Synthesis and Structure of Glycolipids

1/16/07

Review of Projections

HO

CHO

H

HHO

OHH

OHH

CH2OH

HO

O

H

H

HO

OH

OH

HH

H

OH

HO

OH

H

OH

OH

HH

OH

CH2OH

H

1

2

3

4

5

6

1

23

4

5

6

12

3

4

5

6

Fischer Hayworth Stereochemical

1. What monosaccharide is this?

2. Which chiral center defines the stereochemistry?

3. How are axial and equitorial positions related to up/down in Haworth projections?

Fischer Haworth Stereochemical

1. Where is the aglycone?

2. Where is the “reducing” end?

3. How many "-glycosidic linkages?

4. How many !-glycosidic linkages?

5. How many 3-substituted residues?

6. How many 6-substituted residues?

1. How many non-reducing termini?

2. How many glycosidic bonds?

3. Other than the NANA (N-acetylneuraminic acid, a type of sialic acid) residues,how many are !-glycosides?

About 20 years ago, bovine brain GT1b retailed for $300/mg (Matreya Lipids). A graduatestudent purified 75 mg during a single 3-4 month lab rotation (street value = $22,500).

GlycoEconomics lesson #1.

From 2007 Matreya Lipids Catalog:

Bovine Brain GQ1b retailed for $500/10 µg at that time. Same purification yielded

approximately 10 mg pure GQ1b (street value = $500,000).

Graduate student stipend was approximately $12,000/year (or $4,000/rotation). Studentvalue multiplier = 130-fold. Tell your PI how valuable you can be!!!

!Large O-linked

Glycosaminoglycans and poly-

lactosamine structures

!Glycoprotein N-linked and O-

linked oligosaccharides

!Glycolipid oligosaccharides

The "Primary" Sialic Acids

H-C-OH

H-C-OH

H-C-OH

C OH-C-C-N

H

H

C C

C C

H

OH

H

OH

1

2

4 3

6

5

C-O

9

8

7

O

H O H

H

H

-C C

H-C-OH

H-C-OH

H-C-OH

C OHO

H

H

H

C C

H

OH

H

OH

1

2

4 3

6

5

C-O

9

8

7

O

-

!-D-KDN!-D-Neu5Ac

Thought to be the metabolic precursors of all other sialic acids

N-acetyl-neuraminic acid (2-keto-5-acetamido-3,5-dideoxy-

D-glycero-D-galacto-nonulosonic acid)

“Neu5Ac” “NANA”, “NeuAc”

KDN (2-keto-3-deoxy-D-glycero-

D-galacto-nonulosonic acid)

After Varki, A

Phylogenetic

relationships

of enzymes

involved

in the

metabolism

of sialic

acids

HOMOLOGYHOMOLOGY

Neu5Ac/bacteria

GlcNAc-6-phosphate

ManNAc

Neu5Ac

CMP-Neu5Ac

ManNAc-6 -phosphate

Pi

PEP

Pi

CTP

PPi

PhosphatasePhosphatase

GlcNAcGlcNAc-6-phosphate-6-phosphate2-2-epimeraseepimerase

Neu5AcNeu5Acsynthetasesynthetase

CMP-Neu5AcCMP-Neu5Acsynthetasesynthetase

Neu5Ac/Vertebrates

UDP-GlcNAc

ManNAc

Neu5Ac-9-phosphate

Neu5Ac

CMP-Neu5Ac

ATP

ADP

UDP

PEP

Pi

Pi

CTP

PPi

ManNAc-6 -phosphate

ManNAcManNAc kinase kinase

UDP-UDP-GlcNAcGlcNAc2-2-epimeraseepimerase

Neu5Ac-9-Neu5Ac-9-phosphatephosphatesynthetasesynthetase

CMP-Neu5AcCMP-Neu5Acsynthetasesynthetase

Neu5Ac-9-phosphateNeu5Ac-9-phosphatephosphatasephosphatase

Angata and VarkiChemical Reviews

102,439-469, 2002.

After Varki, A

THE SIALIC ACIDS

R2

R2

R5

R5N-acetyl

Hydrogen or:!-linkage to: -Gal (-3 -4 -6) or -GalNAc (-6) or -GlcNAc(-4 -6) or -Sialic Acid (-8 -9) or"-linkage to CMP orAbsent in: - 2,3dehydro or - 2,7anhydro (double-bond when R2 absent)

R5

R5

R5

N-glycolyl

Hydroxyl

Amino

*

-at physio-logical pH,ionized or lactonized

R111

22

33

44

55

66

77

88

99

*

CARBON OXYGEN HYDROGEN

R1

= hydrogen or:

R8

R9

R7

R4

R8R9 R7 R4

Acetyl

R8R9 R7 R4

Phosphate R9

Methyl

R8

Sulfate R8

LactylR9

After Varki, A

Two Major Kinds of Sialic Acids

in Mammalian Cells

N-ACETYLNEURAMINIC ACID

(Neu5Ac)88

77

1122

33

55

66

99

LINKAGE TOUNDERLYING

SUGAR CHAIN

44

N-GLYCOLYLNEURAMINIC ACID

(Neu5Gc)

8877

11

44

22

33

55

66

99

LINKAGE TOUNDERLYING

SUGAR CHAIN

After Varki, A

!Large O-linked

Glycosaminoglycans and poly-

lactosamine structures

!Glycoprotein N-linked and O-

linked oligosaccharides

!Glycolipid oligosaccharides

Glycan synthesis

in a cellular context

Overview

From ER

through

Trans-Golgi and

points

inbetween

On and into the ER

Dolichol-P-X

Glycosyl phosphatidylinositol (GPI)

Glycosphingolipids (GSL)

Schenk, B. et al. Glycobiology 2001 11:61R-70R; doi:10.1093/glycob/11.5.61R

Topological model for the enzymatic reactions leading to Dol-P biosynthesis denovo on the cytoplasmic face of the ER

Biosynthesis of N-Glycans:

Production of GlcNAc-P-P-Dolichol

Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79

GlcNAcMan

GalSia

Fuc

Glc

Dolichol

TunicamycinBlocks - notvery specific!

Schenk, B. et al. Glycobiology 2001 11:61R-70R; doi:10.1093/glycob/11.5.61R

Topological model for lipid intermediate synthesis, translocation and the role forDol-P-P/Dol-P phosphatases in the recycling of Dol-P-P/Dol-P in the ER

ER glycolipid synthesis

Dolichol-P-X

Glycosyl phosphatidylinositol (GPI)

Basic Glycosylphosphatidylinositol (GPI) Anchor

Phospholipid

After Hart, G

Examples of GPI-Anchored Proteins

Cell surface hydrolases

alkaline phosphatase

acetylcholinesterase

5! nucleotidase

Adhesion molecules

neural cell adhesion molecule

heparan sulfate proteoglycan

Others

decay accelerating factor

scrapie prion protein

folate receptor

Protozoal antigens

trypanosome VSG

leishmanial protease

plasmodium antigens

Mammalian antigens

Thy-1

carcinoembryonic antigen

After Hart, G

Structure of the Basic GPI Anchor

E t nP

INOSITOL

P

= Mannose (Man)

= Glucosamine

Etn = Ethanolamine

P = Phosphate

N H2

N H2Pig-A

PNH Defect

GPI-Linked Protein

Cell SurfaceMembrane

After Hart, G

Structural AnalysisStructural Analysis

of the GPI Anchorof the GPI Anchor

Enzymatic and chemical

cleavage sites

are useful in identifying

GPI anchored

membrane proteins

After Hart, G

After Freeze, H

Biosynthesis

of GPI

anchors

• The first step in biosynthesis of the GPIanchor requires at least four genes

• One of them, PIG-A is an X-linked gene

Mutation in PNH

MUTATIONS INDOL-P-MAN SYNAND USE

After Freeze, H

Paroxysmal Nocturnal Hemoglobinuria

"A hematopoietic stem cell disorder characterized by

intravascular hemolytic anemia. 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.

Examples of C-Terminal Sequences Signaling

the Addition of GPI-Anchors

Protein GPI-Signal Sequence

Acetylcholinesterase (Torpedo) NQFLPKLLNATAC DGELSSSGTSSSKGIIFYVLFSILYLIFY

Alkaline Phosphatase (placenta) TACDLAPPAGTTD AAHPGRSVVPALLPLLAGTLLLLETATAP

Decay Accelerating Factor HETTPNKGSGTTS GTTRLLSGHTCFTLTGLLGTLVTMGLLT

PARP (T. Brucei) EPEPEPEPEPEPG AATLKSVALPFAIAAAALVAAF

Prion Protein (hamster) QKESQAYYDGRRS SAVLFSSPPVILLISFLIFLMVG

Thy-1 (rat) KTINVIRDKLVKC GGISLLVQNTSWLLLLLLSLSFLQATDFISI

Variant Surface Glycoprotein (T. Brucei) ESNCKWENNACKD SSILVTKKFALTVVSAAFVALLF

Bold AA is site of GPI attachment Sequence to right iscleaved by the transpeptidase upon Anchor addition

After Hart, G

5-10 hydrophilic, 15-20 hydrophobic

ER glycolipid synthesis

Dolichol-P-X

Glycosyl phosphatidylinositol (GPI)

Glycosphingolipids (GSL)

Minimal Defining Structure of a

Glycosphingolipid

Glycan-O-Ceramide

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

NH OH

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

Hexose !1-

Ceramide (Cer)

Sphingosine

Fatty Acyl group

Glycan *

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

After Varki, A

Biosynthesis of Ceramide and Glucosylceramide

Golgi processing of GlycosphingolipidsMajor Classes of Glycosphingolipids

Series Designation Core Structure

Lacto (LcOSe4) Gal"3GlcNAc"3GalGal""44GlcGlc""1Ceramide1Ceramide

Lactoneo (LcnOSe4) Gal"4GlcNAc"3GalGal""44GlcGlc""1Ceramide1Ceramide

Globo (GbOSe4) GalNAc"3Gal!4GalGal""44GlcGlc""1Ceramide1Ceramide

Isoglobo (GbiOSe4) GalNAc"3Gal!3GalGal""44GlcGlc""1Ceramide1Ceramide

Ganglio (GgOSe4) Gal"3GalNAc"4GalGal""44GlcGlc""1Ceramide1Ceramide

Muco (MucOSe4) Gal"3Gal"3GalGal""44GlcGlc""1Ceramide1Ceramide

Gala (GalOSe2) Gal!4GalGal""1Ceramide1Ceramide

Sulfatides 3-0-Sulfo-GalGal""1Ceramide1Ceramide

Different Core structures generate unique shapesand 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:

Neu5Ac!3Gal"3GalNAc"4Gal"4Glc"1Cer

= GM1b in the Svennerholm nomenclature

OR

II4Neu5Ac-GgOSe4-Cer

in the official IUPAC-IUB designation

After Varki, A

Pathways for Ganglio-series Glycosphingolipid biosynthesis

!4 "3 "8 "8

GM 3 GD 3 GT 3

GM 2 GD 2 GT 2

!4!4 !4!4

GM 1 a GD1b GT1c

!3 !3 !3 !3

GD1a GT1b GQ1c

GA2

GA1

"3 "3"3 "3

GA1

GT1a GQ1b GP 1 cGD1c

"8 "8 "8 "8

A series B series C seriesO series

GalNAc

T rans fe rase

S ia ly l -

Transferase IIS ia ly l -

Transferase I

S ia ly l -

Transferase III

S ia ly l -

Transferase V

Gal

Transferase II

S ia ly l -

Transferase IV

OAc-G D 3 OAc-G T 3

OAc-G D 3

OAc-G Q1b

OAc-G T1b

OAc-G Q 1 b ( ? )

Gm1b

After Varki, A

Turnover and Degradation of Glycosphingolipids

• Internalized from plasma membrane via endocytosis

• Pass through endosomes (some remodelling possible?)

• Terminal degradation in lysosomes - stepwise reactionsby specific enzymes.

• Some final steps involve cleavages close to the cellmembrane, and require facilitation by specificsphingolipid activator proteins (SAPs).

• Individual components, available for re-utilization invarious pathways.

• At least some of glucosylceramide may remain intact andbe recycled

• Human diseases in which specific enzymes or SAPs aregenetically deficient (storage diseases)

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 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

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

PDMP

NB-DJGFumonisin B1

de novo synthesis

Fumonisin B1

D-MAPP

Plasmalolysogalactosphingolipids

PC

DAG

Sulfatides

After Varki, A

Ganglioside synthetic enzyme KOs

–GlcCer synthase (GlcT): Early embryonic lethal,

differentiation and germ layer formation okay

–LacCer synthase (GalT): ??

–GM3 synthase (SiaTI): Increased sensitivity to insulin

(skeletal muscle receptor phosphorylation)

–GM2 synthase (GalNAcT): Male sterility, demyelinating

disorder

–GD2 synthase (SiaTII): Ok

–GM2 synthase/GD2 synthase double KO: sudden death,

auditory seizures, demyelination

General Principles

–The sialic acids are a family of structurally related

compounds that can bear a variety of post-synthetic

modifications

–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

–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

are also utilized in signaling pathways