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Principal Investigator: ANANT K MENON
Organization: WEILL MEDICAL COLL OF CORNELL UNIV
Fiscal Year: 2024
Award: $470,047
Funding agency: National Institute of General Medical Sciences
Protein glycosylation is essential in all eukaryotes, from disease-causing protists such as malaria, to yeast and
mammals. Secretory proteins are N-glycosylated, O- and C-mannosylated, and/or glycosylphosphatidylinositol
(GPI)-anchored as they enter the lumen of the endoplasmic reticulum (ER). Yeast that cannot synthesize N-
glycoproteins or GPI-proteins are inviable, and mice with the same defects die as embryos. Glycosylation is
important in dengue and SARS-CoV-2 viral infections, and defects in glycosylation cause human disease. Thus,
deficient O-mannosylation of dystroglycan is a cause of muscular dystrophy and GPI deficiency in
hematopoietic human stem cells underlies the hemolytic disease paroxysmal nocturnal hemoglobinuria.
Congenital Disorders of Glycosylation (CDGs) are severe inherited diseases with neurological symptoms.
Protein glycosylation reactions require the glycolipids mannosyl- and glucosyl-phosphoryl dolichol (MPD,
GPD) to act as sugar donors in the lumen of the ER. As these lipids are synthesized on the cytoplasmic side,
they must be flipped across the ER membrane to function in the lumen, a process requiring specific
transporters, termed scramblases, that have yet to be identified. Assays of the two scramblases in microsomes
and reconstituted vesicles, using natural lipids and short-chain analogs as reporters, reveal that transport is
bidirectional, ATP-independent, and highly structure specific, discriminating between structural isomers.
We will identify the MPD and GPD scramblases using chemo-proteomic and bioinformatic approaches.
Deploying novel photo-clickable probes synthesized by the Häner group (University of Bern) we will determine
the MPD and GPD interactomes, that we hypothesize will include the scramblases. Our preliminary results
validate this approach: the MPD probe functions in ER mannosylation and photo-identifies specific yeast
microsomal proteins. Photo-adducted proteins will be identified by quantitative proteomics and tested for
scramblase activity in our reconstitution-based assays. Promising candidates will be validated in vivo by
evaluating phenotypes of yeast mutants. For GPD scramblase we will also identify candidates via phylogenetic
profiling, a bioinformatics method for assignment of protein function. This approach complements the photo-
identification strategy and has already yielded a list of GPD scramblase candidates for testing.
This is a consequential proposal to discover critical players in ER protein glycosylation. Our extensive
experience in studying scramblases puts us in a strong position to tackle this objective. We discovered the
scramblase activity of Class A GPCRs and were the first to show lipid scrambling by a TMEM16 ion channel.
We now deploy in silico, biochemical and biophysical methods to elucidate their mechanism. We will use this
expertise in future work to reveal the molecular mechanism of structure-specific lipid scrambling mediated by
the MPD and GPD scramblases that we predict to be distinct from that of the currently known phospholipid
scramblases. At a biological level, our discoveries will reveal new genetic loci associated with CDGs.
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><Alkynes><Allelism Test><Antibodies><Assay><Azides><Benzophenones><Bio-Informatics><Bioassay><Biochemical><Bioinformatics><Biological><Biological Assay><COVID-19 virus><COVID19 virus><Carrier Proteins><Cell Body><Cell-Adhesion Molecule Receptors><Cell-Adhesion Protein Receptors><Cell-Extracellular Matrix><Cells><Cellular Matrix><Cellular biology><Characteristics><CoV-2><CoV2><Complementation Test><Complex><Congenital disorders of glycosylation><Cranin><Cytoplasm><Cytoskeletal System><Cytoskeleton><D-Glucose><D-Mannopyranosyldolichyl><D-Mannose><Data><Defect><Dengue><Dextrose><Disease><Disorder><Distant><Dolichol><Dolichol Monophosphate Mannose><Dolicholphosphate Mannose><Dolichyl Mannopyranosyl Phosphate><Dolichyl Mannosyl Phosphate><Dystroglycan><ECM><Embryo><Embryonic><Endoplasmic Reticulum><Ergastoplasm><Eukaryota><Eukaryote><Extracellular Matrix><Face><Future><G Protein-Complex Receptor><G Protein-Coupled Receptor Genes><G-Protein-Coupled Receptors><GPCR><GPI Membrane Anchors><Genetic Complementation Test><Genome><Glucose><Gly-PtdIns><Glycans><Glycoinositol Phospholipid Membrane Anchor><Glycolipids><Glycoproteins><Glycosyl-Phosphatidylinositol><Glycosyl-Phosphatidylinositol Membrane Protein Anchors><Glycosylated Phosphatidylinositols><Glycosylphosphatidylinositol Anchors><Glycosylphosphatidylinositols><Goals><Hematopoietic><Hereditary Disease><Inborn Genetic Diseases><Inherited disorder><Ion Channel><Ionic Channels><Isomerism><Learning><Link><Lipids><Malaria><Mammalia><Mammals><Mannolipid><Mannopyranose><Mannopyranoside><Mannose><Mannosyl Phosphoryl Dolichol><Mediating><Membrane><Membrane Channels><Membrane Protein Gene><Membrane Proteins><Membrane-Associated Proteins><Metabolic Glycosylation><Methods><Mice><Mice Mammals><Microsomes><Molecular><Murine><Mus><Muscular Dystrophies><Myodystrophica><Myodystrophy><Neurologic Manifestations><Neurologic Signs and Symptoms><Neurologic Symptoms><Neurological Manifestations><Neurological Signs and Symptoms><Organism><Paludism><Phenotype><Phylogenetic Analysis><Phylogenetics><Physiologic><Physiological><Plant Resins><Plasmodium Infections><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><Procedures><Process><Proliferating><Protein Glycosylation><Protein Modification><Proteins><Proteomics><Reaction><Reporter><SARS corona virus 2><SARS-CO-V2><SARS-COVID-2><SARS-CoV-2><SARS-CoV2><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><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-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome related corona virus 2><Side><Source><Specificity><Structure><Surface Proteins><Testing><Trans Test><Transport Protein Gene><Transport Proteins><Transporter Protein><Trypanosoma><Trypanosome><Universities><Vesicle><Viral Diseases><Virus><Virus Diseases><Work><Wuhan coronavirus><Yeasts><adduct><analog><biologic><biophysical approaches><biophysical methodology><biophysical methods><biophysical techniques><candidate identification><cell biology><chemoproteomics><complementation analysis><complementation approach><coronavirus disease 2019 virus><coronavirus disease-19 virus><crosslink><experience><experiment><experimental research><experimental study><experiments><faces><facial><gene locus><genetic locus><genomic location><genomic locus><glycosylation><hCoV19><hemopoietic><hereditary disorder><heritable disorder><human disease><human progenitor><human stem cells><in silico><in vivo><inborn error><inherited diseases><inherited genetic disease><inherited genetic disorder><innovate><innovation><innovative><interest><intracellular skeleton><isomer><living system><membrane structure><muscle dystrophy><mutant><nCoV2><neural manifestation><nonspecific phospholipid transfer proteins><novel><paroxysmal nocturnal hemoglobinuria><phospholipid scramblase><protein function><reconstitute><reconstitution><resin><secretory protein><sugar><viral infection><virus infection><virus-induced disease>