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Principal Investigator: KELLY G TEN HAGEN
Organization: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
Fiscal Year: 2022
Award: $1,873,672
Funding agency: National Institute of Dental and Craniofacial Research
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.
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><2019-nCoV S protein><2019-nCoV spike glycoprotein><2019-nCoV spike protein><Acetylgalactosamine><Adipose tissue><Affect><Allergens><Animal Model><Animal Models and Related Studies><Animals><Autoregulation><B.1.617.2><BMI><BMI percentile><BMI z-score><Biological><Body Tissues><Body fat><Body mass index><Bone Density><Bone Mineral Density><COVID crisis><COVID epidemic><COVID pandemic><COVID-19 S protein><COVID-19 crisis><COVID-19 epidemic><COVID-19 global health crisis><COVID-19 global pandemic><COVID-19 health crisis><COVID-19 infection><COVID-19 pandemic><COVID-19 public health crisis><COVID-19 spike glycoprotein><COVID-19 spike protein><COVID-19 virus><COVID19 S protein><COVID19 crisis><COVID19 epidemic><COVID19 global health crisis><COVID19 global pandemic><COVID19 health crisis><COVID19 infection><COVID19 pandemic><COVID19 public health crisis><COVID19 spike glycoprotein><COVID19 spike protein><COVID19 virus><Cell Body><Cell Communication and Signaling><Cell Signaling><Cells><CoV-2><CoV2><Colon Cancer><Colon Carcinoma><Complex><Congenital Cardiac Defects><Congenital Heart Defects><Coronaviridae><Coronavirus><Cytoplasmic Granules><Defect><Delta variant><Development><Diathesis><Dimensions><Disease><Disease Progression><Disease susceptibility><Disorder><Drosophila><Drosophila genus><Energy-Generating Resources><Environment><Enzyme Gene><Enzymes><Event><Familial tumoral calcinosis><Family><Family member><Fatty Tissue><Foundations><GWA study><GWAS><Gene Expression><Genes><Genetic><Genetic Alteration><Genetic Change><Genetic defect><Giant Cells><Glycans><HDL Cholesterol><HDL Cholesterol Lipoproteins><High Density Lipoprotein Cholesterol><Homeostasis><Human><Infection><Infectious Agent><Injury><Insulin Receptor><Insulin Receptor Protein-Tyrosine Kinase><Insulin-Dependent Tyrosine Protein Kinase><Intermediary Metabolism><Intracellular Communication and Signaling><Ions><Kidney Diseases><Knowledge><L-Proline><L-Serine><L-Threonine><Link><Lipids><Lung damage><Lung infections><Mammalia><Mammals><Mediating><Membrane><Metabolic Glycosylation><Metabolic Processes><Metabolism><Metastasis><Metastasize><Metastatic Lesion><Metastatic Mass><Metastatic Neoplasm><Metastatic Tumor><Mice><Mice Mammals><Modern Man><Molecular><Morphology><Mucins><Mucus Glycoprotein><Multimodal Imaging><Multinucleated Giant Cells><Murine><Mus><Mutation><N acetylgalactosamine><Neoplasm Metastasis><Nephropathy><Obesity><Pattern><Phase><Physiological Homeostasis><Polykaryocytes><Polysaccharides><Position><Positioning Attribute><Post-Translational Modification Protein/Amino Acid Biochemistry><Post-Translational Modifications><Post-Translational Protein Modification><Post-Translational Protein Processing><Posttranslational Modifications><Posttranslational Protein Processing><Process><Proline><Protein Modification><Proteins><Pulmonary Body System><Pulmonary Organ System><Quetelet index><Racial Segregation><Renal Disease><Research><Respiratory System><Respiratory Tracts><Respiratory tract structure><Role><SARS corona virus 2><SARS-CO-V2><SARS-COVID-2><SARS-CoV-2><SARS-CoV-2 B.1.617.2><SARS-CoV-2 S protein><SARS-CoV-2 delta><SARS-CoV-2 epidemic><SARS-CoV-2 global health crisis><SARS-CoV-2 global pandemic><SARS-CoV-2 infection><SARS-CoV-2 pandemic><SARS-CoV-2 spike glycoprotein><SARS-CoV-2 spike protein><SARS-CoV2><SARS-CoV2 S protein><SARS-CoV2 epidemic><SARS-CoV2 infection><SARS-CoV2 pandemic><SARS-CoV2 spike glycoprotein><SARS-CoV2 spike protein><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-coronavirus-2 epidemic><SARS-coronavirus-2 pandemic><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><Salivary Glands><Salivary Glands Head and Neck><Secondary Neoplasm><Secondary Tumor><Secretory Cell><Secretory Granules><Secretory Vesicles><Serine><Severe Acute Respiratory Coronavirus 2><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome CoV 2 epidemic><Severe Acute Respiratory Syndrome CoV 2 pandemic><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome coronavirus 2 S protein><Severe acute respiratory syndrome coronavirus 2 epidemic><Severe acute respiratory syndrome coronavirus 2 infection><Severe acute respiratory syndrome coronavirus 2 pandemic><Severe acute respiratory syndrome coronavirus 2 spike glycoprotein><Severe acute respiratory syndrome coronavirus 2 spike protein><Severe acute respiratory syndrome related corona virus 2><Signal Transduction><Signal Transduction Systems><Signaling><Site><Structure><Syncytium><Syndrome><Threonine><Tissues><Toxin><Transferase><Transferase Gene><Triacylglycerol><Triglycerides><Tropism><Tzanck Cell><Viral><Virus><Visceral><Work><Wuhan coronavirus><Yang><adipose><adiposity><alpha-Lipoprotein Cholesterol><biologic><biological signal transduction><cancer in the colon><cancer metastasis><cancer progression><congenital cardiac anomalies><corona virus><corona virus disease 2019 epidemic><corona virus disease 2019 pandemic><coronavirus disease 2019 S protein><coronavirus disease 2019 crisis><coronavirus disease 2019 epidemic><coronavirus disease 2019 global health crisis><coronavirus disease 2019 global pandemic><coronavirus disease 2019 health crisis><coronavirus disease 2019 infection><coronavirus disease 2019 pandemic><coronavirus disease 2019 public health crisis><coronavirus disease 2019 spike glycoprotein><coronavirus disease 2019 spike protein><coronavirus disease 2019 virus><coronavirus disease crisis><coronavirus disease epidemic><coronavirus disease pandemic><coronavirus disease-19 global pandemic><coronavirus disease-19 pandemic><coronavirus disease-19 virus><corpulence><developmental><energy source><extracellular><fruit fly><genome mutation><genome wide association><genome wide association scan><genome wide association studies><genome wide association study><genomewide association scan><genomewide association studies><genomewide association study><glycosylation><granule><hCoV19><human disease><hydroxyl group><in vivo><infected with COVID-19><infected with COVID19><infected with SARS-CoV-2><infected with SARS-CoV2><infected with coronavirus disease 2019><infected with severe acute respiratory syndrome coronavirus 2><infectious organism><injuries><insight><insulin signaling><kidney disorder><liability to disease><lung injury><member><membrane structure><model of animal><model organism><mouse model><multi-modal imaging><multi-modality imaging><multimodality imaging><murine model><nCoV2><neoplasm progression><neoplastic progression><novel><organ development><organ growth><pandemic><pandemic disease><pathogen><pulmonary damage><pulmonary infections><pulmonary injury><pulmonary tissue damage><pulmonary tissue injury><renal disorder><respiratory><response><segregation><severe acute respiratory syndrome coronavirus 2 B.1.617.2><severe acute respiratory syndrome coronavirus 2 global health crisis><severe acute respiratory syndrome coronavirus 2 global pandemic><social role><sugar><tumor cell metastasis><tumor progression><white adipose tissue><whole genome association analysis><whole genome association studies><whole genome association study><yellow adipose tissue>