O-glycosylation of cysteine-rich modules

我们研究的总体目标是确定结构、修饰部位、生物合成和功能 与两个富含半胱氨酸的结构域相连的糖(O-聚糖)：表皮生长因子样重复序列(EGF)和 凝血酶原蛋白1型重复序列(TSR)。EGF和TSR都存在于许多细胞表面和 胞外蛋白。我们重点研究了对EGF的两种修饰，O-岩藻糖(添加了O-蛋白- 岩藻糖基转移酶1(POFUT1)和O-葡萄糖(添加蛋白O-葡萄糖基转移酶1，POGLUT1)。我们 还研究了O-岩藻糖(添加了POFUT2)对TSR的影响。这三种酶都可以修饰特定的羟基 丝氨酸或苏氨酸在EGF或TSR内具有良好特征的共同序列中。这些基因的击倒 酶对小鼠的胚胎是致命的，POFUT1或POGLUT1的突变导致人类基因 疾病，证明了这些修饰的生物学重要性。我们建议解决以下几个问题 关于这些O-葡聚糖修饰的蛋白质的问题尚未回答。例如，我们想要确定 O-岩藻糖蛋白质组。为了了解O-岩藻糖的功能，我们需要知道哪些蛋白质是被修饰的。 而使用POFUT1和POFUT2的共同序列进行数据库搜索已经成功 鉴定了许多靶蛋白，最近的数据显示了一个不同的富含半胱氨酸的结构域，由O- Fucose，这是Multimerin1中的一个EMI结构域，在我们的搜索中没有发现。在这里，我们描述了一个不偏不倚的 利用生物正交探针6-炔基岩藻糖(6AF)鉴定O-岩藻糖化蛋白的方法，即 在细胞中高效和优先地结合到O-岩藻糖基化的蛋白质中。我们预计将确认一些 POFUT1和POFUT2的预测底物，这将为研究提供新的靶点，也将识别 没有出现在数据库搜索中的蛋白质。我们还想研究一下它的结构和功能 NOTCH3上的O-岩藻糖和O-葡萄糖修饰。在过去的几年里，我们已经绘制了O-岩藻糖 用糖蛋白组学方法确定糖链与NOTCH1和NOTCH2上的糖链结合 在生物学上具有重要作用。已知NOTCH3在血管内稳态和突变中发挥重要作用 在NOTCH3中引起CADASIL，一种毁灭性的常染色体显性遗传性血管疾病。分子 导致CADASIL的机制尚不清楚。CADASIL突变增加或移除半胱氨酸 NOTCH3 EGF，预计会破坏糖基化。我们将绘制NOTCH3上的糖基化位点图 血管平滑肌细胞中CADASIL基因突变对其糖基化的影响 状态。最后，POFUT1和POFUT2都是位于内质腔的可溶性酶 但它们的供体底物GDP-岩藻糖是在胞浆中合成的。目前还不知道是如何做到的 GDP-岩藻糖被运送到急诊室。在这里，我们描述一个CRISPR-CAS9筛查来识别推定的ER GDP-岩藻糖运输机。这些研究将扩大我们对O-的结构和功能的理解。 富含半胱氨酸结构域的葡聚糖及其在疾病中的潜在作用。

The overall goal of our research is to determine the structures, modification sites, biosynthesis, and functions of sugars (O-glycans) linked to two cysteine-rich domains: Epidermal Growth Factor-like Repeats (EGFs) and Thrombospondin Type 1 Repeats (TSRs). Both EGFs and TSRs are found in numerous cell-surface and extracellular proteins. We focus on two modifications on EGFs, O-fucose (added by Protein O- fucosyltransferase 1, POFUT1) and O-glucose (added by Protein O-glucosyltransferase 1, POGLUT1). We also study O-fucose (added by POFUT2) on TSRs. All three enzymes modify hydroxyl groups of specific serines or threonines in well characterized consensus sequences within EGFs or TSRs. Knockouts of these enzymes are embryonic lethal in mice, and mutations in POFUT1 or POGLUT1 cause human genetic disorders, demonstrating the biological importance of these modifications. We propose to address several unanswered questions about the proteins modified by these O-glycans. For instance, we want to determine the O-fucose proteome. To understand O-fucose function, we need to know which proteins are modified. While database searches with the consensus sequences for POFUT1 and POFUT2 have successfully identified many target proteins, recent data has revealed a different cysteine-rich domain modified with O- fucose, an EMI domain in Multimerin1, which was not identified in our searches. Here we describe an unbiased approach to identify O-fucosylated proteins using a bioorthogonal probe, 6-alkynyl fucose (6AF), that is efficiently and preferentially incorporated into O-fucosylated proteins in cells. We expect to confirm a number of predicted substrates for POFUT1 and POFUT2, which will provide novel targets to study, but also to identify proteins that did not appear in database searches. We also want to examine the structure and function of O-fucose and O-glucose modifications on NOTCH3. In the past few years we have mapped O-fucose glycans to sites on NOTCH1 and NOTCH2 using glycoproteomic methods and determined which sites play biologically important roles. NOTCH3 is known to play important roles in vascular homeostasis, and mutations in NOTCH3 cause CADASIL, a devastating, autosomal dominant vascular disorder. The molecular mechanisms resulting in CADASIL are poorly understood. CADASIL mutations add or remove cysteines in NOTCH3 EGFs, which are predicted to disrupt glycosylation. We will map glycosylation sites on NOTCH3 isolated from vascular smooth muscle cells and evaluate how CADASIL mutations affect its glycosylation status. Finally, POFUT1 and POFUT2 are both soluble enzymes located in the lumen of the endoplasmic reticulum (ER), but their donor substrate, GDP-fucose, is synthesized in the cytosol. It is not known how GDP-fucose is transported into the ER. Here we describe a CRISPR-Cas9 screen to identify the putative ER GDP-fucose transporter. These studies will extend our understanding of the structure and function of O- glycans on cysteine-rich domains and their potential roles in diseases.