Essentials of glycobiology lecture 7 april 12 2002 ajit varki
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Essentials of Glycobiology Lecture 7 April 12, 2002 Ajit Varki. Structure, biosynthesis and general biology of Glycosphingolipids. CHONDROITIN SULFATE. HYALURONAN. GLYCOSAMINO- GLYCANS. HEPARAN SULFATE. N-LINKED CHAINS. O-LINKED CHAIN. GLYCOPHOSPHO- LIPID ANCHOR.

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Essentials of Glycobiology Lecture 7 April 12, 2002 Ajit Varki

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Essentials of glycobiology lecture 7 april 12 2002 ajit varki

Essentials of Glycobiology Lecture 7April 12, 2002Ajit Varki

Structure, biosynthesis and

general biology of

Glycosphingolipids


Major glycan classes in animal cells

CHONDROITIN

SULFATE

HYALURONAN

GLYCOSAMINO-

GLYCANS

HEPARAN SULFATE

N-LINKED CHAINS

O-LINKED

CHAIN

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

Ac

P

Etn

S

P

N

O

N

NH

Asn

Ser/Thr

2

Asn

INOSITOL

Glycoprotein

Ac

OUTSIDE

P

Sialic Acids

INSIDE

O

Ser


Lecture overview

Historical Background

Defining Structures and Major Classes

Other Nomenclature Issues

Biosynthesis

Occurrence & Structural Variations

Isolation and purification

Trafficking, Turnover and Degradation

Lecture Overview


Lecture overview continued

Relationship to biosynthesis, turnover and signalling functions of other Sphingolipids

Antibodies against Glycosphingolipids

Biological Roles

Genetic Disorders in GSL biosynthesis

Perspectives and Future Directions

Lecture Overview (Continued)


Minimal defining structure of a glycosphingolipid

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 Missing Double Bond!


Biosynthesis of ceramide and glucosylceramide

Biosynthesis of Ceramide and Glucosylceramide

Find the Missing

Double Bond!


Nomenclature issues

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:

Neu5Ac3Gal3GalNAc4Gal4Glc1Cer = 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 presented elsewhere

Isolation and purification of Glycosphingolipids


Biosynthesis of different classes of glycans within the er golgi pathway

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


Stepwise elongation of glucosylceramidegenerates unique core structures

Stepwise elongation of Glucosylceramidegenerates unique Core structures


Major classes of glycosphingolipids

SeriesDesignationCore Structure

Lacto(LcOSe4) Gal3GlcNAc3Gal4Glc1Ceramide

Lactoneo(LcnOSe4)Gal4GlcNAc3Gal4Glc1Ceramide

Globo(GbOSe4) GalNAc3Gala4Gal4Glc1Ceramide

Isoglobo(GbiOSe4)GalNAc3Gal3Gal4Glc1Ceramide

Ganglio(GgOSe4)Gal3GalNAc4Gal4Glc1Ceramide

Muco (MucOSe4) GalGal3Gal4Glc1Ceramide

Gala (GalOSe2) Gal4Gal1Ceramide

Sulfatides 3-0-Sulfo-Gal1Ceramide

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

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

Pathways for ganglio-series Glycosphingolipid biosynthesis

Gm1b


Metabolic relationships in the biosynthesis and turnover of sphingolipids

Metabolic relationships in the biosynthesis and turnover of Sphingolipids


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.

Turnover and Degradation of Glycosphingolipids


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..

Monoclonal antibodies (Mabs) against Glycosphingolipids


Biological roles of glycosphingolipids

Biological Roles of Glycosphingolipids


Biological roles of glycosphingolipids1

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


Biological roles of glycosphingolipids2

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

Examples of interactions between glycosphingolipids and bacterial toxins or receptors


Examples of proposed interactions between glycosphingolipids and mammalian 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 dish

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

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


Natural and induced genetic disorders in glycosphingolipid biosynthesis1

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

Natural and induced Genetic Disorders in Glycosphingolipid biosynthesis


Consequences of galnac transferase i gene disruption

Consequences of GalNAc Transferase I gene disruption


Natural and induced genetic disorders in glycosphingolipid biosynthesis2

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


Consequences of sialyltransferase ii gd3 synthase gene disruption

Consequences of SialylTransferase II (GD3 synthase) gene disruption


Consequences of sialyltransferase i gm3 synthase gene disruption

Consequences of SialylTransferase I (GM3 synthase) gene disruption


Consequences of lactosylceramide synthase gene disruption

Consequences of Lactosylceramide Synthase gene disruption


Consequences of glycosylceramide synthase gene disruption

Consequences of Glycosylceramide Synthase gene disruption


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