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What do Glycans Do? – Finding the Rightful Place for Carbohydrates in the Central Dogma of Life! Gerald W. Hart, Professor & Director, Department of Biological.

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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 Chemistry Johns 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 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 No example of a polypeptide that is not modified?
Functional Diversity Gene Ultimate Gene Products exon 1 exon 2 exon 3 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. Genome Sequencing Genomic DNA Microarray Analysis mRNA Traditional Proteomics Protein Functional Modified 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 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: /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 )Glycobiology 12,43R-56R

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

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, (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 6 The Term “Glycosylation” Often Confuses Non-Glycobiologists Essentials of Glycobiology Second Edition Outside FIGURE 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

16 O-GlcNAcylation Is Not Glycation!

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

18 Origins of Glycobiology

19 Who Discovered Protein Glycosylation
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 Department 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) Discoveries 1876 J.L.W. Thudichum Glycosphingolipids (cerebrosides), sphingomyelin & sphingosine 1888 H. Stillmark Lectins as Hemagglutinins 1916 J. MacLean (Howell) a second-year medical student at Johns Hopkins University, Isolation of Heparin as an Anti-coagulant 1934 K. Meyer Hyaluronan and hyaluronidase 1949 L.F. Leloir Discovery of nucleotide sugars and roles in biosynthesis of glycans 1952 Gottschalk Sialic Acids as the Receptor for Influenza virus 1958 H. Muir Mucopolysaccharides (GAGs) covalently attached to protein via Ser G.E. Palade ER-Golgi pathway for glycoprotein biosynthesis 1964 B. Gesner, V. Ginsburg Glycans 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) Discoveries 1966 M. Neutra, C. Leblond Role of Golgi in Protein Glycosylation 1969 L. Warren, M.C. Glick, P.W. Robbins Increased size (branching) of N-glycans in malignantly transformed cancer cells. G. Ashwell, A. Morell Glycans control half-life of circulating glycoproteins 1972 J.F. G. Vliegenthart Power of high-field proton NMR for glycan analysis 1975 V.I. Teichberg The First Galectin A. Kobata First to do N- & O-”Glycomics” 1977 R.L. Hill, R. Barker First Purification of a glycoprotein glycosyltransferase 1981 M.J. Ferguson, I. Silman, M. Low First Structure of a GPI-Anchor 1986 P.K. Qasba, J. Shaper, N. Shaper Cloning of the first animal glycosyltransferase Source: 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 – 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 Mucin Glycans 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

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 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 & Rotovirus eg. – 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, (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 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 Glycomics convened 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 Katherine Bowman, Ph.D. Board on Life Sciences Douglas C. Friedman, Ph.D. Program Officer | Board on Chemical Sciences and Technology

36 We Welcome Your Input Friday, 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

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