2. Review of Projections Fischer Haworth Stereochemical
What monosaccharide is this?
Which chiral center defines the stereochemistry?
How are axial and equitorial positions related to up/down in Haworth projections?

3. Where is the aglycone?
Where is the “reducing” end?
How many b-glycosidic linkages?
How many a-glycosidic linkages?
How many 3-substituted residues?
How many 6-substituted residues?

4. How many non-reducing termini?
How many glycosidic bonds?
Other than the NANA (N-acetylneuraminic acid, a type of sialic acid) residues, how many are a-glycosides?

5. GlycoEconomics lesson #1.
From 2007 Matreya Lipids Catalog:
About 20 years ago, bovine brain GT1b retailed for $300/mg (Matreya Lipids). A graduate student purified 75 mg during a single 3-4 month lab rotation (street value = $22,500).
Bovine Brain GQ1b retailed for $500/10 mg at that time. Same purification yielded approximately 10 mg pure GQ1b (street value = $500,000).
Graduate student stipend was approximately $12,000/year (or $4,000/rotation). Student value multiplier = 130-fold. Tell your PI how valuable you can be!!!

6. 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

7. Large O-linked Glycosaminoglycans and poly-lactosamine structures
Glycoprotein N-linked and O-linked oligosaccharides
Glycolipid oligosaccharides

8. The "Primary" Sialic Acids
C C
H-C-OH
C O HO H
C C OH 1 2 4 3 6 5
C-O 9 8 7 O -
H-C-OH
C O
H-C-C-N H
C C
C C OH 1 2 4 3 6 5
C-O 9 8 7 O
H O H - a
-D-KDN a
-D-Neu5Ac
N-acetyl-neuraminic acid
(2-keto-5-acetamido-3,5-dideoxy-
D-glycero-D-galacto-nonulosonic acid)
“Neu5Ac” “NANA”, “NeuAc”
KDN
(2-keto-3-deoxy-D-glycero-
D-galacto-nonulosonic acid)
Thought to be the metabolic
precursors of all other sialic acids
After Varki, A

9. Neu5Ac/bacteria
GlcNAc-6-phosphate
ManNAc
Neu5Ac
CMP-Neu5Ac
ManNAc-6 -phosphate Pi
PEP
CTP
PPi
Phosphatase
2-epimerase
synthetase
Neu5Ac/Vertebrates
UDP-GlcNAc
Neu5Ac-9-phosphate
ATP
ADP UD P
ManNAc kinase
Neu5Ac-9-
phosphate
phosphatase
Phylogenetic relationships of enzymes involved in the metabolism of sialic acids
HOMOLOGY
Angata and Varki
Chemical Reviews 102,
, 2002.
After Varki, A

10. After Varki, A * * THE SIALIC ACIDS Hydrogen or: -linkage to:
Acetyl R8 R9 R7 R4
-at physio-
logical pH,
ionized or
lactonized 3 4 5 6 7 8 9 R1 R1 1 2 * R5
N-acetyl R2
Phosphate R9
Hydrogen or:
-linkage to:
-Gal ( ) or
-GalNAc (-6) or
-GlcNAc(-4 -6) or
-Sialic Acid (-8 -9) or
-linkage to CMP
Absent in:
- 2,3dehydro or
- 2,7anhydro
(double-bond
when R2 absent) R2
CARBON OXYGEN HYDROGEN
Methyl R8 R5
N-glycolyl
Hydroxyl
Amino
Sulfate R8
Lactyl R9 *
After Varki, A

11. Two Major Kinds of Sialic Acids in Mammalian Cells
N-ACETYLNEURAMINIC ACID
(Neu5Ac) 9 7 8 6 1 2 5 3 4
N-GLYCOLYLNEURAMINIC ACID
(Neu5Gc) 8 7 1 2 3 5 6 9
LINKAGE TO
UNDERLYING
SUGAR CHAIN
LINKAGE TO
UNDERLYING
SUGAR CHAIN
After Varki, A

13. Synthesis and Structure of Glycolipids
1/16/07

14. Large O-linked Glycosaminoglycans and poly-lactosamine structures
Glycoprotein N-linked and O-linked oligosaccharides
Glycolipid oligosaccharides

15. Glycan synthesis
in a cellular context

16. Overview
From ER
through
Trans-Golgi and
points inbetween

17. On and into the ER
Glycosphingolipids (GSL)
Dolichol-P-X
Glycosyl phosphatidylinositol (GPI)

18. Topological model for the enzymatic reactions leading to Dol-P biosynthesis de novo on the cytoplasmic face of the ER
Schenk, B. et al. Glycobiology :61R-70R; doi: /glycob/ R

19. Biosynthesis of N-Glycans: Production of GlcNAc-P-P-Dolichol
Man
Gal
Sia
Fuc
Glc
Biosynthesis of N-Glycans: Production of GlcNAc-P-P-Dolichol
Tunicamycin
Blocks - not
very specific!
Dolichol
Adapted from Marquardt T, Denecke J. Eur J Pediatr Jun;162(6):359-79

20. Topological model for lipid intermediate synthesis, translocation and the role for Dol-P-P/Dol-P phosphatases in the recycling of Dol-P-P/Dol-P in the ER
Schenk, B. et al. Glycobiology :61R-70R; doi: /glycob/ R

21. ER glycolipid synthesis
Dolichol-P-X
Glycosyl phosphatidylinositol (GPI)

22. Basic Glycosylphosphatidylinositol (GPI) Anchor
Phospholipid
After Hart, G

23. Examples of GPI-Anchored Proteins
Cell surface hydrolases
alkaline phosphatase
acetylcholinesterase
5’ nucleotidase
Protozoal antigens
trypanosome VSG
leishmanial protease
plasmodium antigens
Adhesion molecules
neural cell adhesion molecule
heparan sulfate proteoglycan
Mammalian antigens
Thy-1
carcinoembryonic antigen
Others
decay accelerating factor
scrapie prion protein
folate receptor
After Hart, G

24. Structure of the Basic GPI Anchor
After Hart, G

25. Structural Analysis of the GPI Anchor Enzymatic and chemical cleavage sites are useful in identifying GPI anchored membrane proteins
After Hart, G

26. After Freeze, H

27. Biosynthesis of GPI anchors
The first step in biosynthesis of the GPI anchor requires at least four genes
One of them, PIG-A is an X-linked gene
Mutation in PNH
MUTATIONS IN
DOL-P-MAN SYN
AND USE
After Freeze, H

28. Paroxysmal Nocturnal Hemoglobinuria
A hematopoietic stem cell disorder characterized by intravascular hemolytic anemia. Abnormal blood cells lack GPI-anchored proteins due to a mutation in the PIG-A gene.
Lack of GPI-anchored complement regulatory proteins, such as decay-accelerating factor (DAF) and CD59, results in complement-mediated hemolysis and hemoglobinuria.

29. Examples of C-Terminal Sequences Signaling the Addition of GPI-Anchors
5-10 hydrophilic, hydrophobic
Bold AA is site of GPI attachment Sequence to right is cleaved by the transpeptidase upon Anchor addition
After Hart, G

30. ER glycolipid synthesis
Glycosphingolipids (GSL)
Dolichol-P-X
Glycosyl phosphatidylinositol (GPI)

31. Minimal Defining Structure of a Glycosphingolipid
Glycan-O-Ceramide *
Ceramide (Cer)
Sphingosine
Fatty Acyl group
Glycan
*Glucose (All animals)
Galactose (?Vertebrates only)
Inositol-P (fungi)
After Varki, A

32. Biosynthesis of Ceramide and Glucosylceramide

34. Golgi processing of Glycosphingolipids

35. cis
medial
trans

36. Major Classes of Glycosphingolipids
Series Designation Core Structure
Lacto (LcOSe4) Gal3GlcNAc3Gal4Glc1Ceramide
Lactoneo (LcnOSe4) Gal4GlcNAc3Gal4Glc1Ceramide
Globo (GbOSe4) GalNAc3Gala4Gal4Glc1Ceramide
Isoglobo (GbiOSe4) GalNAc3Gal3Gal4Glc1Ceramide
Ganglio (GgOSe4) Gal3GalNAc4Gal4Glc1Ceramide
Muco (MucOSe4) GalGal3Gal4Glc1Ceramide
Gala (GalOSe2) Gal4Gal1Ceramide
Sulfatides Sulfo-Gal1Ceramide
Different Core structures generate unique shapes and are expressed in a cell-type specific manner
After Varki, A

37. Nomenclature Issues Glycosphingolipid (GSL) = Glycan + Sphingolipid
(named after the Egyptian Sphinx)
Glycosphingolipids often just referred to as “Glycolipids”.
“Ganglioside": a GSL one or more sialic acid residues
Example of nomenclature:
Neu5Ac3Gal3GalNAc4Gal4Glc1Cer
= GM1b in the Svennerholm nomenclature OR
II4Neu5Ac-GgOSe4-Cer
in the official IUPAC-IUB designation
After Varki, A

38. Pathways for Ganglio-series Glycosphingolipid biosynthesis
Gm1b
After Varki, A

39. Turnover and Degradation of Glycosphingolipids
Internalized from plasma membrane via endocytosis
Pass through endosomes (some remodelling possible?)
Terminal degradation in lysosomes - stepwise reactions by specific enzymes.
Some final steps involve cleavages close to the cell membrane, and require facilitation by specific sphingolipid activator proteins (SAPs).
Individual components, available for re-utilization in various pathways.
At least some of glucosylceramide may remain intact and be recycled
Human diseases in which specific enzymes or SAPs are genetically deficient (storage diseases)

40. Biological Roles of Glycosphingolipids
Thought to be critical components of the epidermal (skin) permeability barrier
Organizing role in cell membrane. Thought to associate with GPI anchors in the trans-Golgi, forming “rafts” which target to apical domains of polarized epithelial cells
May also be in glycosphingolipid enriched domains (“GEMs”) which are associated with cytosolic oncogenes and signalling molecules
Physical protection against hostile environnments
Binding sites for the adhesion of symbiont bacteria.
Highly specific receptor targets for a variety of bacteria, toxins and viruses.

41. Biological Roles of Glycosphingolipids
Specific association of certain glycosphingolipids with certain membrane receptors.
Can mediate low-affinity but high specificity carbohydrate-carbohydrate interactions between different cell types.
Targets for autoimmune antibodies in Guillian-Barre and Miller-Fisher syndromes following Campylobacter infections and in some patients with human myeloma
Shed in large amounts by certain cancers - these are found to have a strong immunosuppressive effects, via as yet unknown mechanisms

42. Metabolic relationships in the biosynthesis and turnover of Sphingolipids
After Varki, A

43. Consequences of Glycosylceramide Synthase gene disruption
Embryonic Lethal. Embryogenesis proceeded into gastrulation with differentiation into primitive germ layers and embryo patterning but abruptly halted by a major apoptotic process. Deficient embryonic stem cells able to form endodermal, mesodermal, and ectodermal derivatives but were strikingly deficient in ability to form well differentiated tissues. However, hematopoietic and neuronal differentiation could be induced.
After Varki, A

44. Consequences of Lactosylceramide Synthase gene disruption
Result Pending
After Varki, A

45. Consequences of GalNAc Transferase I gene disruption
Male Sterility. Late Onset Peripheral Nerve De-myelination possibly related to loss of ligands for Myelin Associated Glycoprotein (Siglec-4).
Reduction in neural conduction velocity in some nerves.
Compensatory increase in GM3 and GD3 in the brain
After Varki, A

46. Consequences of SialylTransferase II (GD3 synthase) gene disruption
Viable, fertile, normal life span, under further
investigation
After Varki, A

47. Consequences of SialylTransferase I (GM3 synthase) gene disruption
Enhanced sensitivity to insulin. Enhanced insulin receptor phosphorylation in skeletal muscle. Protection from high-fat diet-induced insulin resistance.
Is GM3 ganglioside a negative regulator of insulin signaling?
After Varki, A

48. Double KO : Mice Expressing only GM3 in the Brain
Sudden death phenotype
Extremely susceptible to induction of lethal seizures by loud sounds
Further characterization in progress
After Varki, A

49. Sheikh, KA, et al. (1999) PNAS 96, 7532

50. General Principles
The sialic acids are a family of structurally related compounds that can bear a variety of post-synthetic modifications
Glycan synthesis is compartmentalized within cells
Precursors begin as lipid-linked species on the cytoplasmic face of the ER, requiring that substrates be flipped for further processsing
Donor substrates contribute to more than one class of glycoconjugate
The assembly-line model for glycan extension in the Golgi apparatus may not be as applicable to glycolipid synthesis as it is to glycoprotein glycosylation
Some precursors and intermediates in glycolipid synthesis are also utilized in signaling pathways

51. Overview

53. General Principles Glycan synthesis is compartmentalized within cells
Precursors begin as lipid-linked species on the cytoplasmic face of the ER, requiring that substrates be flipped for further processsing
Addition of N-linked glycans generally occurs co-translationally
ER glycosylation may have evolved as a method for quality control, Golgi modifications are layered on top of this well-conserved function
Within the Golgi and distal secretory pathway, specific glycan structures can impart targeting information
The assembly-line model holds well for N-linked proteins but may not be as applicable to glycolipid and GAG synthesis

55. Ganglioside synthetic enzyme KOs
GlcCer synthase (GlcT): Early embryonic lethal, differentiation and germ layer formation okay
LacCer synthase (GalT): ??
GM3 synthase (SiaTI): Increased sensitivity to insulin (skeletal muscle receptor phosphorylation)
GM2 synthase (GalNAcT): Male sterility, demyelinating disorder
GD2 synthase (SiaTII): Ok
GM2 synthase/GD2 synthase double KO: sudden death, auditory seizures, demyelination

56. xx