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!
Genomics Does Not Explain Biology:
- ~26,000 Genes
Gene Sequences 99.9% Identical!
- ~30,000 Genes
Gene
Ultimate Gene Products
Functional Diversity
~100K Proteins
>millions of Molecular Species
Genome
Sequencing
Microarray
Analysis
Traditional
Proteomics
Functional
Proteomics
Genomic DNA
mRNA
Protein
Modified Protein
exon 1
exon 2
exon 3
-P
-O-GlcNAc
-Ub
-P
-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.
>400 Non-Glyco. PTMs Known; Glycosylation is by far the most abundant!
No example of a polypeptide that is not modified?
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 posttranslational modification
statistics: frequency analysis
and curation of the swissprot database
*
*
Scientific Reports 1,90 doi:10.1038/srep00090
Source: Public Domain: Wikipedia
13 September 2011
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.”
How Non-Glycobiologists
View Glycosylation
Scientists & Editors View “Glycosylation” as Just Another Post-Translational Modification:
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
Hierarchy of Protein Glycosylation:
Typical Biochemistry/Cell Biology Textbook View
Glycocalyx of Human Erythrocyte:
Plasma Membrane
Misleading Depiction
of ß-Adrenergic Receptor:Protein Centric World
Note: Representation of
N-glycans.
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)
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.
The Term “Glycosylation” Often Confuses Non-Glycobiologists
Outside
Inside
Essentials of Glycobiology
Second Edition
Chapter 1, Figure 6
O-GlcNAcylation Is Not Glycation!
Origins of Glycobiology
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)
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
Isolation of Heparin as an Anti-coagulant
second-year medical student
at Johns Hopkins University,
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.
Glycans Generate Structural Diversity that is Plastic with Respect to Biology:
 ~250 Glycosyltransferases in the Human Genome – 2% of the genome (BBA 1792, 925930)
 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??
N-Glycan Biosynthetic Pathway: A System to Generate Diversity.
Why are Mucin Glycans So Complex!!
Protein-Bound Glycans Are Targets
For 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 & NonGlycoscientists.
• 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 antifungal 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