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.

分泌颗粒成熟期间精心策划的重组事件介导了杂志的货物隔离。 使用果蝇唾液腺，我们进一步研究了随着分泌细胞为调节分泌做准备的体内动态细胞内变化。 受调节的分泌是一个必不可少的过程，将蛋白质包装到膜分泌囊泡中，然后在将其含量分泌到细胞外环境中之前等待信号。 但是，货物包装和分泌颗粒成熟的细节在很大程度上是未知的。 我们证明，多种不同的蛋白质（粘蛋白）在体内分泌颗粒成熟期间经过精心策划的内部重组，以允许在同一颗粒内进行空间分离。 此外，通过遗传学和多模式成像（共聚焦，纤维-SEM和TEM）的结合，我们证明了每个独特的内部结构的分子身份。 我们进一步确定了对时间排序的重组事件至关重要的基因，包括控制pH（VHA16.1），CLC和CLC和CLC-C）和Ca2+离子（FWE）的基因。 最后，我们表明货物糖基化改变会影响这些结构的维度，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期间调节的重组事件对货物内而不是晶间隔离涉及的关键步骤和因素。 了解如何将多种不同的蛋白质有效地包装到同一分泌的颗粒中并分泌到分泌缺陷引起的疾病（Syed等，2021，doi：https：//doi.org/10.1101/2021/2021.08.16.16.16.456250）。 目前正在审查这项工作。 GalNAC-T2依赖性糖基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在能量稳态中的损失的影响。 GALNT2 SNP与体重指数，体内脂肪百分比和全身脂肪量的变化有关。 在烧杯的小鼠模型中，小鼠明显较小，并且降低了内脏白脂肪组织（WAT）。 还记录了在没有GALNT2的情况下WAT内AKT/MTORC1信号传导的变化，并与胰岛素受体O-糖基化状态的变化有关。 另外，发现GALNT2缺乏的小鼠优先将脂质用作不活跃期的能源。 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，十个Hagen KG，Chernish A，McCluggage P，Tabak LA，Jonker JW，KuivenHoven JA。 O-糖基化调节SARS-COV-2峰值蛋白的脂蛋白裂解。 负责全球大流行的SARS-COV-2冠状病毒包含尖峰蛋白中的新型脂蛋白裂解位点，可增加病毒感染性，疗法和合成性形成。我们证明，在Furin裂解位点附近的O-糖基化是由GALNT酶家族的特定成员介导的，从而导致脂肪蛋白裂解降低并降低合胞菌的形成。 此外，我们表明O-糖基化取决于位置681（P681）的新型脯氨酸。 在高度可传播的α和三角洲变体中看到的p681突变，消除O-糖基化，增加脂肪蛋白的裂解并增加合胞症的形成。 最后，我们表明，能够糖基化S的GALNT家族成员在人类呼吸细胞中表达，这是SARS-COV-2感染的靶标。 我们的结果表明，宿主O-糖基化可能通过调节S的fur蛋白裂解来影响病毒感染/乳化，并提供机械的洞察力，以了解在高度传播的Alpha和Delta变体中发现的P681突变的作用（Zhang等，2021，PNAS DOI：：PNAS DOI：：：：：：： https://doi.org/10.1101/2021.02.05.429982） 在小鼠呼吸系统中控制粘蛋白型O-糖基化基因的动态表达 COVID-19全球大流行强调了了解病毒和其他病原体如何能够在呼吸系统中感染和复制的需求。 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感染性。 因此，我们着手确定整个小鼠呼吸系统中控制粘蛋白型O-糖基化的特定基因，并确定它们的表达是否在反应感染或损伤的响应中发生变化。 我们表明，GALNT家族的某些成员在某些呼吸道组织/细胞中大量表达，并在各种呼吸道组织中表现出独特的O-糖基化模式。 此外，我们发现在受感染剂，毒素和过敏原挑战的实验小鼠中，在肺部感染和损伤过程中某些GALNT的表达发生了改变。 最后，我们检查了SARS-COV-2感染的小鼠模型中GALNT基因表达的变化。 我们的工作提供了有关在整个呼吸系统中负责O-糖基化的GALNT酶家族的特定成员的基础知识，以及在肺部感染和损伤时如何改变某些GALNT基因的表达。