成年体内稳态过程中控制着大量的生理过程。 Notch 受体的保守翻译后修饰之一是通过蛋白 O-葡萄糖基转移酶 1（POGLUT1；果蝇中的 Rumi）将一个 O-连接的葡萄糖添加到表皮生长因子样（EGF）重复序列中，具有 C-X-S-X-(P/A)-C 基序。果蝇、小鼠中的遗传实验以及果蝇体内结构功能分析表明，O-葡萄糖残基促进 Notch 信号转导。哺乳动物 Notch1 和 Notch2 蛋白上的 O-葡萄糖残基可通过特定的哺乳动物木糖基转移酶的功能添加一个或两个木糖残基而有效地延长。然而，木糖基化对 Notch 信号转导的贡献尚不清楚。在这里，我们确定了负责在 EGF 重复序列上向 O-葡萄糖添加木糖的果蝇酶 Shams。令人惊讶的是，缺失和功能获得实验强烈表明，木糖负调控 Notch 信号转导，与葡萄糖残基的作用相反。对果蝇 Notch 的质谱分析表明，O-葡萄糖基化的 Notch EGF14-20 上添加木糖是有限的。具有 Notch EGF16-20 中 O-葡萄糖基化位点突变的 Notch 转基因可重现 shams 缺失功能表型，并抑制人木糖基转移酶过表达引起的表型。在 Notch 去木糖基化减少的动物中进行抗体染色表明，EGF16-20 上的木糖残基负调控 Notch 受体的表面表达。我们的研究揭示了木糖在调控果蝇 Notch 信号中的特定作用，并提示 Notch 的 EGF16-20 具有以前未被认识到的调节作用。
