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Principal Investigator: KELLY G TEN HAGEN
Organization: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
Fiscal Year: 2021
Award: $2,025,039
Funding agency: National Institute of Dental and Craniofacial Research
Loss of Galnt3 alters the oral microbiome and glycosylation of Muc10.
The importance of the microbiome in health and its disruption in disease is continuing to be elucidated. However, the multitude of host and environmental factors that influence the microbiome are still largely unknown. Here, we examined Galnt3-deficient mice, which serve as a model for the disease hyperphosphatemic familial tumoral calcinosis (HFTC). In HFTC, loss of GALNT3 activity in the bone is thought to lead to altered glycosylation of the phosphate-regulating hormone fibroblast growth factor 23 (FGF23), resulting in hyperphosphatemia and subdermal calcified tumors. However, GALNT3 is expressed in other tissues in addition to bone, suggesting that systemic loss could result in other pathologies. We found that Galnt3 is the major O-glycosyltransferase expressed in the secretory cells of salivary glands and the loss of Galnt3 resulted in changes in the structure, composition and stability of the oral microbiome (Peluso et al., 2020). Moreover, we identified the major secreted salivary mucin, Muc10, as an in vivo substrate of Galnt3. Given that mucins and their O-glycans are known to interact with various microbes, our results suggest that loss of Galnt3 decreases glycosylation of Muc10, which alters the composition and stability of the oral microbiome. Considering that oral findings have been documented in HFTC patients, our study suggests that investigating GALNT3-mediated changes in the oral microbiome may be warranted (Peluso et al., 2020). We are continuing to study the effects of Galnt3 loss throughout the digestive tract.
Splicing within the lectin domain alters peptide and glycopeptide specificity.
We previously discovered that one member of the pgant family undergoes tissue-specific splicing within the region encoding the non-catalytic lectin domain (Ji, Samara et al., 2018). Specifically, we found that the differential splicing event replaces a 30 amino acid region encoding the alpha sub-region of the lectin domain. In collaboration with Drs. Nadine Samara and Lawrence Tabak, the structure of both isoforms was solved at atomic resolution to reveal that the differentially spliced region creates either a positively charged (for PGANT9A) or a negatively charged (for PGANT9B) loop that lies in close proximity to the active site of each enzyme. We further demonstrate that while both PGANT9A and PGANT9B have preferences for oppositely charged peptide substrates, PGANT9A is much more sensitive to charge. Finally, we show that each splice variant has unique glycopeptide preferences as well. This study provides the first demonstration that changes within a subregion of the non-catalytic lectin domain can alter the recognition of both peptide and glycopeptide substrates (May et al., 2020, JBC, in press).
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).
O-glycosylation modulates furin cleavage of the SARS-CoV-2 spike protein
The SARS-CoV-2 coronavirus responsible for the global COVID-19 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, doi: https://doi.org/10.1101/2021.02.05.429982
Ongoing studies by our group continue to focus on the enzymatic details that control substrate specificity of the many members of this enzyme family, as well as the roles of O-glycosylation in tissue and organ function. Our hope is that the cumulative results of our research will elucidate the mechanisms by which this conserved protein modification operates across species and in conserved cellular events.
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><2019-nCoV S protein><2019-nCoV spike glycoprotein><2019-nCoV spike protein><Acetylgalactosamine><Active Sites><Affect><Alimentary Canal><Amino Acids><Animal Model><Animal Models and Related Studies><Animals><B.1.617.2><Biological><Body Tissues><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><Calcified><Cell Body><Cell Communication and Signaling><Cell Signaling><Cells><Charge><CoV-2><CoV2><Collaborations><Colon Cancer><Colon Carcinoma><Colonic Carcinoma><Congenital Cardiac Defects><Congenital Heart Defects><Coronaviridae><Coronavirus><Cytoplasmic Granules><Defect><Delta variant><Development><Diathesis><Digestive Tract><Dimensions><Disease><Disease Progression><Disease model><Disease susceptibility><Disorder><Drosophila><Drosophila genus><EC 2.4><Endocrine Gland Secretion><Environment><Environmental Factor><Environmental Risk Factor><Enzyme Gene><Enzymes><Event><FGF23 gene product><Familial tumoral calcinosis><Family><Family member><GALNT3><GALNT3 gene><GI Tract><GWA study><GWAS><GalNAc Transferase 3><GalNAcT3><Gastrointestinal Tract><Gastrointestinal tract structure><Genes><Genetic><Genetic Alteration><Genetic Change><Genetic defect><Giant Cells><Glycans><Glycopeptides><Glycoside Transferases><HDL Cholesterol><HDL Cholesterol Lipoproteins><Health><High Density Lipoprotein Cholesterol><Hormones><Host Factor><Host Factor Protein><Human><Integration Host Factors><Intracellular Communication and Signaling><Ions><Isoforms><Kidney Diseases><L-Proline><L-Serine><L-Threonine><Lead><Lectin><Link><Mammalia><Mammals><Mediating><Membrane><Metabolic Glycosylation><Metastasis><Metastasize><Metastatic Lesion><Metastatic Mass><Metastatic Neoplasm><Metastatic Tumor><Mice><Mice Mammals><Microbe><Modern Man><Molecular><Morphology><Mucins><Mucus Glycoprotein><Multimodal Imaging><Multinucleated Giant Cells><Murine><Mus><Mutation><N acetylgalactosamine><Neoplasm Metastasis><Nephropathy><Oral><Organ><Pathology><Patients><Pb element><Peptides><Phosphates><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 Isoforms><Protein Modification><Proteins><RNA Splicing><Racial Segregation><Renal Disease><Research><Resolution><Role><SARS corona virus 2><SARS-CoV-2><SARS-CoV-2 B.1.617.2><SARS-CoV-2 S protein><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 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><Specificity><Splicing><Structure><Substrate Specificity><Syncytium><Syndrome><Therapeutic Hormone><Threonine><Tissue-Specific Splicing><Tissues><Triacylglycerol><Triglycerides><Tropism><Tzanck Cell><Variant><Variation><Viral><Wuhan coronavirus><alimentary tract><alpha-Lipoprotein Cholesterol><aminoacid><biological signal transduction><bone><calcification><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><developmental><digestive canal><disorder model><environmental risk><extracellular><fibroblast growth factor 23><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><glycosyltransferase><granule><hCoV19><heavy metal Pb><heavy metal lead><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><inorganic phosphate><insight><kidney disorder><liability to disease><member><membrane structure><microbiome><model of animal><model organism><multi-modal imaging><multi-modality imaging><multimodality imaging><nCoV2><neoplasm progression><neoplastic progression><novel><oral microbiome><organ development><organ growth><preference><renal disorder><respiratory><salivary mucins><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><subcutaneous><subdermal><sugar><tumor><tumor cell metastasis><tumor progression><whole genome association analysis><whole genome association studies><whole genome association study>