1. Essentials of Glycobiology Lecture 7 April 12, 2002 Ajit Varki Structure, biosynthesis and general biology of Glycosphingolipids

2. Major Glycan Classes in Animal Cells O Ser O Ser/Thr N Asn Ser-O- OUTSIDE INSIDE N Asn S SS -O-Ser S S S SS Etn P INOSITOL P NH Ac P NS Ac S 2 P Glycoprotein ProteoglycanGLYCOPHOSPHO-LIPIDANCHOR N-LINKED CHAINS O-LINKEDCHAIN HYALURONAN GLYCOSAMINO-GLYCANS HEPARAN SULFATE CHONDROITIN SULFATE SULFATE Sialic Acids GLYCOSPHINGOLIPID O-LINKED GlcNAc

3. Lecture Overview Historical Background Defining Structures and Major Classes Other Nomenclature Issues Biosynthesis Occurrence & Structural Variations Isolation and purification Trafficking, Turnover and Degradation

4. Lecture Overview (Continued) Relationship to biosynthesis, turnover and signalling functions of other Sphingolipids Antibodies against Glycosphingolipids Biological Roles Genetic Disorders in GSL biosynthesis Perspectives and Future Directions

5. Minimal Defining Structure of a Glycosphingolipid Glycan-O-Ceramide Ceramide (Cer) Sphingosine Fatty Acyl group Glycan * * Glucose (All animals) Galactose (?Vertebrates only) Mannose (Invertebrates) Inositol-P (fungi) Find the Missing Double Bond!

6. Biosynthesis of Ceramide and Glucosylceramide Find the Missing Double Bond!

7. 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 = GM1a in the Svennerholm nomenclature OR II 4 Neu5Ac-GgOSe 4 -Cer in the official IUPAC-IUB designation

8. Isolation and purification of Glycosphingolipids Most glycosphingolipids obtained in good yield from cells and tissues by sequential organic extractions of increasing polarity Some separation by polarity achieved by two-phase extractions Subsequent fractionation away from other lipids in the extract using: DEAE ion exchange chromatography silica gel thin layer and column separations HPLC adaptations of these methods are useful in obtaining complete separations. Principles of structural characterization of these molecules presented elsewhere

9. Biosynthesis of different classes of glycans within the ER-Golgi Pathway

10. Stepwise elongation of Glucosylceramide generates unique Core structures

11. Major Classes of Glycosphingolipids SeriesDesignationCore Structure Gal  4Glc  1Ceramide Lacto(LcOSe4) Gal  3GlcNAc  3Gal  4Glc  1Ceramide Gal  4Glc  1Ceramide Lactoneo(LcnOSe4)Gal  4GlcNAc  3Gal  4Glc  1Ceramide Gal  4Glc  1Ceramide Globo(GbOSe4) GalNAc  3Gal  4Gal  4Glc  1Ceramide Gal  4Glc  1Ceramide Isoglobo(GbiOSe4)GalNAc  3Gal  3Gal  4Glc  1Ceramide Gal  4Glc  1Ceramide Ganglio(GgOSe4)Gal  3GalNAc  4Gal  4Glc  1Ceramide Gal  4Glc  1Ceramide Muco (MucOSe4) Gal  Gal  3Gal  4Glc  1Ceramide Gal  1Ceramide Gala (GalOSe2) Gal  4Gal  1Ceramide Gal  1Ceramide Sulfatides 3-0-Sulfo-Gal  1Ceramide Different Core structures generate unique shapes and are expressed in a cell-type specific manner

12. Examples of outer chains and modifications to Glycosphingolipid Cores Much similarity to outer chains of N- and O-glycans

13. Pathways for ganglio-series Glycosphingolipid biosynthesis Gm1b

14. Metabolic relationships in the biosynthesis and turnover of Sphingolipids

15. 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.

16. Monoclonal antibodies (Mabs) against Glycosphingolipids Many “tumor-specific” MAbs directed against glycans Majority react best with glycosphingolipids. Most MAbs are actually detecting “onco-fetal” antigens Some used for diagnostic and prognostic applications in human diseases Few being exploited for attempts at monoclonal antibody therapy of tumors. Many used to demonstrate cell type-specific regulation of specific GSL structures in a temporal and spatial manner during development. Precise meaning of findings for cancer biology and development being explored..

17. Biological Roles of Glycosphingolipids

18. 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 distinct 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.

19. 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

20. Examples of interactions between glycosphingolipids and bacterial toxins or receptors

21. Examples of proposed interactions between glycosphingolipids and mammalian receptors

22. Natural and induced Genetic Disorders in Glycosphingolipid biosynthesis Large number of known genetic defects in lysosomal enzymes or SAP proteins result in “storage disorders” characterized by the accumulation of specific intermediates. Very few naturally-defined genetic defects in the biosynthesis of glycosphingolipids. A cultured cell line is completely deficient in the glucosylceramide synthase - thus, glycosphingolipids are not essential for growth of single cells in a culture dish Targetted gene disruption of Ceramide Galactosyltransferase loss of Gal Cer and sulfatides in nervous system myelin. Mice form myelin with GlcCer, which replaces GalCer. Despite myelin of relatively normal appearance mice have generalized tremors and mild ataxia, and electrophysiological evidence for conduction deficits. With increasing age, progressive hindlimb paralysis and severe vacuolation of the ventral region of the spinal cord. Transgenic overexpression of the GalNAc transferase I (GM2/GD2 synthase) gives higher expression of complex gangliosides. No gross morphological changes observed, but much stronger inflammatory reactions involving neutrophils Targetted gene disruption of the GalNAc transferase I (GM2/GD2 synthase) no major histological defects in nervous system nor in gross behavior, only a reduction in neural conduction velocity in some nerves. Compensatory increase in GM3 and GD3 in the brain seems sufficient to compensate for lack of complex gangliosides.

23. Natural and induced Genetic Disorders in Glycosphingolipid biosynthesis Targetted gene disruption of Ceramide Galactosyltransferase loss of Gal Cer and sulfatides in nervous system myelin. Mice form myelin with GlcCer, which replaces GalCer. Despite myelin of relatively normal appearance mice have generalized tremors and mild ataxia, and electrophysiological evidence for conduction deficits. With increasing age, progressive hindlimb paralysis and severe vacuolation of the ventral region of the spinal cord. Transgenic overexpression of the GalNAc transferase I (GM2/GD2 synthase) gives higher expression of complex gangliosides. No gross morphological changes observed, but much stronger inflammatory reactions involving neutrophils

24. Consequences of GalNAc Transferase I gene disruption

25. Natural and induced Genetic Disorders in Glycosphingolipid biosynthesis Targetted gene disruption of the GalNAc transferase I (GM2/GD2 synthase) no major histological defects in nervous system nor in gross behavior, only a reduction in neural conduction velocity in some nerves. Compensatory increase in GM3 and GD3 in the brain seems sufficient to compensate for lack of complex gangliosides.

26. Consequences of SialylTransferase II (GD3 synthase) gene disruption

27. Consequences of SialylTransferase I (GM3 synthase) gene disruption

28. Consequences of Lactosylceramide Synthase gene disruption

29. Consequences of Glycosylceramide Synthase gene disruption