1. BMB8130 Glycobiology, 2015
Course Organizers: Lance Wells and Michael Tiemeyer

2. Course Content BCMB8130 Spring '15
Instructors: Mike Tiemeyer and Lance Wells
Essentials of Glycobiology: Chapters
Tuesday
1/13/14
Introduction to Glycobiology (Tiemeyer); Monosaccharides and the Glycosidic Bond (Wells)
1, 2, 3, 13, 14
Thursday
1/15/14
Structure and Synthesis: Glycochemistry (Boons)-?
2, 49, 50
1/20/14
Structure and Synthesis: Glycosyltranferases and Glycosidases (Wells) 5
1/22/14
Structure and Synthesis: Sugar Nucleotides (Bar-Peled)-confirmed 4
1/27/14
Structure and Synthesis: GAGS (Bergmann)-confirmed
15, 16
1/29/14
Structure and Synthesis: Non-GAG glycans in O-linkage to proteins (Wells)
9, 12
2/3/14
Structure and Synthesis: Glycans linked to lipids and lipid precursors (Tiemeyer)
10, 11
2/5/14
Structure and Synthesis: Glycans in N-linkage to proteins (Tiemeyer) 8
2/10/14
EXAM: Glyco-alphabet
2/12/14
Glycoanalytics: MS-based approaches (Wells)
47, 48
2/17/14
Glycoanalytics: NMR methodoligies (York)-confirmed
2/19/14
Cellular Function: Protein Folding, ER Processing, Quality Control (Moremen)-confirmed 36
2/24/14
Cellular Function: Membrane Trafficking through Golgi and endosomal compartments (Steet)-confirmed 3
2/26/14
Cellular Function: Nucleocytoplasmic Glycosylation (Wells)
17, 18
3/3/14
Cellular Function: GAG Functions (Wang)-confirmed
16, 35
3/5/14
WORLD CAFÉ #1
3/10/14
SPRING BREAK (NO CLASS)
3/12/14
3/17/14
Cellular Function: Glycan Binding Proteins (Tiemeyer)
26-35
3/19/14
Cellular Function: Plant Cell Walls (York)-flex 22
3/24/14
Glycans and Development: Fertilization to Gastrulation (Tiemeyer) 38
3/26/14
Glycans and Development: Adhesion, signaling, and differentiation (Tiemeyer)
3/31/14
WORLD CAFÉ #2
4/2/14
Glycan-based Pathophysiology: CDGs and Storage Disease (Steet)-confirmed
41, 42
4/7/14
Glycan-based Pathophysiology: Inflammation and Immunity (Avci)-confirmed
39, 40
4/9/14
WORLD CAFÉ #3
4/14/14
Glycan-based Pathophysiology: CMDs (Wells) 42
4/16/14
Glycan-based Pathophysiology: Cancer (Pierce)-confirmed 44
4/21/14
WORLD CAFÉ #4
4/23/14
Course Overview and Evaluation (Wells and Tiemeyer)

3. Administrative Details
BMB8130 Glycobiology, 2015
Administrative Details
Grade
49% Attendance and Participation in Lectures
30% EXAM (Learning the Language)
20% World Glycobiology Café
1% Instructors’ Discretion
World Glycobiology Café Sessions
Interactive discussions of papers relevant to lecture content; topical papers of basic biochemical or translational biomedical interest
Circulating discussion groups (speed-dating)
Reporters summarize discussions in the context of the major ideas of the paper

4. Administrative Details (cont’d)
BMB8130 Glycobiology, 2013
Administrative Details (cont’d)
World Glycobiology Café (cont’d)
Between 1-3 papers assigned per Café session
The Café will be held in the breakroom
The Café opens with 3-4 tables, depending on the topic
Each table has 1 person assigned to it as a “Reporter”
Everybody will be a Reporter once during the course
All other participants will distribute themselves evenly between the Café tables
Each Café table will have an assigned topic or data figure to discuss
Discussion will continue for 10 minutes
At the end of the discussion period, everybody except the Reporter will move to another table

5. Administrative Details (cont’d)
BMB8130 Glycobiology, 2015
Administrative Details (cont’d)
World Glycobiology Café (cont’d)
After as many discussion periods have elapsed as there are Café tables, the reporters will have 10 minutes to work-up a summary report of the discussions that they heard at their table
Each Reporter will present a brief report (no more than 5 minutes) to the whole group that summarizes the key discussion points raised during the speed-dating period
Reporters will be graded based on their report
Since everybody will be a reporter at some point, all participants should be motivated to help the Reporters develop well-structured ideas and keen observations to report to the whole group--everybody has a stake in the success of the process

6. BMB8130 Glycobiology, 2015 Thematic Content Bootcamp
Glycan Structures and Synthesis (Learning the Language)
EXAM, Tues., Feb. 10.
Compelling Topics
Glycan Structure and Synthesis
Glycoanalytics
Glycans and Cellular Function
Glycan Macromolecules (Plant and Animal)
Glycans and Development
Glycan-based Pathophysiology

7. Course Content EXAM! BCMB8130 Spring '15
Instructors: Mike Tiemeyer and Lance Wells
Essentials of Glycobiology: Chapters
Tuesday
1/13/14
Introduction to Glycobiology (Tiemeyer); Monosaccharides and the Glycosidic Bond (Wells)
1, 2, 3, 13, 14
Thursday
1/15/14
Structure and Synthesis: Glycochemistry (Boons)-?
2, 49, 50
1/20/14
Structure and Synthesis: Glycosyltranferases and Glycosidases (Wells) 5
1/22/14
Structure and Synthesis: Sugar Nucleotides (Bar-Peled)-confirmed 4
1/27/14
Structure and Synthesis: GAGS (Bergmann)-confirmed
15, 16
1/29/14
Structure and Synthesis: Non-GAG glycans in O-linkage to proteins (Wells)
9, 12
2/3/14
Structure and Synthesis: Glycans linked to lipids and lipid precursors (Tiemeyer)
10, 11
2/5/14
Structure and Synthesis: Glycans in N-linkage to proteins (Tiemeyer) 8
2/10/14
EXAM: Glyco-alphabet
2/12/14
Glycoanalytics: MS-based approaches (Wells)
47, 48
2/17/14
Glycoanalytics: NMR methodoligies (York)-confirmed
2/19/14
Cellular Function: Protein Folding, ER Processing, Quality Control (Moremen)-confirmed 36
2/24/14
Cellular Function: Membrane Trafficking through Golgi and endosomal compartments (Steet)-confirmed 3
2/26/14
Cellular Function: Nucleocytoplasmic Glycosylation (Wells)
17, 18
3/3/14
Cellular Function: GAG Functions (Wang)-confirmed
16, 35
3/5/14
WORLD CAFÉ #1
3/10/14
SPRING BREAK (NO CLASS)
3/12/14
3/17/14
Cellular Function: Glycan Binding Proteins (Tiemeyer)
26-35
3/19/14
Cellular Function: Plant Cell Walls (York)-flex 22
3/24/14
Glycans and Development: Fertilization to Gastrulation (Tiemeyer) 38
3/26/14
Glycans and Development: Adhesion, signaling, and differentiation (Tiemeyer)
3/31/14
WORLD CAFÉ #2
4/2/14
Glycan-based Pathophysiology: CDGs and Storage Disease (Steet)-confirmed
41, 42
4/7/14
Glycan-based Pathophysiology: Inflammation and Immunity (Avci)-confirmed
39, 40
4/9/14
WORLD CAFÉ #3
4/14/14
Glycan-based Pathophysiology: CMDs (Wells) 42
4/16/14
Glycan-based Pathophysiology: Cancer (Pierce)-confirmed 44
4/21/14
WORLD CAFÉ #4
4/23/14
Course Overview and Evaluation (Wells and Tiemeyer)
EXAM!

8. BMB8130 Glycobiology, 2015 Key Resources Course Website
Links to lecture slides, papers for World Glycobiology Cafés, Materials from previous years, other background information
Essential Reference
Essentials of Glycobiology, Varki, et al., eds. Second Edition
The authoritative text in Glycobiology
Fully open access

9. What is Glycobiology?
A term frequently attributed to Raymond Dwek, et al (circa 1990) to encompass the body of research that contributes to understanding:
The structure, biosynthesis, and biology of saccharides

10. Or, more explicitly stated (at least more explicit than could have been imagined 30 years ago)--Glycobiology is the human field of endeavor that strives to characterize and understand:
The diversity of glycan structures
The processes by which glycans are synthesized
The determinants of glycan structure
The mechanisms of glycan-protein interactions
The impact of glycans on the structure and function of the molecules to which they are attached
The contribution of glycans to normal cellular function and tissue development
The participation of glycans in diverse pathologies

11. Thus, aspects of glycobiology impact a broad range of disciplines
To name a few--
organic synthetic chemistry neurobiology
protein biochemistry reproductive medicine
enzymology endocrinology
analytic chemistry cell signaling
structural biochemistry stem cell biology
cell biology membrane biophysics
developmental biology microbiology
genetics cancer biology
genomics immunology
proteomics microbiology
parasitology biotechnology
And, in fact, glycobiology impacts life, itself, from conception (sperm-egg interactions) to death (apoptosis, multiple systemic pathologies)

12. How do glycans impact your work?
Depends on who you are:
Protein biochemist: Pesky modifications that impart heterogeneity to my otherwise pure protein.
Structural biochemist: Nasty modifications that make it hard to crystallize some really interesting proteins.
Synthetic chemist: You want to synthesize what? And you want how much?
Geneticist: A family of regulated molecules whose expression is not simply related to the activity of a single gene. Painful pleiotropy.
Molecular Biologist: What’s the big deal--I’ve been doing glycobiology for my whole career? They make a nice backbone structure.

13. How, then, does Glycobiology fit within the context of modern molecular, genetic, structural, and systems biology?

14. The Central Dogma, circa 1970
DNA
RNA
Protein
Rather ignores a role for lipids and carbohydrates, especially at the cell surface
Cell
Organism
After Varki, A

15. An expanded Dogma DNA RNA Protein Cell Organism enzymes Saccharides
and lipids
glycoconjugates
Modified
transcription
factors
After Varki, A

16. Predominant cellular distribution of glycans implies role for carbohydrate in the societal interactions of cells in tissues
Electron micrograph of a human lymphocyte (Ruthenium Red staining)
After Varki, A

17. Carbohydrates - Basic Terms
Monosaccharide
A carbohydrate that can not be broken down into smaller carbohydrates by treatment with acids- a simple sugar
Oligosaccharide
Approximately 4-12 mono units
Polysaccharide
Usually greater then 12 mono units
Often a long linear repeating chain consiting of a single monosaccharide type or a repeating disaccharide with or without small side chains

18. Glycoconjugates Glycoproteins Glycolipids Proteoglycans
Protein + Carbohydrate
Most proteins of biological and therapeutic significance
e.g. TPA, EPO, LH, Immunoglobulins
Glycolipids
Lipid + Carbohydrate
Important tissue and cell type markers
e.g. blood group antigens, tumor associated antigens
Proteoglycans
Complex of a protein and one or more polysaccharides known as glycosaminoglycans (GAGs)
Immense structural diversity, pleiotropic functions

19. Essentials of Glycobiology
The Building Blocks (in Essentials/CFG code)
Essentials of Glycobiology
Second Edition

20. Essentials of Glycobiology
Second Edition

21. Potential for high information content is inherent in glycan structure

22. Essentials of Glycobiology
Built into oligosaccharides or other polymers,
post-synthetic modifications are common, increasing structural diversity
Essentials of Glycobiology
Second Edition

23. Carbohydrate Synthesis - Secondary gene products
Oligosaccharide structures are not encoded in the DNA
Correct synthesis dependent on
Glycosyltransferases
Glycosidases
Availability of monosaccharide and activated forms
Organelle distribution and function
Consequence
Glycosylation is very sensitive to subtle shifts in the enviroment

24. Glycoconjugates- Basic Terms
Microheterogeneity
The variation seen in glycosylation at a given glycosylation site. A site may be unoccupied or may be occupied by different sugars. This fact led early researchers to falsely conclude that carbohydrates were not important.
Glycoforms
A glycoconjugate may have different glycoforms. That is, the non-carbohydrate portion will remain the same, but variances in the carbohydrate portion will create different glycoforms of a glycoconjugate.
Glycone vs. Aglycone
Carbohydrates (glycones) should be considered in the context of what they are linked to (aglycones)

25. Is this the right scale?

26. Is this the right scale?

27. Essentials of Glycobiology
Glycoconjugates of the real world
Thy-1 Glycoprotein
Predicted polypeptide MW = 14 kD
Observed MW by SDS-PAGE = kD
Glycan accounts for almost 50% of total glycoprotein mass.
FIGURE 1.3. Schematic representation of the Thy-1 glycoprotein including the three N-glycans (blue) and a glycosylphosphatidylinositol (GPI-glycan, green) lipid anchor whose acyl chains (yellow) would normally be embedded in the membrane bilayer. Note that the polypeptide (purple) represents only a relatively small portion of the total mass of the protein. (Original art courtesy of Mark Wormald and Raymond Dwek, Oxford Glycobiology Institute.)
Essentials of Glycobiology
Second Edition

28. Thy-1 Microheterogeneity
Dwek, R.A., et al. (1993) Philosophical Transactions: Biological Sciences 342, 43-50

29. The great classes of animal glycans

30. Reasonably well-supported and accepted functions for glycans
Structural support for cells (extracellular matrix, plant cell wall)
Signaling molecules (currently only characterized in plants)
Determinants of protein folding, stability, and half-life
Mediators of cellular interactions with:
Other cells in developing tissues
Specific cell types in mature tissues, especially related to immune response and tissue stability
Viral, bacterial, parasitic pathogens and associated toxins
Modulators of signaling activities that drive cellular differentiation

31. The thrill and the agony of Glycobiology:
Glycobiology Apr;3(2):
Biological roles of oligosaccharides: all of the theories are correct.
Varki A.
Glycobiology Program, UCSD Cancer Center.
Many different theories have been advanced concerning the biological roles of the oligosaccharide units of individual classes of glycoconjugates. Analysis of the evidence indicates that while all of these theories are correct, exceptions to each can also be found. The biological roles of oligosaccharides appear to span the spectrum from those that are trivial, to those that are crucial for the development, growth, function or survival of an organism. Some general principles emerge.
And this is where we begin!

33. Carbohydrate Structure and the Glycosidic Bond
Lance Wells

34. Carbohydrates - Definition and Language
Carbohydrates = “Hydrates” of carbon.
Hexose, e.g.: C6H12O6 = [C(H2O)]6.
Need to develop a system for talking about and/or representing carbohydrates.
Monosaccharides: single sugars; clear language and numerous pictorial forms.
Oligosaccharides (typically 4-10 sugars): need a more complex language, only one of previous pictorial forms remains tractable.
Polysaccharides: systematic language is accurate but cumbersome, new pictorial representation more useful.
Carbonyl containing compounds: Aldehydes RCHO and Ketones RCOR’

35. Glyceraldehyde and “Fischer Projections”
Glyceraldehyde (aldotriose), a 3 carbon aldehyde sugar or “aldotriose,” exists as 2 mirror image isomers.
d-glyceraldehyde l-glyceraldehyde
The origin of D vs. L nomenclature for sugars - does stereocenter farthest from the aldehyde terminus have the configuration of D- or L-glyceraldehyde?
Dihydroxyacetone (ketotriose): No chiral carbon.
All monosaccharides with one exception have at least one chiral carbon with
the total number (k) being equal to the number of internal (CHOH) groups; that is n-2 for Aldoses and n-3 for Ketoses with n=number of carbon atoms in the monosaccharide.
Possible Stereoisomers = 2 raised to the power of k (how many for hexose?)

36. D-”Aldoses” with 4 Carbons
Two possible isomers at each new carbon center.
Mentally insert new carbon center between aldehyde terminus (C1) and what was previously C2.
Note that D configuration is retained.
Two sugars that differ only in the configuration around a single chiral carbon are called EPIMERS

37. D-”Aldoses” with 5 Carbons
Xyl Rib
Which 5 carbon sugars are epimers of D-ribose?

38. D-”Aldoses” with 6 Carbons
** special attention to Gal, Man, and Glc abbreviations
Man
Glc
Gal
6 carbon ketose: fructose fru

39. A Few of the Common non-(CH20)n Building Blocks of Oligosaccharides
Products of esterification, oxidation, reduction, acetylation, etc.
deoxy
lactones
Fuc
N-acetyl
Amino
GlcA
GlcNAc & GalNAc
sialic acids
Sia

40. Common monosaccharides found in vertebrates Essentials of Glycobiology
Chapter 2, Figure 4
Essentials of Glycobiology
Second Edition
FIGURE 2.4. Common monosaccharides found in vertebrates. N-Acetylneuraminic acid is the most common form of sialic acid.

41. Glc, Gal, Man, Fuc, GlcNAc, GalNAc, Neu5Ac(Gc), Xyl, GlcA, IdoA Ones to Remember (in a mammalian central view of the world)!

42. Fischer Structures Does Not Take Into Account that
5 and 6 Carbon Sugars Tend to Cyclize:
RING CONFIRMATION

43. Cyclization Can Produce Multiple Isomers
Fischer Projection is NOT good enough

44. For Hexoses Alpha=anti (anomeric hydroxyl to C6); Beta=beside
From Fischer to Haworth to abbreviated Haworth diagrams.
Looking down the carbon spine from C1 down of the Fischer projection, noting whether hydroxyl groups are to the right (down) or left (up). Now imagine around the periphery of a flat hexagon.
“A new asymetric carbon” is formed so we need more language (great!)
New asymetric carbon is termed the anomeric carbon and a is used to denote the anomer where the absolute stereochemistry of the anomeric position and the most remote sterocenter in the sugar chain are the same, b opposite
For Hexoses Alpha=anti (anomeric hydroxyl to C6); Beta=beside
Where did this pyranose (p) term come from?

45. Pyranose (p) and Furanose (f) both 5 and 6 membered rings are stable so:

46. What about ketoses
Repeating the exercise for a 6 carbon ketose:

47. A Last Bit of Nomenclature for Chair Structures (the most accurate way of drawing the sugars) Haworth suggests that sugars are flat but neither furanose or pyranose rings are actually flat in their lowest energy confirmations
Although D-glucose has a strong preference for one chair conformation, this is not true for all sugars.

48. Mutatrotation In solution anomers can introconvert
% Distribution at 20C
Pyranose Furanose
a b a b
D-Glc
D-Gal
D-Man
D-Fru

50. The Glycosidic Bond
How do we describe molecules containing sugars that are attached to one another? (We’ll limit our discussion to cases where the anomeric center of one sugar is attached to an oxygen atom of another sugar - that is, we’ll discus only “glycosides.”)
There are basically 2 things we need to keep track of: 1) the anomeric configuration of the “glycosidic linkage,” and 2) the identity of the carbon on the next sugar that shares the bridging oxygen.

51. Naming of Oligosaccharides
Here’s a simple example: maltose, or D-Glc-a(1-4)-D-Glc.
How did we come up with that name, how do we know which sugar to name 1st? We always begin naming with the sugar furthest from the “reducing terminus” of the oligosaccharide. The “reducing sugar” is the sugar that still contains a free anomeric carbon. Branches are put in parenthesis. Notice the a or b is now no longer “free” but is fixed in one orientation.
Many “common name” disacharrides (sucrose, lactose, etc.)-see Ess. Glycobiol.
Note: we even keep this nomenclature when the oligosaccharide is attached to an aglycone

52. Example of Naming a Branched Oligosaccharide

54. Monosaccharide symbol set Essentials of Glycobiology
Chapter 1, Figure 5
Monosaccharide symbol set a b c
Essentials of Glycobiology
Second Edition
FIGURE 1.5. Recommended symbols and conventions for drawing glycan structures. (a) The monosaccharide symbol set from the first edition of Essentials of Glycobiology is modified to avoid using the same shape or color, but with different orientation to represent different sugars. Each monosaccharide class (e.g., hexose) now has the same shape, and isomers are differentiated by color. The same shading/color is used for different monosaccharides of the same stereochemical designation (e.g., Gal, GalNAc, and GalA). To minimize variations, sialic acids and uronic acids are in the same shape, and only the major uronic and sialic acid types are represented. When the type of sialic acid is uncertain, the abbreviation Sia can be used instead. Only common monosaccharides in vertebrate systems are assigned specific symbols. All other monosaccharides are represented by an open hexagon or defined in the figure legend. If there is more than one type of undesignated monosaccharide in a figure, a letter designation can be included to differentiate between them. Unless otherwise indicated, all of these vertebrate monosaccharides are assumed to be in the d configuration (except for fucose and iduronic acid, which are in the l configuration), all glycosidically linked monosaccharides are assumed to be in the pyranose form, and all glycosidic linkages are assumed to originate from the 1-position (except for the sialic acids, which are linked from the 2-position). Anomeric notation and destination linkages can be indicated without spacing/dashes. Although color is useful, these representations will survive black-and-white printing. Modifications of monosaccharides are indicated by lowercase letters, with numbers indicating linkage positions, if known (e.g., 9Ac for the 9-O-acetyl group, 3S for the 3-O-sulfate group, 6P for a 6-O-phosphate group, 8Me for the 8-O-methyl group, 9Acy for the 9-O- acyl group, and 9Lt for the 9-O-lactyl group). Esters and ethers are shown attached to the symbol with a number. For N-substituted groups, it is assumed that only one amino group is on the monosaccharide with an already known position (e.g., NS for an N-sulfate group on glucosamine, assumed to be at the 2-position). (b) Typical branched “biantennary” N-glycan with two types of outer termini, depicted at different levels of structural details. (c) Some typical glycosaminoglycan (GAG) chains.

56. xx

57. title