Role of O-glycosylation in Animal Development

National Institute Of Dental & Craniofacial Research

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Abstract

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.