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What do Glycans Do? – Finding the Rightful Place for Carbohydrates in the Central Dogma of Life!

What do Glycans Do? – Finding the Rightful Place for Carbohydrates in the Central Dogma of Life! . Gerald W. Hart , Professor & Director, Department of Biological Chemistry Johns Hopkins University, School of Medicine; email : gwhart@jhmi.edu.

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What do Glycans Do? – Finding the Rightful Place for Carbohydrates in the Central Dogma of Life!

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  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 Chemistry Johns Hopkins University, School of Medicine; email: gwhart@jhmi.edu • 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 Forward • What Are the Major Challenges to Moving Into the “Mainstream” • Some Major Questions for the Future – One Person’s Opinion • The NAS Initiative – “Assessing the Importance and Impact of Glycosciences and Glycomics” – Need your input!

  2. Genomics Does Not Explain Biology: - ~26,000 Genes Gene Sequences 99.9% Identical! - ~30,000 Genes

  3. Gene Ultimate Gene Products exon 1 exon 2 exon 3 Functional Diversity Genome Sequencing Genomic DNA Microarray Analysis mRNA Traditional Proteomics Protein Functional Proteomics Modified Protein -P -O-GlcNAc -P -Ub -O-GlcNAc -P -O-GlcNAc Transcriptional Regulation Alternative Splicing, Cell Type Specific Expression, etc. Translational Regulation Masking, mRNA Stability etc. Post-translational Regulation Modification by O-GlcNAc, Phosphate, Ubiquitin, etc. ~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. 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). Percentageglycosylatedismuchhigher, ifyouinclude O-GlcNAc! Phosphorylation is Not the most Common PTM! * Proteome-wide post-translational modification statistics: frequency analysis and curation of the swiss-prot database * * * Scientific Reports 1,90 doi:10.1038/srep00090 13 September 2011 Source: Public Domain: Wikipedia

  5. Currently, We live in a “Protein and Nucleic Acid Centric World”: # of building blocks is actually Fairly 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 easily assimilated. Imagine a world in which each of us knew only a fraction of the alphabet.”

  6. How Non-Glycobiologists View Glycosylation

  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 )Glycobiology12,43R-56R

  9. Hierarchy of Protein Glycosylation: Hyaluronic Acid Nuclear & Cytoplasmic Glycoproteins Collagens Mucins Proteoglycans N-Linked GPs

  10. Typical Biochemistry/Cell Biology Textbook View

  11. Glycocalyx of Human Erythrocyte: Plasma Membrane

  12. Misleading Depiction of ß-Adrenergic Receptor:Protein Centric World Note: Representation of N-glycans.

  13. Complex Glycans Are Often Very Large: Rivaling the Size of the Polypeptides to which they are attached. Size of a Typical Fc Domain Ann. Rev. Biochem. 57, 785- 838 (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. Essentials of Glycobiology Second Edition The Term “Glycosylation” Often Confuses Non-Glycobiologists Outside Inside Chapter 1, Figure 6

  16. O-GlcNAcylation Is NotGlycation!

  17. Ack…Ack…Ack…GlcNAc….GlcNAc….GlcNAc.GlcNAc..GlcNAc….Ack…Ack…Ack…GlcNAc….GlcNAc….GlcNAc.GlcNAc..GlcNAc….

  18. Origins of Glycobiology

  19. Who Discovered Protein Glycosylation? – “First to Establish the Existence of a Covalent Linkage of Sugar to Protein – A. Neuberger Biochem. J. 32, 1435. Biochem. J. (1960) 77, 239 Carbohydrates 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. NEUBERGER DepartmentofChemicalPathology, St Mary'8 Hospital Medical School, London, W. 2 (Received 15 March 1960)

  20. Some Important Discoveries in the History of Glycobiology: Source: Essentials of Glycobiology. 2nd Edition.

  21. Some Important Discoveries in the History of Glycobiology: Source: Essentials of Glycobiology. 2nd Edition.

  22. Glycans Generate Structural Diversity that is Plastic with Respect to Biology: • ~250 Glycosyltransferases in the Human Genome – 2% of the genome (BBA 1792, 925-930) • Glycan Structures are not encoded on a template – Structure Determined by: • Glycosyltransferase Expression, Localization and Organization – Competition • Expression, localization, activity of glycosidases • Sugar Nucleotide Concentration & Transport • Protein Structure at all Levels – 1o, 2o,3o, 4o • Synthesis, Transport & Folding Rates of Polypeptide • Structures of Glycans on a Polypeptide – Characteristic of Cell Type, Developmental Stage and Environmental Influence. • Glycotypes; Glycoforms • Why 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.

  24. Why are MucinGlycans So Complex!!

  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.

  27. Glycans Permeate Cellular Functions Cell 143, 672-676

  28. Glycansare 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 cells • O-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 Dystrophy • O-GlcNAcylation – Diabetes, Alzheimer’s Disease, Cancer & Cardiovascular Disease. • Regulation of Notch Signaling by Glycans • Selectins and Inflammation • Siglecs and Regulation of Immunity • Galectins role in immunity • Proteoglycans- 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 & Rotoviruseg. – Relenza and Tamiflu • Heparin – 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 Spectrometry • Improved Methods & Sensitivity in NMR • Molecular Genetics – Transgenic Organisms; siRNA • Array Technologies – Lectins, Antibodies, Glycans. • Synthetic Methods – Chemical & Enzymatic. • Availability of Pure Enzymes – GTs & GS • Specific 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, 672-676 (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 glycoformsreally 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 Glycoproteins • Glycan Roles in Viruses – AIDS & Influenza • Glycans in Innate Immunity • Roles in Signaling & Membrane Dynamics • Glycan Roles in Bacteria & Cell Walls • Parasite Glycobiology – Fungi, Malaria, protozoa, worms • Glycans in Stem Cell Biology • Glycans 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 Glycomicsconvened through the National Academy of Sciences; report expected to be released fall 2012 • Explore transformative impacts that advances in glycoscience can have across sectors such as health, energy, and materials science • Articulate a vision for the field of glycoscience and a roadmap for future development • Sponsored by NIH, FDA, DOE, and NSF • Contact us at glyco@nas.edu Douglas C. Friedman, Ph.D. Program Officer | Board on Chemical Sciences and Technology Katherine Bowman, Ph.D. Board on Life Sciences

  36. We Welcome Your Input • Friday, Nov. 11, 1:00 − 2:00pm in Room Grand IIIJoin us for an informal discussion on the committee’s task and share your thoughts. • Website:http://glyco.nas.edu/feedback • Community feedback is crucial to the study’s success

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