Presentation on theme: "What do Glycans Do? – Finding the Rightful Place for Carbohydrates in the Central Dogma of Life! Gerald W. Hart, Professor & Director, Department of Biological."— Presentation transcript:
1 What do Glycans Do? – Finding the Rightful Place for Carbohydrates in the Central Dogma of Life! Gerald W. Hart, Professor & Director, Department of Biological ChemistryJohns Hopkins University, School of Medicine;Glycosciences – Why The Next Big Thing?How Other Scientists View Glycosylation – Part of the Problem.Historical Remarks - Where Did Glycobiology Come From?Preaching to the Choir – Biological Functions of Glycans?Technological Advances Moving the Field ForwardWhat Are the Major Challenges to Moving Into the “Mainstream”Some Major Questions for the Future – One Person’s OpinionThe NAS Initiative – “Assessing the Importance and Impact of Glycosciences and Glycomics” – Need your input!
2 Genomics Does Not Explain Biology: - ~26,000 GenesGene Sequences 99.9% Identical!- ~30,000 Genes
3 No example of a polypeptide that is not modified? Functional DiversityGeneUltimate Gene Productsexon 1exon 2exon 3Transcriptional RegulationAlternative Splicing, CellType Specific Expression, etc.Translational RegulationMasking, mRNAStability etc.Post-translational RegulationModification by O-GlcNAc,Phosphate, Ubiquitin, etc.GenomeSequencingGenomic DNAMicroarrayAnalysismRNATraditionalProteomicsProteinFunctionalModified Protein-O-GlcNAc-Ub-P~100K Proteins >millions of Molecular Species>400 Non-Glyco. PTMs Known; Glycosylation is by far the most abundant!No example of a polypeptide that is not modified?
4 Scientific Reports 1,90 doi:10.1038/srep00090 PTMs Greatly Expand Chemical Diversity of the Genetic Code:“~50% of all proteins are glycosylated”: Apweiler et al. Biochim. Biophys. Acta, Gen. Subj. 1473, 4–8 (1999). Percentage glycosylated is much higher, if you include O-GlcNAc!Phosphorylation is Notthe most Common PTM!*Proteome-wide post-translational modification statistics: frequency analysis and curation of the swiss-prot database***Scientific Reports 1,90 doi: /srep0009013 September 2011Source: Public Domain: Wikipedia
5 Currently, We live in a “Protein and Nucleic Acid Centric World”: # of building blocks is actuallyFairly small.“Scientific discussions that encompass “glycans” remain relatively infrequent in the protein centric world of cell biology. Some scientists lament the ‘complexity of the molecules’. Yet our alphabet of 26 characters, let alone Chinese characters, is rather easilyassimilated. Imagine a world in which each of us knew only a fraction of the alphabet.”
7 Scientists & Editors View “Glycosylation” as Just Another Post-Translational Modification:
8 Pro- & Eukaryotic Glycoproteins: If Consider only the linkage sugar, there are over 41 different chemical bonds, each more different than acetylation is from methylation!(updated from Spiro review )Glycobiology 12,43R-56R
9 Hierarchy of Protein Glycosylation: Nuclear & CytoplasmicGlycoproteinsCollagensMucinsN-Linked GPsProteoglycansHyaluronicAcid
11 Glycocalyx of Human Erythrocyte: Plasma Membrane
12 Misleading Depiction of ß-Adrenergic Receptor:Protein Centric World Note: Representation ofN-glycans.
13 Complex Glycans Are Often Very Large: Rivaling the Size of the Polypeptides to which they are attached.Size of a Typical Fc DomainAnn. Rev. Biochem. 57, (1988)
14 Relative Sizes of pT200 and og189 on CAMKIV Surface models of N-acetylglucosamine (left foreground) and inorganic phosphate (right foreground), along with a cartoon model of the kinase domain from human wild-type CaMKIV (center background) modeled from an X-ray crystal structure of human CaMKIγ. The amino acid residues colored in green and red are those that are modified by GlcNAcylation and phosphorylation, respectively.
15 Chapter 1, Figure 6The Term “Glycosylation” Often Confuses Non-GlycobiologistsEssentials of GlycobiologySecond EditionOutsideFIGURE 1.6. Common classes of animal glycans. (Modified from Varki A FASEB J. 11: 248–255; Fuster M. and Esko J.D Nat. Rev. Can. 7: 526–542.)Inside
19 Who Discovered Protein Glycosylation Who Discovered Protein Glycosylation? – “First to Establish the Existence of a Covalent Linkage of Sugar to Protein – A. NeubergerBiochem. J. 32, 1435.Biochem. J. (1960) 77, 239Carbohydrates in Protein 2. THE HEXOSE, HEXOSAMINE, ACETYL AND AMIDE-NITROGEN CONTENT OF HEN'S-EGG ALBUMIN*BY PATRICIA G. JOHANSEN,t R. D. MARSHALL AND A. NEUBERGERDepartment of Chemical Pathology, St Mary'8 Hospital Medical School, London, W. 2(Received 15 March 1960)
20 Some Important Discoveries in the History of Glycobiology: Year Primary Scientist(s)Discoveries1876J.L.W. ThudichumGlycosphingolipids (cerebrosides), sphingomyelin & sphingosine1888H. StillmarkLectins as Hemagglutinins1916J. MacLean (Howell) a second-year medical student at Johns Hopkins University,Isolation of Heparin as an Anti-coagulant1934K. MeyerHyaluronan and hyaluronidase1949L.F. LeloirDiscovery of nucleotide sugars and roles in biosynthesis of glycans1952GottschalkSialic Acids as the Receptor for Influenza virus1958H. MuirMucopolysaccharides (GAGs) covalently attached to protein via SerG.E. PaladeER-Golgi pathway for glycoprotein biosynthesis1964B. Gesner, V. GinsburgGlycans control migration of leukocytes to target organs.Source: Essentials of Glycobiology. 2nd Edition.
21 Some Important Discoveries in the History of Glycobiology: Year Primary Scientist(s)Discoveries1966M. Neutra, C. LeblondRole of Golgi in Protein Glycosylation1969L. Warren, M.C. Glick, P.W. RobbinsIncreased size (branching) of N-glycans in malignantly transformed cancer cells.G. Ashwell, A. MorellGlycans control half-life of circulating glycoproteins1972J.F. G. VliegenthartPower of high-field proton NMR for glycan analysis1975V.I. TeichbergThe First GalectinA. KobataFirst to do N- & O-”Glycomics”1977R.L. Hill, R. BarkerFirst Purification of a glycoprotein glycosyltransferase1981M.J. Ferguson, I. Silman, M. LowFirst Structure of a GPI-Anchor1986P.K. Qasba, J. Shaper, N. ShaperCloning of the first animal glycosyltransferaseSource: Essentials of Glycobiology. 2nd Edition.
22 Expression, localization, activity of glycosidases Glycans Generate Structural Diversity that is Plastic with Respect to Biology:~250 Glycosyltransferases in the Human Genome – 2% of the genome (BBA 1792, )Glycan Structures are not encoded on a template – Structure Determined by:Glycosyltransferase Expression, Localization and Organization – CompetitionExpression, localization, activity of glycosidasesSugar Nucleotide Concentration & TransportProtein Structure at all Levels – 1o, 2o,3o, 4oSynthesis, Transport & Folding Rates of PolypeptideStructures of Glycans on a Polypeptide – Characteristic of Cell Type, Developmental Stage and Environmental Influence.Glycotypes; GlycoformsWhy do different transferases use distinct donor Sugar Nucleotides??UDP-GlcNAc, UDP-Gal, UDP-GalNAc, GDP-Man, GDP-Fuc, CMP-NeuAc, PAPS – Relationship to Nucleotide Metabolism??
23 N-Glycan Biosynthetic Pathway: A System to Generate Diversity.
25 Protein-Bound Glycans Are Targets For Many Pathogens and Toxins: Mucins are at the “Front Lines”Acting as Decoys
26 Impact of Glycosciences on Society is Huge: Human Health – Nearly every major disease afflicting mankind directly involves glycoconjugates.Renewable Energy – Development of Biofuels requires a better understanding of plant cell wall synthesis and deconstruction.Agriculture – Nitrogen-Fixation; Anti-fungals; food.Industry – Polysaccharide-based materials will replace petroleum. wood fiber, textile, agricultural, and food industries.
28 Glycans are involved in nearly all biological processes & play a major role in human disease: Generalization:Complex Glycans Usually Function at the Multi-Cellular Level – lectin resistant cellsO-GlcNAc Functions at the Intracellular Level in Single Cells.Rarity and severity of genetic diseases (eg. CDGs) illustrate the importance of glycans.Some Examples of Glycans and Disease:Defective O-glycosylation of alpha-dystroglycan in Muscular DystrophyO-GlcNAcylation – Diabetes, Alzheimer’s Disease, Cancer & Cardiovascular Disease.Regulation of Notch Signaling by GlycansSelectins and InflammationSiglecs and Regulation of ImmunityGalectins role in immunityProteoglycans- Regulation of growth factors, microbe binding, tissue morphogenesis and cardiovascular disease.Microbes and Viruses Gain Entry via glycans; Mucins are front-line of defense.Roles of Sialic acids in viral infection – Flu & Rotovirus eg. – Relenza and TamifluHeparin – One of the oldest and most widely used ‘drugs” is a GAG.Monoclonal Therapeutics – Glycoforms are critical to efficacy.Cell Surface Glycans Key to Tumor Metastasis – Cancer Biomarkers.Vaccines to Infectious Organisms – Many (Most) are glycans.
29 Recent Advances Moving the Field Forward Genomics & Proteomics – Allowed the molecular characterization of glyco enzymes & glycoproteins.Rapid Advances in Mass SpectrometryImproved Methods & Sensitivity in NMRMolecular Genetics – Transgenic Organisms; siRNAArray Technologies – Lectins, Antibodies, Glycans.Synthetic Methods – Chemical & Enzymatic.Availability of Pure Enzymes – GTs & GSSpecific Inhibitors of Glyco Enzymes.Bioinformatics & Molecular Modeling.
30 Glycomic Complexity Reflects Cellular Complexity: Functional glycomics also requires the tools of genomics, proteomics, lipidomics, and metabolomics.Cell 143, (modified after Packer et al. 2008)
31 Challenges to the Integration of Glycosciences Into the Mainstream: Lack of Education of Young People & Non-Glycoscientists.Inherent Complexity & Nomenclature of Glycans.Sophistication Required for Structural Analyses.Difficulties in Chemical Synthesis & Analysis – No “PCR”!Lack of Tools to Understand Site-Specific Function of Glycans.Lack of Simple Tools (“kits”) so non-glycoscientists can study glycans on their molecules.Failure to incorporate glycan data into long-term stable, govt. supported databases (eg. NCBI).
32 Some Major Questions/Problems: (A personal opinion) How can we perform a complete molecular species analysis of a glycoprotein with multiple sites? – Top-Down MS?What are the biological functions of site-specific oligosaccharide heterogeneity? Does it still exist in a single cell?How does altered glycan branching contribute to the metastatic properties of a cancer cell?Will specific glycoforms really provide better biomarkers for disease?Glycosciences should have a huge impact on anti-viral, anti-bacterial and anti-fungal therapeutics – how do we get there?Specific Roles of Glycans in Intercellular Communications Regulating Development.How do glycans regulate the lateral organization & function of receptors in the plasma membrane – signalosomes.How do we decode the information content of GAGs & Proteoglycans?Roles of the Crosstalk Between O-GlcNAc and other PTMs in Signaling, Transcription, Diabetes, Alzheimer’s Disease and Cancer?
33 Good News for Young People Trained in Glycoscience: The future is bright! Glycoscience is poised to be the “next big thing”!Industry and Academia – Hire problem solvers willing to use whatever tools are needed.Glycoscientists, by necessity, know how to think about and do a lot of different approaches.By Training, you are multi-lingual in the language of science.
34 This Meeting Illustrates the Remarkable Biological Breadth of Our Field: Production of Recombinant GlycoproteinsGlycan Roles in Viruses – AIDS & InfluenzaGlycans in Innate ImmunityRoles in Signaling & Membrane DynamicsGlycan Roles in Bacteria & Cell WallsParasite Glycobiology – Fungi, Malaria, protozoa, wormsGlycans in Stem Cell BiologyGlycans in Tuberculosis, Cell Adhesion, Fertilization, & Evolution.Glycans in Immunity, muscular dystrophy, biomarkers, and drug development.
35 What is the Future of Glycoscience? Study by the Committee on Assessing the Importance and Impact of Glycosciences and Glycomics convened through the National Academy of Sciences; report expected to be released fall 2012Explore transformative impacts that advances in glycoscience can have across sectors such as health, energy, and materials scienceArticulate a vision for the field of glycoscience and a roadmap for future developmentSponsored by NIH, FDA, DOE, and NSFContact us atKatherine Bowman, Ph.D.Board on Life SciencesDouglas C. Friedman, Ph.D.Program Officer | Board on Chemical Sciences and Technology
36 We Welcome Your InputFriday, Nov. 11, 1:00 − 2:00pm in Room Grand III Join us for an informal discussion on the committee’s task and share your thoughts.Website:Community feedback is crucial to the study’s success