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Principal Investigator: Nadine Samara
Organization: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
Fiscal Year: 2020
Award: $750,000
Funding agency: National Institute of Dental and Craniofacial Research
GalNAc-Ts initiate mucin-type O-glycosylation by transferring N-acetylgalactosamine (GalNAc) from UDP-GalNAc to Thr/Ser acceptors on protein substrates such as mucins. These enzymes do not recognize a consensus sequence or motif and each isoenzyme shows unique specificity towards acceptor sites on a substrate. Although GalNAc-Ts are critical for development and associated with neurological diseases and cancer, the presence of multiple isoenzymes within a species (20 human family members) and substrates within each cell type makes it a challenge to determine the substrates of each isoenzyme. To characterize GalNAc-T substrate specificity and gain insight into their possible biological substrates, we use structural and biochemical methods to investigate the diverse mechanisms used by various isoenzymes to recognize, bind, and position substrates for catalysis. GalNAc-Ts are Golgi-membrane anchored enzymes containing 2 luminal domains that dictate substrate specificity: a catalytic domain and a C-terminal lectin domain consisting of 3 repeats (alpha, beta, gamma) that can potentially recognize and bind extant GalNAcs on glycoprotein or glycopeptide substrates. In collaboration with Dr. Kelly Ten Hagens group at NIDCR, we study the Drosophila melanogaster GalNAc-T isoenzyme PGANT9, which influences secretory granule morphology in the salivary glands and has 2 splicing isoforms, PGANT9A/PGANT9B, that differ in the alpha repeat of their lectin domain. PGANT9A-alpha has a net positive charge, while PGANT9B-alpha has a net negative charge. PGANT9B is highly expressed in the salivary gland and was shown to O-glycosylate the Sgs3 mucin, which contains positively charged residues (Lys) near acceptor sites. We previously showed that PGANT9B prefers to modify a peptide containing a net positive charge, consistent with the overlapping in vivo localization of PGANT9B and Sgs3. In contrast, PGANT9A more efficiently modifies negatively charged peptide substrates. We solved the X-ray crystal structures of both enzymes, revealing that the charged repeats of PGANT9A and PGANT9B form extensive loops that extend towards the active sites of each enzyme. We thus proposed that the charged repeats of PGANT9A and PGANT9B dictate substrate specificity by recruiting peptides containing oppositely charged residues or discriminating against peptides with similarly charged residues.
In recent work in collaboration with the Ten Hagen group (May et al. 2020), we tested our hypothesis by measuring the activity of PGANT9A and PGANT9B against a range of Drosophila mucin peptides containing charged residues, as well as artificial peptides containing residues with opposite charges. We verified that PGANT9A has high specificity towards peptides with net negative charges. Unexpectedly, PGANT9B was less specific and glycosylated peptides containing both positive and negative net charges. To understand the differences between the isoforms, we modeled Sgs3 peptides into the PGANT9A/B active sites, which contain a gating loop (catalytic flexible loop) that interacts with and aligns the acceptor peptide in the active site for catalysis. In most isoenzymes, including PGANT9A/B, this loop has a positive net charge. Thus, in PGANT9A, the alpha repeat and the gating loop are both positively charged, explaining its strong preference for negatively charged peptides. In contrast, PGANT9B contains positive charges near the N-terminus of the peptide via the gating loop, and negative charges near the C-terminus via the alpha repeat, explaining why it can glycosylate peptides with various charges. Overall, these studies highlight a unique mechanism of GalNAc-T substrate specificity that is regulated by alternative splicing and dictated by charges in both the catalytic and lectin domains. Overall, we hope that our structural and biochemical insights will contribute to our understanding of how GalNAc-Ts function at a molecular level, and that this information can be used to understand how we can modulate these enzymes in disease.
This project involves research on human coronavirus, novel coronavirus, COVID-19, Severe Acute Respiratory Syndrome coronavirus disease, SARS coronavirus, SARS-coronavirus-2, SARS-cov-2, SARS-cov2, SARS-related coronavirus 2, Severe acute respiratory syndrome coronavirus 2, SARS-Associated Coronavirus, SARS-cov, or SARS-Related Coronavirus.
Terms: <2019 novel coronavirus><2019-nCoV><Acetylgalactosamine><Active Sites><Alternate Splicing><Alternative RNA Splicing><Alternative Splicing><Assay><Binding><Bioassay><Biochemical><Biologic Assays><Biological><Biological Assay><Body Tissues><C-terminal><CA-15-3 Antigen><COVID-19><COVID19><Cancers><Catalysis><Catalytic Core><Catalytic Domain><Catalytic Region><Catalytic Site><Catalytic Subunit><Charge><CoV emergence><Collaborations><Consensus Sequence><Cryo-electron Microscopy><Cryoelectron Microscopy><Crystallization><DF3 Antigen><Development><Disease><Disorder><Drosophila><Drosophila genus><Drosophila melanogaster><Dysfunction><EC 2.4><Electron Cryomicroscopy><Environment><Enzyme Gene><Enzymes><Episialin><Epithelial><Epithelial Membrane Antigen><Epithelium><Epithelium Part><Eukaryota><Eukaryote><Family><Family member><Functional disorder><GalNAc-T8><GalNAc-transferase><GalNAcT-8><Glycans><Glycopeptides><Glycoproteins><Glycoside Transferases><Glycosylated MUC-1><Golgi><Golgi Apparatus><Golgi Complex><HCoV><Human><Infection><Isoenzymes><Isoforms><Isozymes><Lectin><MUC-1 Antigen><MUC1 antigen><Malignant Neoplasms><Malignant Nervous System Neoplasm><Malignant Tumor><Measures><Membrane><Metabolic Glycosylation><Methods><Modeling><Modern Man><Molecular><Molecular Interaction><Morphology><Mucin Peptide MUC-1><Mucin/Peptide><Mucins><Mucous body substance><Mucus><Mucus Glycoprotein><N acetylgalactosamine><NIDCR><NIDR><National Institute of Dental Research><National Institute of Dental and Craniofacial Research><Nervous System Diseases><Neurologic Disorders><Neurological Disorders><Peptides><Physiopathology><Polymorphic Epithelial Mucin><Polypeptide N-acetylgalactosaminyltransferase><Polysaccharides><Position><Positioning Attribute><Property><Protein Isoforms><Proteins><RNA Splicing><Research><Roentgen Rays><SARS><SARS Virus><SARS corona virus><SARS coronavirus><SARS coronavirus disease><SARS-Associated Coronavirus><SARS-CoV><SARS-CoV disease><SARS-CoV-2><SARS-CoV2><SARS-Related Coronavirus><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related coronavirus 2><Salivary Glands><Salivary Glands Head and Neck><Secretory Granules><Secretory Vesicles><Severe Acute Respiratory Syndrome><Severe Acute Respiratory Syndrome CoV disease><Severe Acute Respiratory Syndrome Virus><Severe Acute Respiratory Syndrome corona virus><Severe Acute Respiratory Syndrome coronavirus><Severe Acute Respiratory Syndrome coronavirus disease><Severe acute respiratory syndrome coronavirus 2><Single Crystal Diffraction><Site><Specificity><Splicing><Structure><Substrate Specificity><Testing><Tissues><UDP-GPAGAT><UDP-GalNAc-polypeptide N-acetylgalactosaminyltransferase><UDP-N-acetylgalactosamine mucin transferase><UDP-N-acetylgalactosamine-polypeptide N-acetylgalactosamine transferase><UDPacetylgalactosamine-protein acetylgalactosaminyltransferase><Work><Wuhan coronavirus><X Ray Crystallographies><X-Radiation><X-Ray Crystallography><X-Ray Diffraction Crystallography><X-Ray Radiation><X-Ray/Neutron Crystallography><X-ray><Xray><Xray Crystallography><cell type><commensal flora><commensal microbes><commensal microbiota><commensal microflora><corona virus disease 2019><coronavirus disease 2019><coronavirus emergence><cryo-EM><cryoEM><developmental><developmental disease><developmental disorder><emergent CoV><emergent coronavirus><emerging CoV><emerging coronavirus><flexibility><flexible><fruit fly><glycosylation><glycosyltransferase><host microbe association><host microbe relationship><host-microbe interactions><host-microbial interactions><host-microorganism interactions><human CoV><human corona virus><human coronavirus><in vivo><insight><malignancy><malignant nervous system tumor><malignant neurologic neoplasms><membrane structure><mucous><nCoV><neoplasm/cancer><nervous system disorder><neurological cancers><neurological disease><new CoV><new coronavirus><novel CoV><novel coronavirus><pathophysiology><preference><protein-UDPacetylgalactosaminyltransferase><recruit><severe acute respiratory syndrome-CoV>