Glycolipids and GPI anchors Glycolipids and GPI anchors Glycolipids and GPI anchors Glycolipids and GPI anchors Learn the structures, and some functions.

Glycolipids and GPI anchors Glycolipids and GPI anchors Glycolipids and GPI anchors Glycolipids and GPI anchors Learn the structures, and some functions of the most abundant vertebrate glycolipidsLearn the structures, and some functions of the most abundant vertebrate glycolipids Glucosylceramide and the skin barrier Galactosylceramide & sulfatide in myelin Glycolipids in cell-cell interactions and membrane receptor modulation Learn the structures, distribution and biosynthesis of GPI anchorsLearn the structures, distribution and biosynthesis of GPI anchors GPI-anchored proteins and their anchor structures in people and parasites GPI anchor biosynthesis and en bloc transfer Glycolipids and GPI anchors in diseaseGlycolipids and GPI anchors in disease Glycolipids and lysosomal storage diseases GPI anchors in anemia Learn the structures, and some functions of the most abundant vertebrate glycolipidsLearn the structures, and some functions of the most abundant vertebrate glycolipids Glucosylceramide and the skin barrier Galactosylceramide & sulfatide in myelin Glycolipids in cell-cell interactions and membrane receptor modulation Learn the structures, distribution and biosynthesis of GPI anchorsLearn the structures, distribution and biosynthesis of GPI anchors GPI-anchored proteins and their anchor structures in people and parasites GPI anchor biosynthesis and en bloc transfer Glycolipids and GPI anchors in diseaseGlycolipids and GPI anchors in disease Glycolipids and lysosomal storage diseases GPI anchors in anemia Objectives

Major membrane lipid classes PLC GLYCEROLIPID

Most vertebrate glycolipids are glycosphingolipids Most vertebrate glycolipids are glycosphingolipids Most vertebrate glycolipids are glycosphingolipids Most vertebrate glycolipids are glycosphingolipids

Glucosylceramide is a key intermediate in skin barrier formation Glucosylceramide is a key intermediate in skin barrier formation Glucosylceramide is a key intermediate in skin barrier formation Glucosylceramide is a key intermediate in skin barrier formation Skin water barrier. Formation and secretion of lamellar bodies (LB) at stratum granulosum – stratum corneum interface. Precursor lipids, including glucosylceramide, sphingomyelin, glycerophospholipids, and cholesterol sulfate, are packaged into LB in the upper epidermis. Fusion of the LB with the apical plasma membrane in the uppermost layer of the epidermis (stratum granulosum), allows extrusion of lipid precursors into the extracellular domain. Enzymatic processing of precursor lipids generates the major lipid classes required for epidermal barrier function. Holleran et al (2006) Febs Letters 23:5456

Normal mouse skin “Gaucher” mouse skin (glucocerebrosidase -/-) Holleran et al (1994) J Clin Invest 93:1756 Glucosylceramide is a key intermediate in skin barrier formation Glucosylceramide is a key intermediate in skin barrier formation Glucosylceramide is a key intermediate in skin barrier formation Glucosylceramide is a key intermediate in skin barrier formation

Normal human skin Human type 2 Gaucher disease skin (glucocerebrosidase- deficient) Holleran et al (1994) J Clin Invest 93:1756 Glucosylceramide is a key intermediate in skin barrier formation Glucosylceramide is a key intermediate in skin barrier formation Glucosylceramide is a key intermediate in skin barrier formation Glucosylceramide is a key intermediate in skin barrier formation

galactosylceramide and it’s 3-sulfated from, sulfatide, are major lipids in the brain galactosylceramide and it’s 3-sulfated from, sulfatide, are major lipids in the brain galactosylceramide and it’s 3-sulfated from, sulfatide, are major lipids in the brain galactosylceramide and it’s 3-sulfated from, sulfatide, are major lipids in the brain Together, GalCer and sulfatide comprise up to 1.9% of brain fresh weight!

galactosylceramide & sulfatide are the major glycans in the brain, and major lipids in myelin galactosylceramide & sulfatide are the major glycans in the brain, and major lipids in myelin galactosylceramide & sulfatide are the major glycans in the brain, and major lipids in myelin galactosylceramide & sulfatide are the major glycans in the brain, and major lipids in myelin Glycan ratios in the brain

Delcomyn, F. (1998) Foundations of Neurobiology. W.H. Freeman, New York.

Mutant mice lacking GalCer synthetase (Ugt8a) or GalCer 3-sulfotransferase (Gal3st1) display myelin defects and associated behavioral deficits Mutant mice lacking GalCer synthetase (Ugt8a) or GalCer 3-sulfotransferase (Gal3st1) display myelin defects and associated behavioral deficits Mutant mice lacking GalCer synthetase (Ugt8a) or GalCer 3-sulfotransferase (Gal3st1) display myelin defects and associated behavioral deficits Mutant mice lacking GalCer synthetase (Ugt8a) or GalCer 3-sulfotransferase (Gal3st1) display myelin defects and associated behavioral deficits Honke et al (2002) PNAS 99:4227 Popko (2000) Glia 29:149

Glycoglycerolipids are not widely distributed in vertebrates, but 3-O-sulfated galactosyldiacylglycerol (seminolipid) makes up 90% of the glycolipid in mammalian testes. Gal3st1-null males are infertile due to an early block in spermatogenesis. Glycoglycerolipids are not widely distributed in vertebrates, but 3-O-sulfated galactosyldiacylglycerol (seminolipid) makes up 90% of the glycolipid in mammalian testes. Gal3st1-null males are infertile due to an early block in spermatogenesis. Glycoglycerolipids are not widely distributed in vertebrates, but 3-O-sulfated galactosyldiacylglycerol (seminolipid) makes up 90% of the glycolipid in mammalian testes. Gal3st1-null males are infertile due to an early block in spermatogenesis. Glycoglycerolipids are not widely distributed in vertebrates, but 3-O-sulfated galactosyldiacylglycerol (seminolipid) makes up 90% of the glycolipid in mammalian testes. Gal3st1-null males are infertile due to an early block in spermatogenesis.

GlcCer based glycolipid families: GlcCer based glycolipid families: -All share a lactosylceramide (LacCer) core All share a lactosylceramide (LacCer) coreAll share a lactosylceramide (LacCer) core -Nomenclature is based on the next sugars in the neutral tetrasaccharide core Nomenclature is based on the next sugars in the neutral tetrasaccharide coreNomenclature is based on the next sugars in the neutral tetrasaccharide core -Classified as “neutral glycosphingolipids”, “sulfated glycosphingolipids” or “gangliosides” (sialylated glycosphingolipids) Classified as “neutral glycosphingolipids”, “sulfated glycosphingolipids” or “gangliosides” (sialylated glycosphingolipids)Classified as “neutral glycosphingolipids”, “sulfated glycosphingolipids” or “gangliosides” (sialylated glycosphingolipids) -Nomenclature describes the length of the core and any substituents, number the sugars from the reducing (Cer) end out by roman numerals Nomenclature describes the length of the core and any substituents, number the sugars from the reducing (Cer) end out by roman numeralsNomenclature describes the length of the core and any substituents, number the sugars from the reducing (Cer) end out by roman numerals -Biosynthesized stepwise by individual glycosphingolipids, some of which are glycolipid-specific, others of which also make glycoprotein glycans Biosynthesized stepwise by individual glycosphingolipids, some of which are glycolipid-specific, others of which also make glycoprotein glycansBiosynthesized stepwise by individual glycosphingolipids, some of which are glycolipid-specific, others of which also make glycoprotein glycans GlcCer based glycolipid families: GlcCer based glycolipid families: -All share a lactosylceramide (LacCer) core All share a lactosylceramide (LacCer) coreAll share a lactosylceramide (LacCer) core -Nomenclature is based on the next sugars in the neutral tetrasaccharide core Nomenclature is based on the next sugars in the neutral tetrasaccharide coreNomenclature is based on the next sugars in the neutral tetrasaccharide core -Classified as “neutral glycosphingolipids”, “sulfated glycosphingolipids” or “gangliosides” (sialylated glycosphingolipids) Classified as “neutral glycosphingolipids”, “sulfated glycosphingolipids” or “gangliosides” (sialylated glycosphingolipids)Classified as “neutral glycosphingolipids”, “sulfated glycosphingolipids” or “gangliosides” (sialylated glycosphingolipids) -Nomenclature describes the length of the core and any substituents, number the sugars from the reducing (Cer) end out by roman numerals Nomenclature describes the length of the core and any substituents, number the sugars from the reducing (Cer) end out by roman numeralsNomenclature describes the length of the core and any substituents, number the sugars from the reducing (Cer) end out by roman numerals -Biosynthesized stepwise by individual glycosphingolipids, some of which are glycolipid-specific, others of which also make glycoprotein glycans Biosynthesized stepwise by individual glycosphingolipids, some of which are glycolipid-specific, others of which also make glycoprotein glycansBiosynthesized stepwise by individual glycosphingolipids, some of which are glycolipid-specific, others of which also make glycoprotein glycans Subfamily seriesStructureAbbreviation LactoGalβ1-3GlcNAcβ1-3Galβ1-4GlcβCerLc 4 Cer NeolactoGalβ1-4GlcNAcβ1-3Galβ1-4GlcβCernLc 4 Cer GanglioGalβ1-3GalNAcβ1-4Galβ1-4GlcβCerGg 4 Cer GloboGalNAcβ1-3Galα1-4Galβ1-4GlcβCerGb 4 Cer Lactosylceramide (LacCer)

NeuAc  3 Gal  3 GalNAc  4 (NeuAc  8 NeuAc  3) Gal  4 Glc  Cer Ganglioside GT1b Gal GalNAc Gal Glc NeuAc

Gangliosides are embedded in the extracellular leaflet of the plasma membrane with their glycans extending outward DeMarco & Woods (2009) Glycobiology 19, 344

Rat cerebellar granule cell axons in culture immunostained for ganglioside GD1a HIC (phase) image overlay imageanti-GD1a mAb Gangliosides are on ALL vertebrate cells, but are the dominant glycan structures on nerve cells * non-neuronal cells

The same four structures comprise the great majority of gangliosides in the brains of all mammals Values are for human; Gangliosides shown constitute 97% of total brain gangliosides Tettamanti et al. (1973) Biochim Biophys Acta 296, 160

Brain ganglioside biosynthesis ST3GAL5: “Infantile-onset symptomatic epilepsy syndrome caused by a homozygous loss-of- function mutation of GM3 synthase” Simpson MA et al. Nature Genet 36, 1225 (2004) B4GALNT1: “Alteration of ganglioside biosynthesis responsible for complex hereditary spastic paraplegia” Boukhris et al. Am J Human Genet 93, 118 (2013)

Concepts of glycolipid function: cis and trans Concepts of glycolipid function: cis and trans Concepts of glycolipid function: cis and trans Concepts of glycolipid function: cis and trans cis: regulation by lateral association cis: regulation by lateral association cis: regulation by lateral association cis: regulation by lateral association trans: regulation by cell-cell recognition trans: regulation by cell-cell recognition trans: regulation by cell-cell recognition trans: regulation by cell-cell recognition Note: Because of their long unsaturated lipid chains, glycolipids cluster together, along with cholesterol, selected other lipids, and selected proteins, in “lipid rafts” Note: Because of their long unsaturated lipid chains, glycolipids cluster together, along with cholesterol, selected other lipids, and selected proteins, in “lipid rafts” Note: Because of their long unsaturated lipid chains, glycolipids cluster together, along with cholesterol, selected other lipids, and selected proteins, in “lipid rafts” Note: Because of their long unsaturated lipid chains, glycolipids cluster together, along with cholesterol, selected other lipids, and selected proteins, in “lipid rafts” Regina Todeschini & Hakomori (2008) Biochim Biophys Acta 1780:421

Glycolipid purification and analysis: Solvent extract, partition/chromatography, TLC Glycolipid purification and analysis: Solvent extract, partition/chromatography, TLC Glycolipid purification and analysis: Solvent extract, partition/chromatography, TLC Glycolipid purification and analysis: Solvent extract, partition/chromatography, TLC Homogenize cells or tissuesHomogenize cells or tissues Add methanol and chloroformAdd methanol and chloroform Remove precipitated protein and DNA by centrifugationRemove precipitated protein and DNA by centrifugation Isolate glycolipids by partition and/or adsorptive chromatographyIsolate glycolipids by partition and/or adsorptive chromatography Analyze by TLCAnalyze by TLC Mouse brain gangliosides – silica TLC – resorcinol sialic acid stain Mouse sialyltransferase mutants origin -

GlcCer based glycolipid families: GlcCer based glycolipid families: -All share a lactosylceramide (LacCer) core All share a lactosylceramide (LacCer) coreAll share a lactosylceramide (LacCer) core -Nomenclature is based on the next sugars in the neutral tetrasaccharide core Nomenclature is based on the next sugars in the neutral tetrasaccharide coreNomenclature is based on the next sugars in the neutral tetrasaccharide core -Classified as “neutral glycosphingolipids”, “sulfated glycosphingolipids” or “gangliosides” (sialylated glycosphingolipids) Classified as “neutral glycosphingolipids”, “sulfated glycosphingolipids” or “gangliosides” (sialylated glycosphingolipids)Classified as “neutral glycosphingolipids”, “sulfated glycosphingolipids” or “gangliosides” (sialylated glycosphingolipids) -Nomenclature describes the length of the core and any substituents, number the sugars from the reducing (Cer) end out by roman numerals Nomenclature describes the length of the core and any substituents, number the sugars from the reducing (Cer) end out by roman numeralsNomenclature describes the length of the core and any substituents, number the sugars from the reducing (Cer) end out by roman numerals -Biosynthesized stepwise by individual glycosphingolipids, some of which are glycolipid-specific, others of which also make glycoprotein glycans Biosynthesized stepwise by individual glycosphingolipids, some of which are glycolipid-specific, others of which also make glycoprotein glycansBiosynthesized stepwise by individual glycosphingolipids, some of which are glycolipid-specific, others of which also make glycoprotein glycans GlcCer based glycolipid families: GlcCer based glycolipid families: -All share a lactosylceramide (LacCer) core All share a lactosylceramide (LacCer) coreAll share a lactosylceramide (LacCer) core -Nomenclature is based on the next sugars in the neutral tetrasaccharide core Nomenclature is based on the next sugars in the neutral tetrasaccharide coreNomenclature is based on the next sugars in the neutral tetrasaccharide core -Classified as “neutral glycosphingolipids”, “sulfated glycosphingolipids” or “gangliosides” (sialylated glycosphingolipids) Classified as “neutral glycosphingolipids”, “sulfated glycosphingolipids” or “gangliosides” (sialylated glycosphingolipids)Classified as “neutral glycosphingolipids”, “sulfated glycosphingolipids” or “gangliosides” (sialylated glycosphingolipids) -Nomenclature describes the length of the core and any substituents, number the sugars from the reducing (Cer) end out by roman numerals Nomenclature describes the length of the core and any substituents, number the sugars from the reducing (Cer) end out by roman numeralsNomenclature describes the length of the core and any substituents, number the sugars from the reducing (Cer) end out by roman numerals -Biosynthesized stepwise by individual glycosphingolipids, some of which are glycolipid-specific, others of which also make glycoprotein glycans Biosynthesized stepwise by individual glycosphingolipids, some of which are glycolipid-specific, others of which also make glycoprotein glycansBiosynthesized stepwise by individual glycosphingolipids, some of which are glycolipid-specific, others of which also make glycoprotein glycans Subfamily seriesStructureAbbreviation LactoGalβ1-3GlcNAcβ1-3Galβ1-4GlcβCerLc 4 Cer NeolactoGalβ1-4GlcNAcβ1-3Galβ1-4GlcβCernLc 4 Cer GanglioGalβ1-3GalNAcβ1-4Galβ1-4GlcβCerGg 4 Cer GloboGalNAcβ1-3Galα1-4Galβ1-4GlcβCerGb 4 Cer Lactosylceramide (LacCer)

Example: Total neolactoseries monsialylated glycolipids from human neutrophils – diversity within a structural theme Example: Total neolactoseries monsialylated glycolipids from human neutrophils – diversity within a structural theme Example: Total neolactoseries monsialylated glycolipids from human neutrophils – diversity within a structural theme Example: Total neolactoseries monsialylated glycolipids from human neutrophils – diversity within a structural theme Nimrichter et al (2008) Blood, 112:3744

Neolactoseries monsialylated glycolipids on human neutrophils Neolactoseries monsialylated glycolipids on human neutrophils Neolactoseries monsialylated glycolipids on human neutrophils Neolactoseries monsialylated glycolipids on human neutrophils Nimrichter et al (2008) Blood, 112:3744

Separation of human neutrophil gangliosides by HPLC Separation of human neutrophil gangliosides by HPLC Separation of human neutrophil gangliosides by HPLC Separation of human neutrophil gangliosides by HPLC Nimrichter et al (2008) Blood, 112:3744

Concepts of glycolipid function: trans recognition in health and disease Concepts of glycolipid function: trans recognition in health and disease Concepts of glycolipid function: trans recognition in health and disease Concepts of glycolipid function: trans recognition in health and disease Bacterial toxins bind to glycolipidsBacterial toxins bind to glycolipids –Shigatoxin (verotoxin) binds to the neutral glycosphingolipid Gb 3 (Galα1-4Galβ1-4Glcβ1-Cer) –Cholera and E. Coli enterotoxins bind to the ganglioside GM1 (Galβ1-3GalNAcβ1-4(NeuAcα2-3)Galβ1-4Glcβ1-Cer) –Tetanus and botulinum neurotoxins toxins bind to nervous system gangliosides with different specificities A brain lectin, myelin-associated glycoprotein (MAG), binds to gangliosides GD1a and GT1b to meidate axon-myelin interactions*A brain lectin, myelin-associated glycoprotein (MAG), binds to gangliosides GD1a and GT1b to meidate axon-myelin interactions* An endothelial cell lectin, E-selectin, binds to sialylated fucosylated glycolipids on human neutrophils to initiate inflammation*An endothelial cell lectin, E-selectin, binds to sialylated fucosylated glycolipids on human neutrophils to initiate inflammation* * These lectins will be discussed in a later lecture

Research correlation: Multivalent toxin inhibitor mimics glycolipid in the cell membrane Research correlation: Multivalent toxin inhibitor mimics glycolipid in the cell membrane Research correlation: Multivalent toxin inhibitor mimics glycolipid in the cell membrane Research correlation: Multivalent toxin inhibitor mimics glycolipid in the cell membrane LEFT: AB 5 bacterial toxins (cholera toxin shown) have 5 glycolipid cell surface binding subunits surrounding a “payload” toxic subunit (not shown) that is inserted into the cell only after binding. LEFT: AB 5 bacterial toxins (cholera toxin shown) have 5 glycolipid cell surface binding subunits surrounding a “payload” toxic subunit (not shown) that is inserted into the cell only after binding. LEFT: AB 5 bacterial toxins (cholera toxin shown) have 5 glycolipid cell surface binding subunits surrounding a “payload” toxic subunit (not shown) that is inserted into the cell only after binding. LEFT: AB 5 bacterial toxins (cholera toxin shown) have 5 glycolipid cell surface binding subunits surrounding a “payload” toxic subunit (not shown) that is inserted into the cell only after binding. Merritt et al. (1994) Protein Sci 3:166 RIGHT: Taking advantage of the AB 5 structure of vertotoxin, and its Gb 3 specific binding, Kitov et al synthesized a “starfish” shaped inhibitor with five arms, each carrying two copies of Gb 3. RIGHT: Taking advantage of the AB 5 structure of vertotoxin, and its Gb 3 specific binding, Kitov et al synthesized a “starfish” shaped inhibitor with five arms, each carrying two copies of Gb 3. RIGHT: Taking advantage of the AB 5 structure of vertotoxin, and its Gb 3 specific binding, Kitov et al synthesized a “starfish” shaped inhibitor with five arms, each carrying two copies of Gb 3. RIGHT: Taking advantage of the AB 5 structure of vertotoxin, and its Gb 3 specific binding, Kitov et al synthesized a “starfish” shaped inhibitor with five arms, each carrying two copies of Gb 3. Kitov et al. (2000) Nature 403:669

Research correlation: Multivalent toxin inhibitor mimics glycolipid in the cell membrane Research correlation: Multivalent toxin inhibitor mimics glycolipid in the cell membrane Research correlation: Multivalent toxin inhibitor mimics glycolipid in the cell membrane Research correlation: Multivalent toxin inhibitor mimics glycolipid in the cell membrane ABOVE: “Starfish”Gb 3 blocks verotoxin binding at sub-nM concentrations – a million times more potent than Gb 3 ABOVE: “Starfish”Gb 3 blocks verotoxin binding at sub-nM concentrations – a million times more potent than Gb 3 ABOVE: “Starfish”Gb 3 blocks verotoxin binding at sub-nM concentrations – a million times more potent than Gb 3 ABOVE: “Starfish”Gb 3 blocks verotoxin binding at sub-nM concentrations – a million times more potent than Gb 3 Kitov et al. (2000) Nature 403:669 Right: “Starfish”Gb 3 crystallized with verotoxin – a perfect pentomeric sandwich Right: “Starfish”Gb 3 crystallized with verotoxin – a perfect pentomeric sandwich Right: “Starfish”Gb 3 crystallized with verotoxin – a perfect pentomeric sandwich Right: “Starfish”Gb 3 crystallized with verotoxin – a perfect pentomeric sandwich

A small but diverse group of proteins are attached to the cell surface by a “glycosylphosphatidylinositol” (GPI) anchor A small but diverse group of proteins are attached to the cell surface by a “glycosylphosphatidylinositol” (GPI) anchor A small but diverse group of proteins are attached to the cell surface by a “glycosylphosphatidylinositol” (GPI) anchor A small but diverse group of proteins are attached to the cell surface by a “glycosylphosphatidylinositol” (GPI) anchor GPI anchors have a minimal (core) structure (black font) with variable additional glycan, fatty acid ester, and phosphoethanolamine groups (blue font) Taylor and Drikamer (2011) Introduction to Glycobiology

Essentials of Glycobiology Second Edition GPI anchors are complex & diverse GPI anchors are complex & diverse GPI anchors are complex & diverse GPI anchors are complex & diverse Glucosamine with free amine – rareGlucosamine with free amine – rare Phosphodiesters at each endPhosphodiesters at each end Diverse modifications along the coreDiverse modifications along the core R3

A group of select proteins are GPI anchored* A group of select proteins are GPI anchored* A group of select proteins are GPI anchored* A group of select proteins are GPI anchored*Mammals Erythrocyte CD59 and decay acceleration factor (DAF) complement regulation Alkaline phosphatase cell-surface hydrolase 5′-Nucleotidase Renal dipeptidase cell-surface hydrolase Trehalase Neural cell adhesion molecule 120 (NCAM-120) adhesion molecule Neural cell adhesion molecule TAG-1 adhesion molecule CD58 FcγIII receptor Fc receptor Ciliary neurotrophic factor receptor (CNTFR) α subunit neural receptor Glial-cell-derived neurotrophic factor receptor (GDNFR) α subunit neural receptor CD14 LPS receptor Prion protein (PrP) unknown Glypican family of GPI- anchored proteoglycans extracellular matrix component Trypanosoma brucei variant surface glycoprotein (VSG) protective coat Leishmania major promastigote surface protease (PSP) bound complement degradation Trypanosoma cruzi GPI- anchored mucins host cell invasion Plasmodium falciparum merozoite surface protein 1 (MSP-1) erythrocyte invasion Toxoplasma gondii surface antigen 1 (SAG-1) host cell invasion Entamoeba histolytica GPI proteophosphoglycans virulence factor Parasites *plus some in yeast, plants, some other species

Variety is the spice of… GPI anchors Variety is the spice of… GPI anchors Variety is the spice of… GPI anchors Variety is the spice of… GPI anchors Essentials of Glycobiology Second Edition

Preassembled GPI anchors are covalently transferred en bloc via amide linkage to an amino acid near the carboxy terminus of a nacient protein, at the same time releasing the C-terminal protein fragment. This occurs in the ER. Preassembled GPI anchors are covalently transferred en bloc via amide linkage to an amino acid near the carboxy terminus of a nacient protein, at the same time releasing the C-terminal protein fragment. This occurs in the ER. Although there is no “consensus sequence,” per se, for GPI transfer, likely sites are identified by their structural features. Although there is no “consensus sequence,” per se, for GPI transfer, likely sites are identified by their structural features. Preassembled GPI anchors are covalently transferred en bloc via amide linkage to an amino acid near the carboxy terminus of a nacient protein, at the same time releasing the C-terminal protein fragment. This occurs in the ER. Preassembled GPI anchors are covalently transferred en bloc via amide linkage to an amino acid near the carboxy terminus of a nacient protein, at the same time releasing the C-terminal protein fragment. This occurs in the ER. Although there is no “consensus sequence,” per se, for GPI transfer, likely sites are identified by their structural features. Although there is no “consensus sequence,” per se, for GPI transfer, likely sites are identified by their structural features. Essentials of Glycobiology Second Edition

Like N-linked glycan biosynthesis, the precursor starts assembly on the cytoplasmic face of the ER, then is flipped inside for further processing. GlcNAc transfer and deacetylation, inositol fatty acid esterification, and multiple ethanolamine phosphate group transfers by separate enzymes are noteworthy. Like N-linked glycan biosynthesis, the precursor starts assembly on the cytoplasmic face of the ER, then is flipped inside for further processing. GlcNAc transfer and deacetylation, inositol fatty acid esterification, and multiple ethanolamine phosphate group transfers by separate enzymes are noteworthy. Like N-linked glycan biosynthesis, the precursor starts assembly on the cytoplasmic face of the ER, then is flipped inside for further processing. GlcNAc transfer and deacetylation, inositol fatty acid esterification, and multiple ethanolamine phosphate group transfers by separate enzymes are noteworthy. Like N-linked glycan biosynthesis, the precursor starts assembly on the cytoplasmic face of the ER, then is flipped inside for further processing. GlcNAc transfer and deacetylation, inositol fatty acid esterification, and multiple ethanolamine phosphate group transfers by separate enzymes are noteworthy. Essentials of Glycobiology Second Edition

GPI anchor synthesis has been studied most extensively in parasites, which express millions of GPI-anchored proteins and glycans on the surface of each cell. GPI anchor synthesis has been studied most extensively in parasites, which express millions of GPI-anchored proteins and glycans on the surface of each cell. GPI anchor synthesis has been studied most extensively in parasites, which express millions of GPI-anchored proteins and glycans on the surface of each cell. GPI anchor synthesis has been studied most extensively in parasites, which express millions of GPI-anchored proteins and glycans on the surface of each cell. http://www.lifesci.dundee.ac.uk/people/mike-ferguson African sleeping sicknessChagas diseaseleishmaniasis

GPI anchor synthesis in parasites is complex with some interesting twists, such as fatty acid remodeling. Since GPI anchored coat proteins are virulence factors, they are being exploited as targets for therapy. GPI anchor synthesis in parasites is complex with some interesting twists, such as fatty acid remodeling. Since GPI anchored coat proteins are virulence factors, they are being exploited as targets for therapy. GPI anchor synthesis in parasites is complex with some interesting twists, such as fatty acid remodeling. Since GPI anchored coat proteins are virulence factors, they are being exploited as targets for therapy. GPI anchor synthesis in parasites is complex with some interesting twists, such as fatty acid remodeling. Since GPI anchored coat proteins are virulence factors, they are being exploited as targets for therapy. Essentials of Glycobiology Second Edition

To study the structures and functions of GPI anchored proteins, they can be released under biologically compatible conditions using bacterial phosphatidylinositol-specific phospholipase C (PIPLC), or decomposed specifically by nitrous acid, which cleaves at the glucosamine residue forming a 2,5-anhydromannose residue. To study the structures and functions of GPI anchored proteins, they can be released under biologically compatible conditions using bacterial phosphatidylinositol-specific phospholipase C (PIPLC), or decomposed specifically by nitrous acid, which cleaves at the glucosamine residue forming a 2,5-anhydromannose residue. To study the structures and functions of GPI anchored proteins, they can be released under biologically compatible conditions using bacterial phosphatidylinositol-specific phospholipase C (PIPLC), or decomposed specifically by nitrous acid, which cleaves at the glucosamine residue forming a 2,5-anhydromannose residue. To study the structures and functions of GPI anchored proteins, they can be released under biologically compatible conditions using bacterial phosphatidylinositol-specific phospholipase C (PIPLC), or decomposed specifically by nitrous acid, which cleaves at the glucosamine residue forming a 2,5-anhydromannose residue. Essentials of Glycobiology Second Edition

Clinical correlation: defects in glycolipid degradation Clinical correlation: defects in glycolipid degradation Clinical correlation: defects in glycolipid degradation Clinical correlation: defects in glycolipid degradation Disease name (glycolipid) Enzyme or protein deficiency Clinical symptoms Tay–Sachs (GM2) β-hexosaminidase A neurodegeneration, death by 4 years Sandhoff (GM2) β-hexosaminidase A and B neurodegeneration, death by 4 years GM1 gangliosidosis (GM1) β-galactosidase progressive neurological disease and skeletal dysplasia in severe infantile form Fabry*(Gb3) α-galactosidase severe pain, angiokeratoma, corneal opacities, death from renal or cerebrovascular disease Gaucher’s*(GlcCer) β-glucoceramidase hepatosplenomegaly neurodegeneration (severe form only) Krabbe(GalCer) β-galactoceramidase early onset with progression to severe mental and motor deterioration Metachromatic leukodystrophy (sulfatide) arylsulfatase A (cerebroside sulfatase) mental regression, peripheral neuropathy, seizures, dementia Saposin deficiency (many) saposin precursor neurodegeneration, death by 4 years *enzyme replacement therapy available Essentials of Glycobiology Second Edition

Gaucher disease – cause, symptoms and treatment Gaucher disease – cause, symptoms and treatment Gaucher disease – cause, symptoms and treatment Gaucher disease – cause, symptoms and treatment Hepatosplenomegaly: Grossly enlarged liver and spleen http://www.gauchercare.com/ The cure: enzyme replacement therapy Andersson et al.. (2008) Pediatrics 122:1182

A new direction – substrate reduction therapy A new direction – substrate reduction therapy A new direction – substrate reduction therapy A new direction – substrate reduction therapy n-butyl-deoxynojirimycin = Miglustat = Zavesca®

Clinical correlation: Paroxysmal nocturnal hemoglobinuria Clinical correlation: Paroxysmal nocturnal hemoglobinuria Clinical correlation: Paroxysmal nocturnal hemoglobinuria Clinical correlation: Paroxysmal nocturnal hemoglobinuria http://www.bioch.ox.ac.uk/glycob/archive/ CD59, a GPI-anchored “complement defense protein” terminal complement complex http://trialx.com/ The cause, somatic mutation of the PIG-A gene resulting in failure to transfer GlcNAc to phosphatidylinositol, and loss of multiple GPI- anchored complement control proteins. Essentials of Glycobiology

Clinical correlation: Paroxysmal nocturnal hemoglobinuria Clinical correlation: Paroxysmal nocturnal hemoglobinuria Clinical correlation: Paroxysmal nocturnal hemoglobinuria Clinical correlation: Paroxysmal nocturnal hemoglobinuria Kelly R J et al. Blood (2011)117:6786 Anti-complement protein 5 treatment improves outcomes Anti-complement protein 5 treatment improves outcomes Anti-complement protein 5 treatment improves outcomes Anti-complement protein 5 treatment improves outcomes per year

Glycolipids and GPI anchors Glycolipids and GPI anchors Glycolipids and GPI anchors Glycolipids and GPI anchors Learn the structures, and some functions.

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Glycolipids and GPI anchors Glycolipids and GPI anchors Glycolipids and GPI anchors Glycolipids and GPI anchors Learn the structures, and some functions.

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