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Principal Investigator: Nadine Samara
Organization: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
Fiscal Year: 2021
Award: $467,822
Funding agency: National Institute of Dental and Craniofacial Research
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. One of our projects focuses on understanding the role of the luminal domains in substrate binding and recognition.
We collaborate with Dr. Kelly Ten Hagens group at NIDCR to study how alternative splicing regulates O-glycosylation. Our recent work shed light on the Drosophila melanogaster GalNAc-T isoenzyme PGANT9, which influences secretory granule morphology in the salivary glands. PGANT9 has 2 splicing isoforms PGANT9A and 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. We previously showed that PGANT9B prefers to modify a salivary gland mucin peptide from Sgs3 with 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. The X-ray crystal structures reveal that the charged repeats of PGANT9A and PGANT9B form a loop that extends towards the active site 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. We tested our hypothesis and verified that while PGANT9A has high specificity towards peptides with net negative charges, 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 catalytic flexible 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. We are currently continuing to collaborate with Dr. Kelly Ten Hagen to understand how alternative splicing influences the function of other isoenyzmes in the family.
We study the role of deactivating GALNT12 mutations in patients with colorectal cancer (CRC). The association of GalNAc-T12 with cancer is not clear because its substrates have not been identified. To further our understanding of the downstream effects of aberrant O-glycosylation that arise due to mutations in GALNT12, we collaborate with Dr. Benjamin Schumanns group at Imperial college of London to identify the in vivo substrates of human GalNAc-T12. We are also conducting biochemical studies to follow up on published work and further understand how CRC mutations affect the function of GalNAc-T12.
We collaborated with Dr. Kelly Ten Hagen and Dr. Lawrence Tabaks groups at NIDCR, and Dr. Darryl Zeldins group at NIEHS to investigate the role of O-glycosylation on SARS-CoV-2 infectivity. We show that O-glycosylation of the SARS-CoV-2 spike protein by GalNAc-T1 influences the furin cleavage of spike, which influences the infectivity and transmissibility of SARS-CoV-2. This is of current interest because both Alpha and Delta variants contain a mutation in spike (P681H and P681R) that abolishes GalNAc-T1 mediated O-glycosylation.
Overall, we hope that these studies provide insight into the specific targeting and modulation (inhibition/activation) of individual GalNAc-Ts 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 corona virus><2019 novel coronavirus><2019-nCoV><2019-nCoV S protein><2019-nCoV spike glycoprotein><2019-nCoV spike protein><Acetylgalactosamine><Active Follow-up><Active Sites><Affect><Alternate Splicing><Alternative RNA Splicing><Alternative Splicing><B.1.617.2><Binding><Biochemical><C-terminal><CA-15-3 Antigen><COVID-19><COVID-19 S protein><COVID-19 spike glycoprotein><COVID-19 spike protein><COVID-19 virus><COVID19><COVID19 S protein><COVID19 spike glycoprotein><COVID19 spike protein><COVID19 virus><CV-19><CV19><Cancers><Catalysis><Catalytic Core><Catalytic Domain><Catalytic Region><Catalytic Site><Catalytic Subunit><Charge><CoV emergence><CoV-2><CoV2><Colo-rectal Cancer><Colorectal Cancer><Crystallization><DF3 Antigen><Delta variant><Development><Disease><Disorder><Drosophila melanogaster><Dysfunction><Enzyme Gene><Enzymes><Episialin><Epithelial><Epithelial Membrane Antigen><Family><Functional disorder><GALNT12><GALNT12 gene><GalNAc Transferase 12><GalNAc-T8><GalNAc-transferase><GalNAcT-8><GalNAcT12><Genetic Alteration><Genetic Change><Genetic defect><Glycopeptides><Glycoproteins><Glycosylated MUC-1><Goals><Golgi><Golgi Apparatus><Golgi Complex><HCoV><Human><Individual><Infection><Isoenzymes><Isoforms><Isozymes><Lectin><Light><London><MUC-1 Antigen><MUC1 antigen><Malignant Neoplasms><Malignant Tumor><Mediating><Membrane><Metabolic Glycosylation><Modeling><Modern Man><Modification><Molecular Interaction><Morphology><Mucin Peptide MUC-1><Mucin/Peptide><Mucins><Mucous body substance><Mucus><Mucus Glycoprotein><Mutation><N acetylgalactosamine><NIDCR><NIDR><NIEHS><National Institute of Dental Research><National Institute of Dental and Craniofacial Research><National Institute of Environmental Health Sciences><Nervous System Diseases><Neurologic Disorders><Neurological Disorders><Patients><Peptides><Photoradiation><Physiopathology><Polymorphic Epithelial Mucin><Polypeptide N-acetylgalactosaminyltransferase><Property><Protein Glycosylation><Protein Isoforms><Proteins><Publishing><RNA Splicing><Regulation><Research><Roentgen Rays><Role><SARS><SARS Virus><SARS corona virus><SARS corona virus 2><SARS coronavirus><SARS coronavirus disease><SARS-Associated Coronavirus><SARS-CoV><SARS-CoV disease><SARS-CoV-2><SARS-CoV-2 B.1.617.2><SARS-CoV-2 S protein><SARS-CoV-2 spike glycoprotein><SARS-CoV-2 spike protein><SARS-CoV2><SARS-CoV2 S protein><SARS-CoV2 spike glycoprotein><SARS-CoV2 spike protein><SARS-Related Coronavirus><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><Salivary Glands><Salivary Glands Head and Neck><Secretory Granules><Secretory Vesicles><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><Severe Acute Respiratory Syndrome CoV 2><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-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 spike glycoprotein><Severe acute respiratory syndrome coronavirus 2 spike protein><Severe acute respiratory syndrome related corona virus 2><Specificity><Splicing><Structure><Substrate Specificity><Testing><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-Radiation><X-Ray Radiation><X-ray><Xray><active followup><biochemical tools><biochemistry tools><college><collegiate><corona virus disease 2019><corona virus emergence><coronavirus disease 2019><coronavirus disease 2019 S protein><coronavirus disease 2019 spike glycoprotein><coronavirus disease 2019 spike protein><coronavirus disease 2019 virus><coronavirus emergence><developmental><developmental disease><developmental disorder><emergent CoV><emergent corona virus><emergent coronavirus><emerging CoV><emerging corona virus><emerging coronavirus><flexibility><flexible><follow up><follow-up><followed up><followup><genome mutation><glycosylation><hCoV19><human CoV><human corona virus><human coronavirus><in vivo><insight><interest><malignancy><membrane structure><microbial consortia><microbial flora><microbiota><microflora><mucous><multispecies consortia><nCoV><nCoV2><neoplasm/cancer><nervous system disorder><neurological disease><new CoV><new corona virus><new coronavirus><novel CoV><novel corona virus><novel coronavirus><pathophysiology><preference><protein-UDPacetylgalactosaminyltransferase><recruit><severe acute respiratory syndrome coronavirus 2 B.1.617.2><severe acute respiratory syndrome-CoV><social role>