Role of O-glycosylation in Animal Development

O-糖基化在动物发育中的作用

• 批准号: 10703881
• 负责人: KELLY G TEN HAGEN
• 金额: $ 187.37万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10703881
• 关键词: 2019-nCoV; Acetylgalactosamine; Adipose tissue; Affect; Allergens; Animal Model; Animals; Biological; Body fat; Body mass index; Bone Density; COVID-19 pandemic; Cells; Colon Carcinoma; Complex; Congenital Heart Defects; Coronavirus; Cytoplasmic Granules; Defect; Development; Dimensions; Disease; Disease Progression; Disease susceptibility; Drosophila genus; Energy-Generating Resources; Environment; Enzymes; Event; Familial tumoral calcinosis; Family; Family member; Foundations; Gene Expression; Genes; Genetic; Giant Cells; High Density Lipoprotein Cholesterol; Homeostasis; Human; Infection; Infectious Agent; Injury; Insulin Receptor; Ions; Kidney Diseases; Knowledge; Link; Lipids; Lung infections; Mammals; Mediating; Membrane; Metabolism; Molecular; Morphology; Mucins; Multimodal Imaging; Mus; Mutation; Neoplasm Metastasis; Obesity; Pattern; Phase; Polysaccharides; Positioning Attribute; Post-Translational Protein Processing; Process; Proline; Proteins; Research; Respiratory System; Role; SARS-CoV-2 B.1.617.2; SARS-CoV-2 infection; SARS-CoV-2 spike protein; Salivary Glands; Secretory Cell; Secretory Vesicles; Serine; Signal Transduction; Site; Structure; Syndrome; Threonine; Tissues; Toxin; Transferase; Triglycerides; Tropism; Viral; Virus; Visceral; Work; Yang; extracellular; genome wide association study; glycosylation; human disease; hydroxyl group; in vivo; insight; insulin signaling; lung injury; member; mouse model; novel; organ growth; pandemic disease; pathogen; respiratory; response; segregation; sugar; tumor progression

Orchestrated restructuring events during secretory granule maturation mediate intragranular cargo segregation. Using Drosophila salivary glands, we further investigated the dynamic intracellular changes occurring in vivo as secretory cells prepare for regulated secretion. Regulated secretion is an essential process where proteins are packaged into membranous secretory vesicles, which then await a signal before secreting their contents into the extracellular environment. However, the details of cargo packaging and secretory granule maturation are largely unknown. We demonstrate that multiple distinct proteins (mucins) undergo orchestrated intragranular restructuring during secretory granule maturation in vivo, to allow spatial segregation of distinct components within the same granule. Furthermore, through a combination of genetics and multimodality imaging (confocal, FIB-SEM and TEM), we demonstrate the molecular identity of each distinct intragranular structure. We further identify genes that are essential for the temporally-ordered restructuring events, including those controlling pH (vha16.1), Cl- ions (Clic and ClC-c) and Ca2+ ions (fwe). Finally, we show that altered cargo glycosylation influences dimensions of these structures, thereby affecting secretory granule morphology. This study elucidates key steps and factors involved in intragranular, rather than intergranular, segregation of cargo through regulated restructuring events during secretory granule maturation. Understanding how multiple distinct proteins are efficiently packaged into and secreted from the same secretory granule may provide insight into diseases resulting from defects in secretion (Syed et al., 2021, doi: https://doi.org/10.1101/2021.08.16.456250). This work is currently under review. A novel role for GalNAc-T2 dependent glycosylation in energy homeostasis. This was a collaborative study with the Kuivenhoven group examining the effects of the loss of Galnt2 in energy homeostasis. GALNT2 SNPs were associated with changes in body mass index, body fat percentage and whole-body fat mass. In mouse models where Galnt2 was ablated, mice are significantly smaller and had reduced visceral white adipose tissue (WAT). Changes in Akt/mTORC1 signaling within WAT in the absence of Galnt2 was also noted and associated with changes in the O-glycosylation status of the insulin receptor. Additionally, Galnt2-deficient mice were found to preferentially use lipids as an energy source during inactive phase. This study identifies the insulin receptor as a functional target of Galnt2 and highlights the complex role of this transferase in insulin signaling, adiposity, energy utilization and metabolism (Verzijl CRC, Oldoni F, Loaiza N, Wolters JC, Rimbert A, Tian E, Yang W, Struik D, Smit M, Kloosterhuis NJ, Fernandez AJ, Samara NL, Ten Hagen KG, Dalal K, Chernish A, McCluggage P, Tabak LA, Jonker JW, Kuivenhoven JA. 2022. A novel role for GalNAc-T2 dependent glycosylation in energy homeostasis. Molec. Metab. In DOI: 10.1016/j.molmet.2022.101472.) O-glycosylation modulates furin cleavage of the SARS-CoV-2 spike protein. The SARS-CoV-2 coronavirus responsible for the global pandemic contains a novel furin cleavage site in the spike protein (S) that increases viral infectivity, tropism and syncytia formation. We demonstrate that O-glycosylation near the furin cleavage site is mediated by specific members of the GALNT enzyme family, resulting in decreased furin cleavage and decreased syncytia formation. Moreover, we show that O-glycosylation is dependent on the novel proline at position 681 (P681). Mutations of P681 seen in the highly transmissible Alpha and Delta variants, abrogate O-glycosylation, increase furin cleavage and increase syncytia formation. Finally, we show that GALNT family members capable of glycosylating S are expressed in human respiratory cells that are targets for SARS-CoV-2 infection. Our results suggest that host O-glycosylation may influence viral infectivity/tropism by modulating furin cleavage of S and provide mechanistic insight into the role of the P681 mutations found in the highly transmissible Alpha and Delta variants (Zhang et al., 2021, PNAS doi: https://doi.org/10.1101/2021.02.05.429982) Dynamic expression of the genes controlling mucin-type O-glycosylation within the mouse respiratory system The COVID-19 global pandemic has underscored the need to understand how viruses and other pathogens are able to infect and replicate within the respiratory systems. Recent studies have highlighted the role of highly O-glycosylated mucins in the protection of the respiratory system as well as how mucin-type O-glycosylation may be able to modify viral infectivity. Therefore, we set out to identify the specific genes controlling mucin-type O-glycosylation throughout the mouse respiratory system and determine whether their expression changes in response to infection or injury. We show that certain members of the Galnt family are abundantly expressed in certain respiratory tissues/cells and demonstrate unique patterns of O-glycosylation across diverse respiratory tissues. Moreover, we found that the expression of certain Galnts was altered during lung infection and injury in experimental mice challenged with infectious agents, toxins and allergens. Finally, we examine Galnt gene expression changes in a mouse model of SARS-CoV-2 infection. Our work provides foundational knowledge regarding the specific members of the Galnt enzyme family responsible for O-glycosylation throughout the respiratory system and how expression of certain Galnt genes is altered upon lung infection and injury.

分泌颗粒成熟过程中精心策划的重组事件介导颗粒内货物分离。 使用果蝇唾液腺，我们进一步研究了当分泌细胞准备调节分泌时体内发生的动态细胞内变化。 调节分泌是蛋白质被包装到膜分泌囊泡中的一个重要过程，然后膜分泌囊泡等待信号，然后将其内容物分泌到细胞外环境中。 然而，货物包装和分泌颗粒成熟的细节很大程度上未知。 我们证明，多种不同的蛋白质（粘蛋白）在体内分泌颗粒成熟过程中经历精心策划的颗粒内重组，以允许同一颗粒内不同成分的空间分离。 此外，通过遗传学和多模态成像（共焦、FIB-SEM 和 TEM）的结合，我们证明了每种不同颗粒内结构的分子特性。 我们进一步鉴定了对于按时间排序的重组事件至关重要的基因，包括那些控制 pH (vha16.1)、Cl- 离子 (Clic 和 ClC-c) 和 Ca2+ 离子 (fwe) 的基因。 最后，我们表明改变的货物糖基化会影响这些结构的尺寸，从而影响分泌颗粒的形态。 这项研究阐明了通过分泌颗粒成熟过程中受调节的重组事件参与颗粒内而不是颗粒间货物分离的关键步骤和因素。 了解多种不同的蛋白质如何有效地包装到同一分泌颗粒中并从中分泌出来，可以深入了解分泌缺陷引起的疾病（Syed 等人，2021，doi：https://doi.org/10.1101/2021.08.16.456250） 。 这项工作目前正在审查中。 GalNAc-T2 依赖性糖基化在能量稳态中的新作用。 这是与 Kuivenhoven 小组的一项合作研究，研究了 Galnt2 缺失对能量稳态的影响。 GALNT2 SNP 与体重指数、体脂百分比和全身脂肪量的变化相关。 在 Galnt2 被消除的小鼠模型中，小鼠明显变小，内脏白色脂肪组织 (WAT) 减少。 在 Galnt2 缺失的情况下，WAT 内 Akt/mTORC1 信号传导的变化也被注意到，并且与胰岛素受体 O-糖基化状态的变化相关。 此外，发现 Galnt2 缺陷的小鼠在非活动期优先使用脂质作为能量来源。 这项研究将胰岛素受体确定为 Galnt2 的功能靶标，并强调了这种转移酶在胰岛素信号转导、肥胖、能量利用和代谢中的复杂作用（Verzijl CRC、Oldoni F、Loaiza N、Wolters JC、Rimbert A、Tian E、Yang W、Struik D、Smit M、Kloosterhuis NJ、Fernandez AJ、Samara NL、十哈根 KG、Dalal K、Chernish A、 McCluggage P、Tabak LA、Jonker JW、Kuivenhoven JA。GalNAc-T2 依赖性糖基化在能量稳态中的新作用。Molec Metab：10.1016/j.molmet.2022.101472。 O-糖基化调节 SARS-CoV-2 刺突蛋白的弗林蛋白酶切割。 造成全球大流行的 SARS-CoV-2 冠状病毒在刺突蛋白 (S) 中含有一个新的弗林蛋白酶切割位点，可增加病毒的感染性、趋向性和合胞体形成。我们证明弗林蛋白酶切割位点附近的 O-糖基化是由 GALNT 酶家族的特定成员介导的，导致弗林蛋白酶切割减少和合胞体形成减少。 此外，我们发现 O-糖基化依赖于第 681 位（P681）的新型脯氨酸。 在高度传播的 Alpha 和 Delta 变体中发现的 P681 突变会消除 O-糖基化，增加弗林蛋白酶裂解并增加合胞体形成。 最后，我们发现能够糖基化 S 的 GALNT 家族成员在作为 SARS-CoV-2 感染目标的人类呼吸细胞中表达。 我们的结果表明，宿主 O-糖基化可能通过调节 S 的弗林蛋白酶切割来影响病毒的感染性/向性，并为高度传播的 Alpha 和 Delta 变体中发现的 P681 突变的作用提供机制见解（Zhang 等人，2021，PNAS doi） ：https://doi.org/10.1101/2021.02.05.429982） 小鼠呼吸系统内控制粘蛋白型O-糖基化的基因的动态表达 COVID-19 全球大流行凸显了了解病毒和其他病原体如何在呼吸系统内感染和复制的必要性。 最近的研究强调了高度 O-糖基化的粘蛋白在保护呼吸系统中的作用，以及粘蛋白型 O-糖基化如何能够改变病毒的感染性。 因此，我们着手鉴定控制整个小鼠呼吸系统粘蛋白型O-糖基化的特定基因，并确定它们的表达是否因感染或损伤而发生变化。 我们发现 Galnt 家族的某些成员在某些呼吸组织/细胞中大量表达，并在不同的呼吸组织中表现出独特的 O-糖基化模式。 此外，我们发现，在受到传染源、毒素和过敏原攻击的实验小鼠肺部感染和损伤期间，某些 Galnts 的表达发生了改变。 最后，我们检查了 SARS-CoV-2 感染小鼠模型中 Galnt 基因表达的变化。 我们的工作提供了有关 Galnt 酶家族特定成员的基础知识，这些成员负责整个呼吸系统的 O-糖基化，以及某些 Galnt 基因的表达在肺部感染和损伤时如何改变。