Essentials of Glycobiology Lecture 7 April 8, 2004 Ajit Varki Glycosphingolipids (Glycolipids)

Essentials of Glycobiology

Lecture 7

April 8, 2004

Ajit Varki

Glycosphingolipids

(Glycolipids)

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

O-LINKED O-LINKED CHAINCHAIN

HYALURONANHYALURONAN

GLYCOSAMINO-GLYCOSAMINO-GLYCANSGLYCANS HEPARAN SULFATEHEPARAN SULFATE

CHONDROITINCHONDROITIN SULFATESULFATE

Sialic AcidsSialic Acids

GLYCOSPHINGOLIPIDGLYCOSPHINGOLIPID

O-LINKED GlcNAcO-LINKED GlcNAc

N-LINKED CHAINSN-LINKED CHAINS

Lecture Outline

• 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

Minimal Defining Structure of a Glycosphingolipid

Glycan-O-Ceramide

1'O-CH 2 -CH-CH=CH-(CH 2 )12 -CH 3

NH OH

O=CH-(CH 2 )x-CH 3

Hexose β1-

Ceramide (Cer)

Sphingosine

Fatty Acyl group

Glycan

*

*Glucose (All animals) Galactose (?Vertebrates only) Mannose (Invertebrates) Inositol-P (fungi)

Find the Mislocated Double Bond!

Serine + Palmitoyl-CoA

D- erythro -SphinganineHO-CH 2 -CH-CH-CH-(CH 2 )12 -CH 3

NH 2 OH

HO-CH 2 -CH-CH=CH-(CH 2 )12 -CH 3

NH OH

O=CH-(CH 2 )x-CH 3

Ceramide (Cer)

UDP-Glc Ceramide Glucosyltransferase

Glucosyl-Ceramide

Pyridoxal phosphate Serine palmitoyltransferase

NADPH 3-Dehydrosphinganine reductase

Fatty Acyl-CoA Sphinganine N-acyltransferase

Dihydroceramide desaturase

1'O-CH 2 -CH-CH=CH-(CH 2 )12 -CH 3

NH OH

O=CH-(CH 2 )x-CH 3

Glucose β1-

Sphingosine

Fatty Acyl group

Biosynthesis of Ceramide and

Glucosylceramide

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:

Neu5Ac3Galβ3GalNAcβ4Galβ4Glcβ1Cer

= 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 to be presented elsewhere

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

SECRETORYGRANULE LYSOSOMEENDOSOME

GOLGIAPPARATUS

ROUGH ER

N-GlcNAc LINKED

O-GalNAc LINKED

Glc-Cer LINKED

O-Xyl LINKED

GPI- LINKED

*

**

** ******

?

**

********

**

*

**

G

G

G

G

G

G**

G

G******

*

*

Stepwise elongation of

Glucosylceramidegenerates unique Core structures

β3

Isoglobo

*α3

β3

Muco

k

l

m

Neo-lacto

Lacto

UDP-

ß4

α3

β4

β3

β4

β3

α4

β3

Globo

α8 Ganglio(b)

Ganglio(a)

*

*

β4*

= ceramide

b

c

d

e

g

e

f

h

i

j

n

GlcNAc

Gal

GalNac

Residues that

can have

alternate

outer chains

*

OUTER CHAINS

MODIFICATIONS

Major Classes of Glycosphingolipids

Series Designation Core Structure

Lacto (LcOSe4) Galβ3GlcNAcβ3GalGalββ44GlcGlcββ1Ceramide1Ceramide

Lactoneo (LcnOSe4) Galβ4GlcNAcβ3GalGalββ44GlcGlcββ1Ceramide1Ceramide

Globo (GbOSe4) GalNAcβ3Gal4GalGalββ44GlcGlcββ1Ceramide1Ceramide

Isoglobo (GbiOSe4) GalNAcβ3Gal3GalGalββ44GlcGlcββ1Ceramide1Ceramide

Ganglio (GgOSe4) Galβ3GalNAcβ4GalGalββ44GlcGlcββ1Ceramide1Ceramide

Muco (MucOSe4) GalβGalβ3GalGalββ44GlcGlcββ1Ceramide1Ceramide

Gala (GalOSe2) Gal4GalGalββ1Ceramide1Ceramide

Sulfatides 3-0-Sulfo-GalGalββ1Ceramide1Ceramide

Different Core structures generate unique shapes and are expressed in a cell-type specific manner

Examples of outer chains and modifications to

Glycosphingolipid Cores

3

Isoglobo

4

α2

ß(3)4

α3

α6

α3

α3

α3(4)

α3(4)

β4

v

v

v

u

z

y

*

α3

α3 or

α3(4)

β6

α3 or

α3

α3(4)q,s t,u

α3

β3

β3 *

α3

α8

Ac

Ac

*

α8

t

t

w

wr

t

v

β3 3S

β4β4

*

*

v

Neo-lacto

Lacto

β4

β3

Globo

α8 Ganglio(b)

Ganglio(a)

β3

*

β3

*

β4*

Muco

β3

residues that can have alternate outer chains

GlcNAc Gal GalNac*

Much similarity to outer chains

of N- and O-glycans

Pathways for Ganglio-series Glycosphingolipid biosynthesis

β4 α3 α8 α8GM3 GD3 GT3

GM2 GD2 GT2

β4β4 β4β4

GM1a GD1b GT1c

β3 β3 β3 β3

GD1a GT1b GQ1c

GA2

GA1

α3 α3α3 α3

GA1

GT1a GQ1b GP1cGD1c

α8 α8 α8 α8

A series B series C seriesO series

GalNAc Transferase

Sialyl-Transferase II

Sialyl-Transferase I

Sialyl-Transferase III

Sialyl-Transferase V

Gal Transferase II

Sialyl-Transferase IV

OAc-G D3 OAc-G T3

OAc-G D3

OAc-G Q1b

OAc-G T1b

OAc-G Q1b(?)

Gm1b

Metabolic relationships in the biosynthesis and turnover of Sphingolipids

Gal-Ceramide

CeramideGlc-Ceramide

Sphingosine

Ceramide-1-phosphate

Sphingosine-1-phosphate

Sphingomyelin

Glycosphingolipids

Di-methyl-sphingosine

Tri-methyl-sphingosine

Lysogalactosphingolipids

Sphingo-myelinase

PDMPNB-DJG

Fumonisin B1

de novo synthesis

Fumonisin B1

D-MAPP

Plasmalolysogalactosphingolipids

PC

DAG

Sulfatides

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 (“storage disorders”

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..

Biological Roles of Glycosphingolipids

*

ENDOGENOUS LECTIN

SELF

=OLIGOSACCHARIDE

(*Pathogen, Toxin or

Symbiont)

EXOGENOUS LECTIN

EndogenousRecognition

(Self)

Structural/Physical

ExogenousRecognition(Non-self)

SELF


• 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.


• 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

Binding Proteins Ganglioside(s)

Bacterial Toxins

V. Cholerae Toxin

E.Coli heat labile toxin

GM1

C.Tetani (Tetanus)

Toxin

GD1b/GT1b

C. Botulinum A,E

toxins

GD1b/GT1b/GD1a

C. Botulinum B,C,F

toxins

GT1b/GQ1b

Clostridium δ Toxin G 2M

.V Parahemolyticus toxin

G 1T b

Staphylococcus

Toxin

II 5Neu AcnLc 4

Bacterial Adhesins

.E Coli -S adhesin G M

.N Gonorrhoeaeadhesin

II 5Neu AcnLc 4

Helicobacter pyloriadhesins

G M , sulfatides

Various Gut anerobes -Gal1-4 -Gal

Sialic AcidsSialic Acids

O-LINKED GlcNAcO-LINKED GlcNAcOSer

GLYCOPHOSPHO-GLYCOPHOSPHO-LIPIDLIPID

ANCHORANCHOR

OSer/Thr

NAsn

Ser-O-

N-LINKED CHAINSN-LINKED CHAINS

O-LINKED O-LINKED CHAINCHAIN

OUTSIDE

NAsn

S S S

-O-SerS SSS S

GLYCOSAMINO-GLYCOSAMINO-GLYCANSGLYCANS

EtnP

INOSITOL

P

NH

P

NS NS

Ac

S

2

P

GlycoproteinGlycoprotein

ProteoglycanProteoglycan

HYALURONANHYALURONAN

HEPARAN SULFATEHEPARAN SULFATE

CHONDROITINCHONDROITIN SULFATESULFATE

B19 Parvovirus and Uropathogenic E.coli Binding

INSIDE

GLYCOSPHINGOLIPIDGLYCOSPHINGOLIPID

The P Blood Group System

Examples of proposed interactions between glycosphingolipids and mammalian receptors

Binding Proteins Ganglioside(s) Proposed function(s)

Epidermal growth

factor (EGF) Receptor

GM3 Diminishes tyrosine phosphophorylation

upon receptor activation, possibly by

inhibiting dimerization

De-NAc-GM3 Activates tyrosine phosphophorylation

upon receptor activation - mechanism

unknown

Insulin receptor SPG Diminishes tyrosine phosphophorylation

upon receptor activation, mechanism

unknown

Platelet-derived

growth factor (PDGF)

receptor

GM1 Diminishes tyrosine phosphophorylation

upon receptor activation, decreased cell

growth

Vitronectin Receptor GD3, GD2 Alters adhesive action of the receptor,

mechanism unknown

TSH Receptor GD1a lactone Physical association, significance

unknown

NGF Receptor (?)

Other neuronal

kinase?

GQ1b Induces/facilitates neurite outgrowth in

certain neuronal cells. Mechanism

unknown

Natural and induced Genetic Disorders in Glycosphingolipid biosynthesis

• 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

Consequences of GalNAc Transferase I gene disruption


GM2 GD2 GT2

β4β4 β4β4

GM1a GD1b GT1c

β3 β3 β3 β3

GD1a GT1b GQ1c

GA2

GA1

α3 α3α3 α3

GA1

GT1a GQ1b GP1cGD1c

α8 α8 α8 α8


GalNAc Transferase





Gal Transferase II


OAc-G D3 OAc-G T3

OAc-G D3

OAc-G Q1b

OAc-G T1b

OAc-G Q1b(?)

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


GM2 GD2 GT2

β4β4 β4β4

GM1a GD1b GT1c

β3 β3 β3 β3

GD1a GT1b GQ1c

GA2

GA1

α3 α3α3 α3

GA1

GT1a GQ1b GP1cGD1c

α8 α8 α8 α8


GalNAc Transferase





Gal Transferase II


OAc-G D3 OAc-G T3

OAc-G D3

OAc-G Q1b

OAc-G T1b

OAc-G Q1b(?)

Viable, fertile, normal life span, under further

investigation

Consequences of SialylTransferase I (GM3 synthase) gene disruption


GM2 GD2 GT2

β4β4 β4β4

GM1a GD1b GT1c

β3 β3 β3 β3

GD1a GT1b GQ1c

GA2

GA1

α3 α3α3 α3

GA1

GT1a GQ1b GP1cGD1c

α8 α8 α8 α8


GalNAc Transferase





Gal Transferase II


OAc-G D3 OAc-G T3

OAc-G D3

OAc-G Q1b

OAc-G T1b

OAc-G Q1b(?)

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


GM2 GD2 GT2

β4β4 β4β4

GM1a GD1b GT1c

β3 β3 β3 β3

GD1a GT1b GQ1c

GA2

GA1

α3 α3α3 α3

GA1

GT1a GQ1b GP1cGD1c

α8 α8 α8 α8


GalNAc Transferase





Gal Transferase II


OAc-G D3 OAc-G T3

OAc-G D3

OAc-G Q1b

OAc-G T1b

OAc-G Q1b(?)

Sudden death phenotype Extremely susceptible to induction of lethal seizures by loud sounds

Further characterization in progress

Consequences of Lactosylceramide Synthase gene disruption


GM2 GD2 GT2

β4β4 β4β4

GM1a GD1b GT1c

β3 β3 β3 β3

GD1a GT1b GQ1c

GA2

GA1

α3 α3α3 α3

GA1

GT1a GQ1b GP1cGD1c

α8 α8 α8 α8


GalNAc Transferase





Gal Transferase II


OAc-G D3 OAc-G T3

OAc-G D3

OAc-G Q1b

OAc-G T1b

OAc-G Q1b(?)

Result Pending

Consequences of Glycosylceramide Synthase gene disruption


GM2 GD2 GT2

β4β4 β4β4

GM1a GD1b GT1c

β3 β3 β3 β3

GD1a GT1b GQ1c

GA2

GA1

α3 α3α3 α3

GA1

GT1a GQ1b GP1cGD1c

α8 α8 α8 α8


GalNAc Transferase





Gal Transferase II


OAc-G D3 OAc-G T3

OAc-G D3

OAc-G Q1b

OAc-G T1b

OAc-G Q1b(?)

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 defects

in Ganglioside Biosynthesis so rare?

GalCer and Ganglio-series gangliosides well studied.

All other GlcCer derived glycolipids less well studied.

Documents

Essentials of Glycobiology Lecture 7 April 8, 2004 Ajit Varki Glycosphingolipids (Glycolipids)