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; 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 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!

Genomics Does Not Explain Biology:

- ~26,000 Genes

- ~30,000 Genes

Gene Sequences 99.9% Identical!

Functional Diversity

Gene Ultimate Gene Products

exon 1 exon 2 exon 3

Transcriptional Regulation Alternative Splicing, Cell Type Specific Expression, etc.

Translational Regulation Masking, mRNAStability etc.

Post-translational Regulation Modification by O-GlcNAc, Phosphate, Ubiquitin, etc.

GenomeSequencing

Genomic DNA

MicroarrayAnalysis

mRNA

TraditionalProteomics

Protein

FunctionalProteomics

Modified Protein

-O-GlcNAc

-O-GlcNAc

-O-GlcNAc

-Ub -P

-P

-P

>400 Non-Glyco. PTMs Known; Glycosylation is by far the most abundant! No example of a polypeptide that is not modified?

~100K Proteins >millions of Molecular Species

Scientific Reports 1,90 doi:10.1038/srep0009013 September 2011

PTMs Greatly Expand Chemical Diversity of the Genetic Code:

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Source: Public Domain: Wikipedia

“~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!

Proteome-wide post-translational modification statistics: frequency analysis and curation of the swiss-prot database

Phosphorylation is Not the most Common PTM!

“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 .”

Currently, We live in a “Protein and Nucleic Acid Centric World”:

# of building blocks is actually Fairly small.

How Non-Glycobiologists View Glycosylation

Scientists & Editors View “Glycosylation” as Just Another Post-Translational Modification:

(updated from Spiro review )Glycobiology 12,43R-56R

Pro- & Eukaryotic Glycoproteins: If Consider only the linkage sugar, there are over 41 different chemical bonds, each more different than acetylation is from methylation!

Hierarchy of Protein Glycosylation:

Nuclear &

Cytoplasmic

Glycoproteins

Collagens

Mucins

N-Linked GPs

Proteoglycans

Hyaluronic

Acid

Typical Biochemistry/Cell Biology Textbook View

Glycocalyx of Human Erythrocyte:

Plasma Membrane

Misleading Depiction of ß-Adrenergic Receptor:Protein Centric

WorldNote: Representation of N-glycans.

Ann. Rev. Biochem. 57, 785- 838 (1988)

Complex Glycans Are Often Very Large: Rivaling the Size of the Polypeptides to which they are attached.

Size of a Typical Fc Domain

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.

Chapter 1, Figure 6

The Term “Glycosylation” Often Confuses Non-Glycobiologists

Essentials of Glycobiology Second Edition

Outside

Inside

O-GlcNAcylation Is Not Glycation!

Ack…Ack…Ack…

GlcNAc….GlcNAc….GlcNAc.

GlcNAc..GlcNAc….

Origins of Glycobiology

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)

Biochem. J. 32, 1435.

Who Discovered Protein Glycosylation? – “First to Establish the Existence of a Covalent Linkage of Sugar to Protein – A. Neuberger

Year Primary Scientist(s) Discoveries1876 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 A. Gottschalk Sialic Acids as the Receptor for Influenza virus

1958 H. Muir Mucopolysaccharides (GAGs) covalently attached to protein via Ser

1961-1965 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.

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.

1969 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

1975-1980 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.

Some Important Discoveries in the History of Glycobiology:

~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 – CompetitionExpression, localization, activity of glycosidasesSugar 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??

Glycans Generate Structural Diversity that is Plastic with Respect to Biology:

N-Glycan Biosynthetic Pathway: A System to Generate Diversity.

Why are Mucin Glycans So Complex!!

Protein-Bound Glycans Are TargetsFor Many Pathogens and Toxins:

Mucins are at the “Front Lines”

Acting as Decoys

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.

Glycans Permeate Cellular Functions

Cell 143, 672-676

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.

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.

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)

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).

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?

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.

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.

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 glyco@nas.edu

Katherine Bowman, Ph.D.Board on Life Sciences

Douglas C. Friedman, Ph.D.Program Officer | Board on Chemical Sciences and Technology

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: http://glyco.nas.edu/feedback

• Community feedback is crucial to the study’s success