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Essentials of Glycobiology Lecture 7 April 8, 2004 Ajit Varki Glycosphingolipids (Glycolipids) Major Glycan Classes in Animal Cells CHONDROITIN SULFATE HYALURONAN P GLYCOSAMINOGLYCANS HEPARAN SULFATE S S S S S NS NS -O-Ser Proteoglycan N-LINKED CHAINS Ac O-LINKED CHAIN GLYCOPHOSPHOLIPID ANCHOR P S O Ser/Thr GLYCOSPHINGOLIPID N Asn N Asn INOSITOL OUTSIDE Sialic Acids O-LINKED GlcNAc INSIDE O Ser Etn P NH 2 Glycoprotein Ac S S Ser-O- P S Lecture Outline • • • • • • • • Historical Background Defining Structures and Major Classes Other Nomenclature Issues Biosynthesis Occurrence & Structural Variations Isolation and purification Trafficking, Turnover and Degradation Relationship to biosynthesis, turnover and signalling functions of other Sphingolipids • Antibodies against Glycosphingolipids • Biological Roles • Genetic Disorders in GSL biosynthesis Minimal Defining Structure of a Glycosphingolipid Glycan-O-Ceramide * Glycan Hexose 1-1'O-CH 2 -CH-CH=CH-(CH 2 ) 12 -CH 3 Sphingosine NH OH *Glucose (All animals) O=CH-(CH 2 ) x -CH 3 Fatty Acyl group Galactose (?Vertebrates only) Mannose (Invertebrates) Inositol-P (fungi) Find the Mislocated Double Bond! Ceramide (Cer) Serine + Palmitoyl-CoA Pyridoxal phosphate NADPH Serine palmitoyltransferase 3-Dehydrosphinganine reductase D-erythro -Sphinganine HO-CH 2 -CH-CH-CH-(CH 2 ) 12 -CH 3 NH 2 OH Fatty Acyl-CoA Sphinganine N-acyltransferase Dihydroceramide desaturase Ceramide (Cer) HO-CH 2 -CH-CH=CH-(CH NH OH O=CH-(CH 2 ) 12 -CH 3 Sphingosine 2 ) x -CH 3 Fatty Acyl group UDP-Glc Ceramide Glucosyltransferase Glucosyl-Ceramide Glucose 1- 1'O-CH 2 -CH-CH=CH-(CH NH OH O=CH-(CH 2 ) x -CH 3 2 ) 12 -CH 3 Biosynthesis of Ceramide and Glucosylceramide 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: Neu5Ac3Gal3GalNAc4Gal4Glc1Cer = GM1a in the Svennerholm nomenclature OR II4Neu5Ac-GgOSe4-Cer in the official IUPAC-IUB designation 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 to be presented elsewhere Biosynthesis of different classes of glycans within the ER-Golgi Pathway ROUGH ER N-GlcNAc LINKED O-GalNAc LINKED GPILINKED O-Xyl LINKED Glc-Cer LINKED G ? G G *G GOLGI APPARATUS * ** ** ** **** SECRETORY GRANULE * * *** * ** ** ** G * ** ENDOSOME *G ** G * * ** * G * LYSOSOME Stepwise elongation of Glucosylceramide generates unique Core structures Ganglio(a) * 4 e * 4 8 Ganglio(b) 3 n OUTER CHAINS MODIFICATIONS * e 4 d Neo-lacto = ceramide ß 4 3 f * 3 b Lacto * 3 4 alternate Globo m k Muco GalNac can have UDP- 3 * Residues that j 3 Gal * g i GlcNAc h * c * * l 3 Isoglobo outer chains Major Classes of Glycosphingolipids Series Designation Lacto (LcOSe4) Gal3GlcNAc3Gal4Glc1Ceramide Lactoneo (LcnOSe4) Gal4GlcNAc3Gal4Glc1Ceramide Globo (GbOSe4) GalNAc3Gal4Gal4Glc1Ceramide Isoglobo (GbiOSe4) GalNAc3Gal3Gal4Glc1Ceramide Ganglio (GgOSe4) Gal3GalNAc4Gal4Glc1Ceramide Muco (MucOSe4) Gala (GalOSe2) Sulfatides Core Structure GalGal3Gal4Glc1Ceramide Gal4Gal1Ceramide 3-0-Sulfo-Gal1Ceramide Different Core structures generate unique shapes and are expressed in a cell-type specific manner Examples of outer chains and modifications to Glycosphingolipid Cores * GlcNAc * Gal * GalNac residues that can have alte rnate outer chains Much similarity to outer chains of N- and O-glycans Pathways for Ganglio-series Glycosphingolipid biosynthesis SialylTransferase I 4 SialylTransferase II GM3 GA2 4 GD3 8 3 OAc-G D3 GM2 4 GD2 4 SialylTransferase III GT3 8 OAc-G T3 GT2 4 OAc-G D3 GA1 3 GM1a 3 3 OAc-G Q1b(?) GGm1b A1 3 GD1a 3 8 O series GT1b OAc-G T1b 3 8 A series OAc-G Q1b 8 B series Gal Transferase II SialylTransferase IV GQ1c SialylTransferase V 3 GQ1b GT1a GD1c GT1c GD1b 3 GalNAc Transferase GP1c 8 C series Metabolic relationships in the biosynthesis and turnover of Sphingolipids de novo synthesis Gal-Ceramide Fumonisin B1 Glc-Ceramide Ceramide Ceramide-1-phosphate PDMP NB-DJG Sulfatides PC Fumonisin B1 Sphingomyelinase DAG Sphingomyelin Glycosphingolipids D-MAPP Di-methyl-sphingosine Sphingosine Lysogalactosphingolipids Plasmalolysogalactosphingolipids Tri-methyl-sphingosine Sphingosine-1-phosphate • • • • • • • 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 disorders” 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.. Biological Roles of Glycosphingolipids Structural/Physical Endogenous Recognition (Self) ENDOGENOUS LECTIN Exogenous Recognition (Non-self) EXOGENOUS LECTIN SELF * SELF =OLIGOSACCHARIDE (*Pathogen, Toxin or Symbiont) 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. Biological Roles of Glycosphingolipids • Specific association of certain glycosphingolipids with certain membrane receptors. • Can mediate low-affinity but high specificity carbohydratecarbohydrate 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 Binding Proteins Ganglioside(s) Bacterial Toxins V. Cholerae Toxin GM1 E.Coli heat labile toxin C.Tetani Toxin (Tetanus) C. Botulinum GD1b /G T1b A,E GD1b /G T1b /G D1a toxins C. Botulinum B,C,F GT1b /G Q1b toxins Clostridium Toxin GM2 V.Parahemolyticus GT1b toxin Staphylococcus II 3 Neu5AcnLc 4 Toxin Bacterial Adhesins E.Coli S-adhesin N.Gonorrhoeae GM3 II 3 Neu5AcnLc adhesin Helicobacter pylori GM3 , sulfatides adhesins Various Gut anerobes -Gal 1-4Gal- 4 Examples of interactions between glycosphingolipids and bacterial toxins or receptors B19 Parvovirus and Uropathogenic E.coli Binding CHONDROITIN HYALURONAN SULFATE P GLYCOSAMINOGLYCANS HEPARAN SULFATE S S S S S NS The P Blood Group System NS -O-Ser Proteoglycan N-LINKED CHAINS Ac O-LINKED CHAIN GLYCOPHOSPHOLIPID ANCHOR P S O Ser/Thr N Asn N Asn INOSITOL OUTSIDE Sialic Acids INSIDE O Ser Etn P NH 2 Glycoprotein GLYCOSPHINGOLIPID O-LINKED GlcNAc S S Ser-O- P S Examples of proposed interactions between glycosphingolipids and mammalian receptors Binding Proteins Epidermal growth Ganglioside(s) GM3 factor (EGF) Receptor Propos ed function(s) Diminishes tyrosine phosphophorylation upon receptor activation, possibly by inhibiting dimerization De-NAc-G M3 Activates tyrosine phosphophorylation upon receptor activation - mechanism unknown Insulin receptor SPG Diminishes tyrosine phosphophorylation upon receptor activation, mechanism unknown Platelet-derived GM1 Diminishes tyrosine phosphophorylation growth factor (PDGF) upon receptor activation, decreased cell receptor growth Vitronectin Receptor GD3 , GD2 Alters adhesive action of the receptor, mechanism unknown TSH Receptor GD1a lactone Physical association, significance unknown NGF Receptor (?) GQ1b Induces/facilitates neurite outgrowth in Other neuronal certain neuronal cells. Mechanism kinase? unknown Natural and induced Genetic Disorders in Glycosphingolipid biosynthesis • One cultured cell line completely deficient in GlcCer synthase - thus, GSLs not essential for growth of single cells in a culture dish • Many human genetic defects in GSL degradation result in “storage disorders” with accumulation of specific intermediates. In contrast, human genetic defects in the biosynthesis of GSLs seem very rare. • Targetted gene disruption of Ceramide Galactosyltransferase in mice: loss of GalCer and SulfatedGalCer (Sulfatide) in myelin. Mice form myelin with GlcCer, which replaces GalCer. Generalized tremors and mild ataxia, electrophysiological evidence for conduction deficits. With increasing age, progressive hindlimb paralysis and severe vacuolation of the ventral region of the spinal cord. Terminal differentiation and morphological maturation of oligodendrocytes is enhanced. • Cerebroside sulfotransferase-null mice lack Sulfatides but express GalC. two- to threefold enhancement in number of terminally differentiated oligodendrocytes both in culture and in vivo Consequences of GalNAc Transferase I gene disruption SialylTransferase I 4 GM3 3 GA2 4 GM2 SialylTransferase II GD3 8 OAc-G D3 4 GD2 SialylTransferase III GT3 8 OAc-G T3 4 GT2 4 OAc-G D3 GA1 3 3 GM1a 8 O series A series 3 OAc-G Q1b 8 B series Gal Transferase II SialylTransferase IV GQ1c SialylTransferase V 3 GQ1b GT1a 8 GT1c GD1b Male Sterility. Late Onset Peripheral Nerve De-myelination possibly related to loss of 3 3 ligands for Myelin OAc-G Q1b(?) Associated Glycoprotein (Siglec-4). GA1 GD1a GT1b Reduction in neural conduction velocity in some nerves. 3 Compensatory increase GM3 and GD3 OAc-G T1b in3 in the brain GD1c GalNAc Transferase GP1c 8 C series Consequences of SialylTransferase II (GD3 synthase) gene disruption SialylTransferase I 4 SialylTransferase II GM3 GA2 4 GD3 8 3 OAc-G D3 GM2 4 GD2 4 SialylTransferase III GT3 8 OAc-G T3 GT2 4 OAc-G D3 GA1 3 GM1a OAc-G GA1 3 GD1a 3 8 O series Viable, fertile, normal life 3span, under further3 Q1b(?) investigation GT1b OAc-G T1b 3 8 A series OAc-G Q1b 8 B series Gal Transferase II SialylTransferase IV GQ1c SialylTransferase V 3 GQ1b GT1a GD1c GT1c GD1b 3 GalNAc Transferase GP1c 8 C series Consequences of SialylTransferase I (GM3 synthase) gene disruption SialylTransferase I 4 GM3 3 GA2 4 GM2 4 GA1 3 3 GA1 3 OAc-G D3 GD2 SialylTransferase III GT3 8 OAc-G T3 GT2 OAc-G T1b 8 3 A series 8 B series GT1c GQ1c SialylTransferase V GP1c 8 C series Gal Transferase II SialylTransferase IV 3 GQ1b OAc-G Q1b GalNAc Transferase 4 Enhanced sensitivity to insulin. OAc-G D3 GM1a Enhanced insulin GD1breceptor phosphorylation in skeletal muscle. Protection from high-fat diet3 3 OAc-G Q1b(?) insulin resistance. induced GD1aIs GM3 ganglioside GT1ba negative regulator of insulin signaling? GT1a GD1c O series GD3 8 4 3 8 SialylTransferase II Double KO : Mice Expressing only GM3 in the Brain SialylTransferase I 4 GM3 3 GA2 4 GM2 SialylTransferase II GD3 8 OAc-G D3 4 GD2 SialylTransferase III GT3 8 OAc-G T3 4 GT2 4 OAc-G D3 GA1 3 GM1a 3 8 O series OAc-G T1b 3 8 A series 3 OAc-G Q1b 8 B series Gal Transferase II SialylTransferase IV GQ1c SialylTransferase V 3 GQ1b GT1a GD1c GT1c GD1b Sudden death phenotype 3 3to induction of Extremely susceptible OAc-G Q1b(?) lethal seizures by loudGsounds GA1 GD1a T1b Further characterization in progress 3 GalNAc Transferase GP1c 8 C series Consequences of Lactosylceramide Synthase gene disruption SialylTransferase I 4 SialylTransferase II GM3 GA2 4 GD3 8 3 OAc-G D3 GM2 4 GD2 4 SialylTransferase III GT3 8 OAc-G T3 GT2 4 OAc-G D3 GM1a Result Pending GD1b GA1 3 3 3 3 GD1a 3 8 O series GT1b OAc-G T1b 3 8 A series OAc-G Q1b 8 B series Gal Transferase II SialylTransferase IV GQ1c SialylTransferase V 3 GQ1b GT1a GD1c GT1c 3 OAc-G Q1b(?) GA1 GalNAc Transferase GP1c 8 C series Consequences of Glycosylceramide Synthase gene disruption SialylTransferase I 4 GM3 3 SialylTransferase II 8 GD3 SialylTransferase III GT3 8 Embryonic Lethal. OAc-G Embryogenesis D3 OAc-G T3 GT2 GA2 GM2 GD2 proceeded into gastrulation with 4 4 4 4 differentiation into primitive germ layers and OAc-G D3 GA1 GM1a GD1b embryo patterning but abruptly halted by a GT1c 3 major apoptotic process. Deficient 3 3 3 OAc-G Q1b(?) stem cells able to form GA1 embryonic GD1a GT1b GQ1c endodermal, mesodermal, and ectodermal deficient in3 3 derivatives3but were 3 OAc-G strikingly T1b ability to formGT1a well differentiated tissues. GP1c GQ1b GD1c However, hematopoietic and neuronal differentiation could be induced. OAc-G Q1b 8 8 8 8 O series A series B series C series GalNAc Transferase Gal Transferase II SialylTransferase IV SialylTransferase V GalCer and Ganglio-series gangliosides well studied. All other GlcCer derived glycolipids less well studied. Why are Naturally-occurring Human Genetic defects in Ganglioside Biosynthesis so rare?
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