290 likes | 440 Views
Essentials of Glycobiology Lecture 7 April 8, 2004 Ajit Varki. Glycosphingolipids (Glycolipids). CHONDROITIN SULFATE. HYALURONAN. GLYCOSAMINO- GLYCANS. HEPARAN SULFATE. GLYCOPHOSPHO- LIPID ANCHOR. GLYCOSPHINGOLIPID. O-LINKED GlcNAc. Major Glycan Classes in Animal Cells. P. S. S.
E N D
Essentials of Glycobiology Lecture 7April 8, 2004Ajit Varki Glycosphingolipids (Glycolipids)
CHONDROITIN SULFATE HYALURONAN GLYCOSAMINO- GLYCANS HEPARAN SULFATE GLYCOPHOSPHO- LIPID ANCHOR GLYCOSPHINGOLIPID O-LINKED GlcNAc Major Glycan Classes in Animal Cells P S S S Ser-O- S S S S S -O-Ser NS NS Proteoglycan N-LINKED CHAINS Ac O-LINKED CHAIN P Etn S P N O N NH Asn Ser/Thr 2 Asn INOSITOL Glycoprotein Ac OUTSIDE P Sialic Acids INSIDE O Ser
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 Lecture Outline
Glycan Sphingosine Ceramide (Cer) *Glucose (All animals) Galactose (?Vertebrates only) Mannose (Invertebrates) Inositol-P (fungi) Fatty Acyl group Minimal Defining Structure of a Glycosphingolipid * Glycan-O-Ceramide Find the Mislocated Double Bond!
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
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 Isolation and purification of Glycosphingolipids
Biosynthesis of different classes of glycans within the ER-Golgi Pathway
Stepwise elongation of Glucosylceramidegenerates unique Core structures
Series Designation Core Structure Lacto (LcOSe4) Gal3GlcNAc3Gal4Glc1Ceramide Lactoneo (LcnOSe4) Gal4GlcNAc3Gal4Glc1Ceramide Globo (GbOSe4) GalNAc3Gala4Gal4Glc1Ceramide Isoglobo (GbiOSe4) GalNAc3Gal3Gal4Glc1Ceramide Ganglio (GgOSe4) Gal3GalNAc4Gal4Glc1Ceramide Muco (MucOSe4) GalGal3Gal4Glc1Ceramide Gala (GalOSe2) Gal4Gal1Ceramide Sulfatides 3-0-Sulfo-Gal1Ceramide Major Classes of Glycosphingolipids Different Core structures generate unique shapes and are expressed in a cell-type specific manner
Examples of outer chains and modifications to Glycosphingolipid Cores Much similarity to outer chains of N- and O-glycans
Pathways for Ganglio-series Glycosphingolipid biosynthesis Gm1b
Metabolic relationships in the biosynthesis and turnover of Sphingolipids
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” Turnover and Degradation of 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.. Monoclonal antibodies (Mabs) against 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 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 Biological Roles of Glycosphingolipids
Examples of interactions between glycosphingolipids and bacterial toxins or receptors
The P Blood Group System GLYCOSPHINGOLIPID CHONDROITIN SULFATE HYALURONAN B19 Parvovirus and Uropathogenic E.coli Binding GLYCOSAMINO- GLYCANS P S S S Ser-O- HEPARAN SULFATE S S S S S -O-Ser NS NS Proteoglycan N-LINKED CHAINS Ac O-LINKED CHAIN GLYCOPHOSPHO- LIPID ANCHOR P Etn S P N O N NH Asn Ser/Thr 2 Asn INOSITOL Glycoprotein OUTSIDE P Sialic Acids INSIDE O-LINKED GlcNAc O Ser
Examples of proposed interactions between glycosphingolipids and mammalian receptors
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 Natural and induced Genetic Disorders in Glycosphingolipid biosynthesis
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
Consequences of SialylTransferase II (GD3 synthase) gene disruption Viable, fertile, normal life span, under further investigation
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?
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
Consequences of Lactosylceramide Synthase gene disruption Result Pending
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.
Why are Naturally-occurring Human Genetic defectsin Ganglioside Biosynthesis so rare? GalCer and Ganglio-series gangliosides well studied. All other GlcCer derived glycolipids less well studied.