Essentials of Glycobiology Lecture 7 April 12, 2002 Ajit Varki

Essentials of Glycobiology

Lecture 7

April 12, 2002

Ajit Varki

Structure, biosynthesis and general biology of Glycosphingolipids

Major Glycan

Classes in Animal Cells

OSer

OSer/Thr

NAsn

Ser-O-

OUTSIDE

INSIDE

NAsn

S S S

-O-SerS SSS S

EtnP

INOSITOL

P

NH

Ac

P

NS NS

Ac

S

2

P

GlycoproteinGlycoprotein

ProteoglycanProteoglycan

GLYCOPHOSPHO-GLYCOPHOSPHO-LIPIDLIPID

ANCHORANCHOR

N-LINKED CHAINSN-LINKED CHAINS

O-LINKED O-LINKED CHAINCHAIN

HYALURONANHYALURONAN

GLYCOSAMINO-GLYCOSAMINO-GLYCANSGLYCANS HEPARAN SULFATEHEPARAN SULFATE

CHONDROITINCHONDROITIN SULFATESULFATE

Sialic AcidsSialic Acids

GLYCOSPHINGOLIPIDGLYCOSPHINGOLIPID

O-LINKED GlcNAcO-LINKED GlcNAc

Lecture Overview

Historical BackgroundDefining Structures and Major ClassesOther Nomenclature IssuesBiosynthesisOccurrence & Structural VariationsIsolation and purificationTrafficking, Turnover and Degradation

Lecture Overview (Continued)

Relationship to biosynthesis, turnover and signalling functions of other Sphingolipids

Antibodies against GlycosphingolipidsBiological RolesGenetic Disorders in GSL biosynthesisPerspectives and Future Directions

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!

Biosynthesis of Ceramide and

Glucosylceramide

Find the Missing 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

Isolation and purification of Glycosphingolipids Most glycosphingolipids obtained in good yield from cells and

tissues by sequential organic extractions of increasing polaritySome separation by polarity achieved by two-phase extractionsSubsequent 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

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

Stepwise elongation of

Glucosylceramidegenerates unique Core structures

Major Classes of Glycosphingolipids

Series Designation Core Structure

Lacto (LcOSe4) Gal3GlcNAc3GalGal44GlcGlc1Ceramide1Ceramide

Lactoneo (LcnOSe4) Gal4GlcNAc3GalGal44GlcGlc1Ceramide1Ceramide

Globo (GbOSe4) GalNAc3Gal4GalGal44GlcGlc1Ceramide1Ceramide

Isoglobo (GbiOSe4) GalNAc3Gal3GalGal44GlcGlc1Ceramide1Ceramide

Ganglio (GgOSe4) Gal3GalNAc4GalGal44GlcGlc1Ceramide1Ceramide

Muco (MucOSe4) GalGal3GalGal44GlcGlc1Ceramide1Ceramide

Gala (GalOSe2) Gal4GalGal1Ceramide1Ceramide

Sulfatides 3-0-Sulfo-GalGal1Ceramide1Ceramide

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

Turnover and Degradation of Glycosphingolipids Internalized from plasma membrane via endocytosisPass 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 recycledHuman diseases in which specific enzymes

or SAPS are genetically deficient.

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 diseasesFew 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

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 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.

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

Examples of interactions between

glycosphingolipids and bacterial toxins

or receptors

Examples of proposed interactions between glycosphingolipids and mammalian receptors

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 dishTargetted 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.

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

Consequences of GalNAc Transferase I gene disruption

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.

Consequences of SialylTransferase II (GD3 synthase) gene disruption

Consequences of SialylTransferase I (GM3 synthase) gene disruption

Consequences of Lactosylceramide Synthase gene disruption

Consequences of Glycosylceramide Synthase gene disruption