Role of O-glycosylation in Animal Development

在分泌颗粒成熟过程中发生的重组事件介导了颗粒内货物分离。 利用果蝇唾液腺，我们进一步研究了体内分泌细胞为调节分泌做准备时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重要过程，然后膜分泌囊泡在将其内容物分泌到细胞外环境之前等待信号。 然而，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过程中进行协调的颗粒内重组，以允许在同一颗粒内的不同组分的空间分离。 此外，通过遗传学和多模态成像（共聚焦，FIB-SEM和TEM）的组合，我们证明了每个不同的颗粒内结构的分子身份。 我们进一步鉴定了对时间顺序重组事件至关重要的基因，包括控制pH（vha16.1）、Cl-离子（Clic和ClC-c）和Ca 2+离子（fwe）的基因。 最后，我们表明，改变货物糖基化影响这些结构的尺寸，从而影响分泌颗粒形态。 这项研究阐明了关键步骤和因素参与颗粒内，而不是颗粒间，通过调节重组事件在分泌颗粒成熟的货物隔离。 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同一分泌颗粒中分泌，可以提供对由分泌缺陷引起的疾病的深入了解。 这项工作发表在PNAS上。(Syed例如，2022）https://www.pnas.org/doi/10.1073/pnas.2209750119。 小鼠呼吸系统中控制粘蛋白型O-糖基化的基因的动态表达。 COVID-19全球大流行强调了了解病毒和其他病原体如何能够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会因感染或损伤而发生变化。 我们发现，Galnt家族和粘蛋白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，某些粘蛋白和Galnts的表达在肺部感染和损伤的实验小鼠与感染因子，毒素和过敏原的挑战改变。 最后，我们研究粘蛋白和Galnt基因表达的变化，在小鼠模型的SARS-CoV-2感染。 我们的工作提供了关于负责整个呼吸系统O-糖基化的Galnt酶家族的特定成员以及某些粘蛋白和Galnt基因的表达在肺部感染和损伤时如何改变的基础知识。这项工作发表在《糖生物学》杂志上（DOI：10.1093/glycob/cwad 031）。 小鼠组织中体内O-GalNAc糖蛋白组的定量作图鉴定代谢紊乱中的GalNAc-T2 O-糖位点。 我们参与了由Tabak实验室领导的一项合作，创建了一个O-糖蛋白和多个小鼠组织中聚糖添加位点的图谱。 定量糖蛋白质组学和蛋白质组学鉴定了糖基化的组织特异性调节。 野生型和Galnt 2缺陷组织之间的糖蛋白和糖位点的比较揭示了Galnt 2特异性底物和位点，其可能参与在Galnt 2缺陷动物中观察到的复杂代谢紊乱。 这项工作正在PNAS出版。 我们将继续研究调节粘蛋白生物合成和分泌的因素。 我们还将继续研究单个Galnt在器官发育、功能和体内平衡中的特定生物学作用。

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. This work was published in PNAS. (Syed et al., 2022) https://www.pnas.org/doi/10.1073/pnas.2209750119. 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 and mucins 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 mucins and Galnts was altered during lung infection and injury in experimental mice challenged with infectious agents, toxins and allergens. Finally, we examine mucin and 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 mucins and Galnt genes is altered upon lung infection and injury. This work was published in the journal Glycobiology (DOI: 10.1093/glycob/cwad031). Quantitative mapping of the in vivo O-GalNAc glycoproteome in mouse tissues identifies GalNAc-T2 O-glycosites in a metabolic disorder. We have been involved in a collaboration led by the Tabak lab creating an atlas of O-glycoproteins and sites of glycan addition across multiple mouse tissues. Quantitative glycoproteomics and proteomics identified tissue-specific regulation of glycosylation. Comparison of glycoproteins and glycosites between wild type and Galnt2-deficient tissues revealed Galnt2-specific substrates and sites that may be involved in the complex metabolic disorder observed in Galnt2-deficient animals. This work is in press at PNAS. We continue to interrogate the factors that regulate mucin biosynthesis and secretion. We also continue to investigate the specific biological roles of individual Galnts in organ development, function and homeostasis.