Review of Projections

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. H 9 H-C-OH H 9 8 H-C-OH H-C-OH 8 7 H-C-OH H-C-OH O 7 6 - H-C-OH 1 C O C-O O HO H O H - 6 1 2 C O C C 5 C-O H-C-C-N H H 4 3 2 5 H C C OH C C H H H 4 3 H OH H OH C C OH H -D-Neu5Ac a -D-KDN a The "Primary" Sialic Acids 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 Neu5Ac/Vertebrates GlcNAc-6-phosphate UDP-GlcNAc GlcNAc-6-phosphate 2-epimerase UDP-GlcNAc 2-epimerase UD P ManNAc-6 -phosphate ManNAc ATP Phosphatase ManNAc kinase ADP Pi ManNAc-6 -phosphate ManNAc PEP PEP Neu5Ac-9- phosphate synthetase Neu5Ac synthetase Pi Pi Neu5Ac-9-phosphate Neu5Ac-9-phosphate phosphatase Pi Neu5Ac Neu5Ac CTP CTP CMP-Neu5Ac synthetase CMP-Neu5Ac synthetase PPi PPi CMP-Neu5Ac CMP-Neu5Ac Phylogenetic relationships of enzymes involved in the metabolism of sialic acids HOMOLOGY Angata and Varki Chemical Reviews 102, 439-469, 2002. After Varki, A

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

11. 4 9 7 8 6 1 N-GLYCOLYLNEURAMINIC ACID (Neu5Gc) 2 5 3 4 LINKAGE TO UNDERLYING SUGAR CHAIN Two Major Kinds of Sialic Acidsin Mammalian Cells N-ACETYLNEURAMINIC ACID (Neu5Ac) 9 7 8 6 1 2 5 3 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. Dolichol-P-X Glycosyl phosphatidylinositol (GPI) Glycosphingolipids (GSL) On and into the ER

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 2001 11:61R-70R; doi:10.1093/glycob/11.5.61R

19. GlcNAc Man Glc Gal Sia Fuc Dolichol Biosynthesis of N-Glycans:Production of GlcNAc-P-P-Dolichol Tunicamycin Blocks - not very specific! Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 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 2001 11:61R-70R; doi:10.1093/glycob/11.5.61R

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

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

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

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

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

26. After Freeze, H

27. Mutation in PNH MUTATIONS IN DOL-P-MAN SYN AND USE • The first step in biosynthesis of the GPI anchor requires at least four genes • One of them, PIG-A is an X-linked gene Biosynthesis of GPI anchors 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, 15-20 hydrophobic Bold AA is site of GPI attachment Sequence to right is cleaved by the transpeptidase upon Anchor addition After Hart, G

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

31. Glycan Sphingosine Ceramide (Cer) *Glucose (All animals) Galactose (?Vertebrates only) Inositol-P (fungi) Fatty Acyl group Minimal Defining Structure of a Glycosphingolipid * Glycan-O-Ceramide 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 3-0-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