O-glycosylation of cysteine-rich modules

我们研究的总体目标是确定其结构、修饰位点、生物合成和功能 糖（O-聚糖）连接到两个富含半胱氨酸的结构域：表皮生长因子样重复序列（EGF）和 凝血酶敏感蛋白1型重复序列（TSR）。EGF和TSR都存在于许多细胞表面， 胞外蛋白质我们专注于EGF的两种修饰，O-岩藻糖（由蛋白O- 岩藻糖基转移酶1，POFUT 1）和O-葡萄糖（通过蛋白质O-葡萄糖基转移酶1，POGLUT 1添加）。我们 还研究了O-岩藻糖（通过POFUT 2添加）对TSR的影响。所有三种酶都修饰特定的 在EGF或TSR内充分表征的共有序列中的丝氨酸或苏氨酸。敲除这些 酶在小鼠胚胎中是致命的，POFUT 1或POGLUT 1的突变导致人类遗传 疾病，证明了这些修饰的生物学重要性。我们建议解决几个 关于这些O-聚糖修饰的蛋白质的未解答的问题。例如，我们想确定 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被修饰。 虽然用POFUT 1和POFUT 2的共有序列进行的数据库搜索已经成功地 鉴定了许多靶蛋白，最近的数据揭示了一个不同的富含半胱氨酸的结构域， 岩藻糖，多聚体蛋白1中的EMI结构域，在我们的检索中未发现。在这里，我们描述了一个公正的 使用生物正交探针6-炔基岩藻糖（6AF）鉴定O-岩藻糖基化蛋白质的方法，即 高效且优先地掺入细胞中的O-岩藻糖基化蛋白质中。我们希望确认一些 POFUT 1和POFUT 2的预测底物，这将提供新的研究目标，但也可以识别 在数据库搜索中没有出现的蛋白质。我们还想检查的结构和功能 NOTCH 3上的O-岩藻糖和O-葡萄糖修饰。在过去的几年里，我们绘制了O-岩藻糖 使用糖蛋白质组学方法将聚糖与NOTCH 1和NOTCH 2上的位点结合，并确定哪些位点起作用。 生物学上的重要角色。已知NOTCH 3在血管稳态中起重要作用，并且突变 在NOTCH 3中，导致CADASIL，一种毁灭性的常染色体显性血管疾病。分子 导致CADASIL的机制知之甚少。CADASIL突变增加或去除半胱氨酸 NOTCH 3 EGF，其被预测破坏糖基化。我们将绘制NOTCH 3上的糖基化位点， 从血管平滑肌细胞中分离，并评估CADASIL突变如何影响其糖基化 status.最后，POFUT 1和POFUT 2都是位于内质网内腔的可溶性酶。 内质网（ER），但它们的供体底物GDP-岩藻糖在胞质溶胶中合成。它不知道如何 GDP-岩藻糖被转运到ER中。在这里，我们描述了一种CRISPR-Cas9筛选来鉴定推定的ER GDP-岩藻糖转运蛋白。这些研究将扩展我们对O- 富含半胱氨酸结构域上的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.