Glycans linked to lipids and lipid precursors

Transcription

1 Glycobiology BCMB 8130 Clinical Correlations! -sign up for 1st and 2nd choice (1/2)! *********************************! Glycosylation in Diabetes! O-Mannosylation in Human Pathophysiology! Glycoconjugate Trafficking and Disorders! Evolution of Immunity and Sialic Acid/Siglec Biology! Glycan Signaling and Development (Notch/Fringe)! Glycosylation in Metastasis and as Biomarkers! Glycans linked to lipids and lipid precursors 1/27/10 1

2 Large O-linked Glycosaminoglycans and polylactosamine structures Glycoprotein N-linked and O- linked oligosaccharides Glycolipid oligosaccharides Glycan synthesis in a cellular context 2

3 Overview From ER through Trans-Golgi and points inbetween On and into the ER Dolichol-P-X Glycosyl phosphatidylinositol (GPI) Glycosphingolipids (GSL) 3

4 ER glycolipid synthesis Dolichol-P-X Glycosyl phosphatidylinositol (GPI) Glycosphingolipids (GSL) Minimal Defining Structure of a Glycosphingolipid Glycan-O-Ceramide Essentials of Glycobiology Second Edition 4

5 Biosynthesis of Ceramide and Glucosylceramide 5

6 Golgi processing of Glycosphingolipids cis medial trans 6

7 Major Classes of Glycosphingolipids Different Core structures generate unique shapes and are expressed in a cell-type specific manner After Varki, A 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: Galβ3GalNAcβ4(Neu5Acα3)Galβ4Glcβ1Cer = GM1 in the Svennerholm nomenclature OR II 3 Neu5Ac-GgOSe 4 -Cer in the official IUPAC-IUB designation After Varki, A 7

8 Pathways for Ganglio-series Glycosphingolipid biosynthesis Ceramide is utilized for Ganglioside synthesis and for GalCer synthesis GM3 is a branching substrate for production of all complex gangliosides Biosynthesis exhibits branch exclusivity (sialyltransferases cannot take a-series to b-series or b-series to c- series Extension with neutral residues utilizes the same transferases regardless of branch ST-III ST-II ST-I GalT-I GalNAcT GalT-II ST-IV ST-V Essentials of Glycobiology Second Edition 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, also known as liftases ). 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) 8

9 Biological Roles of Glycosphingolipids Thought to be critical components of the epidermal (skin) permeability barrier (Glc-Cer delivers Cer to stratum corneum) 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 9

10 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. Consequences of Lactosylceramide Synthase gene disruption Early embryonic lethal, associated primarily with extra-embryonic tissues (trophoblast, extraembryonic membranes) 10

11 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? Consequences of SialylTransferase II (GD3 synthase) gene disruption Viable, fertile, normal life span, sensory deficits especially related to pain sensation 11

12 Consequences of GalNAc Transferase I gene disruption Male Sterility. Late Onset Peripheral Nerve Demyelination 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 Sheikh, KA, et al. (1999) PNAS 96,

13 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 On and into the ER Glycosyl phosphatidylinositol (GPI) 13

14 Examples of GPI-Anchored Proteins Cell surface hydrolases alkaline phosphatase acetylcholinesterase 5ʼ nucleotidase Adhesion molecules neural cell adhesion molecule heparan sulfate proteoglycan Protozoal antigens trypanosome VSG leishmanial protease plasmodium antigens Mammalian antigens carcinoembryonic antigen Thy-1 Others scrapie prion protein folate receptor decay accelerating factor Structure of the Basic GPI Anchor GPI-Linked Protein Etn P NH 2 = Mannose (Man) = Glucosamine = Ethanolamine = Phosphate PNH Defect Etn P Pig-A NH 2 INOSITOL P Cell Surface Membrane After Hart, G 14

15 Chemical and enzymatic reactions of GPI anchors Essentials of Glycobiology Second Edition GPIbiosynthetic pathways of mammalian cells and Trypanosoma brucei Essentials of Glycobiology Second Edition 15

16 After Freeze, H After Freeze, H 16

17 Essentials of Glycobiology Second Edition Examples of C-Terminal Sequences Signaling the Addition of GPI-Anchors 5-10 hydrophilic, hydrophobic Essentials of Glycobiology Second Edition 17

18 PIG-A is mutated in PNH The first step in biosynthesis of the GPI anchor requires at least four genes One of them, PIG-A is an X-linked gene After Freeze, H Paroxysmal Nocturnal Hemoglobinuria A hematopoietic stem cell disorder characterized by intravascular hemolytic anemia (sudden onset, intermittent/episodic). 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. 18

19 Nocturnal hemoglobinuria, not clear why morning urine is enriched in hemoglobin breakdown products (variable course) Morning Report, Toronto General Hospital; GPI-linked proteins protect RBCs from complementmediated lysis Rother RP, et al. (2007) Nature Biotechnology 25,

20 Accumulation of hemoglobin breakdown products in kidney tubule eventually leads to tubule pathology Tsai C-W, et al. (2007) Kidney International 71, 1187 On and into the ER Dolichol-P-X 20

21 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 GlcNAc Man Glc Gal Sia Fuc 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):

22 GlcNAc Man Glc Gal Sia Fuc Biosynthesis of the N-Glycan Precursor on the Cytosolic Leaflet of the Endoplasmic Reticulum (ER) CDG = Congenital Disorder of Glycosylation in Humans Adapted from Marquardt T, Denecke J. Eur J Pediatr Jun;162(6): GlcNAc Man Glc Gal Sia Fuc Biosynthesis of the N-Glycan Precursor on Lumenal Leaflet of ER Adapted from Marquardt T, Denecke J. Eur J Pediatr Jun;162(6):

23 GlcNAc Man Glc Gal Sia Fuc Completion of Biosynthesis of N-Glycan Precursor on Lumenal Leaflet of ER - and Transfer to Protein Adapted from Marquardt T, Denecke J. Eur J Pediatr Jun;162(6): 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 23

24 General Principles Regarding Lipid-Linked Glycans 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 Nucleotide sugar donors are used for cytoplasmic face extension and for Golgi extension; Dolichol-linked donors are used for ER extension of N-linked glycan precursors 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 influence signaling pathways Glycosphingolipids and GPI-anchored proteins associate in membrane microdomains (rafts, GEMs) Processing and maturation of an N-glycan Essentials of Glycobiology Second Edition 24