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 (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.