O-glycosylation of cysteine-rich modules

富含半胱氨酸的模块的 O-糖基化

• 批准号: 10559833
• 负责人: Robert S. Haltiwanger
• 金额: $ 43.79万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-01 至 2027-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10559833
• 关键词: Address; Affect; Anabolism; Biological; Blood Vessels; CADASIL; CRISPR screen; Cell surface; Cells; Consensus Sequence; Cysteine; Cysteine-Rich Domain; Cytosol; Data; Databases; Defect; Development; Disease; Embryo; Endoplasmic Reticulum; Enzymes; Epidermal Growth Factor; Extracellular Protein; Fucose; Fucosyltransferase 1; Genes; Genetic; Genetic Diseases; Glucosyltransferase; Goals; Guanosine Diphosphate Fucose; Homeostasis; Human Genetics; Knock-out; Learning; Link; Maps; Methods; Modification; Molecular; Mus; Mutation; NOTCH1 gene; NOTCH3 gene; Play; Polysaccharides; Proteins; Proteome; Research; Role; Serine; Site; Smooth Muscle Myocytes; Structure; Tertiary Protein Structure; Threonine; Thrombospondins; Vascular Diseases; Vascular Smooth Muscle; autosome; glycoproteomics; glycosylation; hydroxyl group; novel; sugar

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.

我们研究的总体目标是确定结构、修饰位点、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 血小板反应蛋白 1 型重复序列 (TSR)。 EGF 和 TSR 均存在于许多细胞表面和 细胞外蛋白。我们重点关注 EGF 的两种修饰，O-岩藻糖（由 Protein O- 添加） 岩藻糖基转移酶 1，POFUT1) 和 O-葡萄糖（由蛋白 O-葡萄糖基转移酶 1，POGLUT1 添加）。我们 还研究 TSR 上的 O-岩藻糖（由 POFUT2 添加）。所有三种酶都修饰特定的羟基基团 EGF 或 TSR 内已明确表征的共有序列中的丝氨酸或苏氨酸。淘汰这些 酶对小鼠胚胎具有致死性，POFUT1或POGLUT1突变导致人类遗传 疾病，证明了这些修饰的生物学重要性。我们建议解决几个问题 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被修饰。 虽然使用 POFUT1 和 POFUT2 的共有序列进行数据库搜索已成功 识别出许多目标蛋白，最近的数据揭示了一个不同的富含半胱氨酸的结构域，用 O- 修饰 岩藻糖是 Multimerin1 中的一个 EMI 结构域，我们的搜索中未发现该结构域。这里我们描述一个无偏的 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 有效且优先地掺入细胞中的O-岩藻糖基化蛋白质中。我们预计将确认一些 预测 POFUT1 和 POFUT2 的底物，这将提供新的研究目标，同时也可以识别 数据库搜索中未出现的蛋白质。我们还想研究一下其结构和功能 NOTCH3 上的 O-岩藻糖和 O-葡萄糖修饰。在过去的几年里，我们绘制了 O-岩藻糖 使用糖蛋白质组学方法将聚糖连接到 NOTCH1 和 NOTCH2 上的位点，并确定哪些位点发挥作用 生物学上的重要作用。已知 NOTCH3 在血管稳态和突变中发挥重要作用 NOTCH3 中的 CADASIL 会导致 CADASIL，这是一种破坏性的常染色体显性血管疾病。分子 导致 CADASIL 的机制尚不清楚。 CADASIL 突变添加或删除了半胱氨酸 NOTCH3 EGF，预计会破坏糖基化。我们将绘制 NOTCH3 上的糖基化位点 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 地位。最后，POFUT1和POFUT2都是位于内质腔内的可溶性酶 网状细胞 (ER)，但它们的供体底物 GDP-岩藻糖是在细胞质中合成的。不知道如何 GDP-岩藻糖被转运至内质网。在这里，我们描述了 CRISPR-Cas9 筛选来识别假定的 ER GDP-岩藻糖转运蛋白。这些研究将扩展我们对O-结构和功能的理解 富含半胱氨酸结构域上的聚糖及其在疾病中的潜在作用。