Glyco Rebecca Holley

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Glycosaminoglycan (GAG)

Back 1

long linear chain of repeating disaccharide units joined by glycosidic bonds

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Disaccharides in GAGs =

Back 2

Amino sugar (GlcNAc/GalNAc)
+
Uronic Acid (GlcA/IdoA)

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GAGs can be divided into 4 groups according to:

Types of sugar
Types of linkages
No. & location of S group

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1) Hyaluronan (HA)
2) Herperan Sulphate (HS) & Heparin
3) Keratan Sulphate (KS)
4)Chondroitin Sulphate (CS) & Dermatan Sulphate (DS)

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Hyaluronan (HA)

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GlcA-GlcNAc

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HS/Heparin

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GlcA/IdoA-GlcNAc/NS

2S-6/3S

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KS

*no uronic acid

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GalNAc-GlcNAc

6S-6S

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CS

DS

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GlcA-GalNAc

IdoA-GalNAc

2S-4/6S

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Proteoglycan (PG)

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core protein to which 1 or more GAG chains are added

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Membrane bound PGs

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Glypicans
Syndecans

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PGs secreted to ECM

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Aggrecan
Perlecan
Decorin & Biglycan

*in pericellular domain

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all PGs expressed in distinct ____, _____ and cell _________ stage specific ______

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cell
tissue
developmental
patterns

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PG functions

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cell behaviour
tissue morphogenesis
patho-physiological phenomena

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HS biosynthesis

Step 1) Chain initiation

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@ golgi
GAGs attached to specific S in PG
target site : S - G/A - X - G
sequential addition by 4 glycosyltransferases

S-Xyl-Gal-Gal-GlcA

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Enzymes in HS/CS/DS chain initiation

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Xyl Transferase
Gal Transferase 1
Gal Transferase 2
GlcA Transferase

UDP-sugar = substrate

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HS attachment site = consecutive S-G/A _______ flanked by _________ and acidic residues

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sequences
hydrophobic

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CS/DS attachment site = ______

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default?

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HA synthesis

Step 2/3) Polymerisation and ________

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Modification

>20 enzymes involved
Each modification creates substrate for further modification

modification is incomplete

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EXT1/EXT2

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heterodimer

have GlcA T and GlcNAc T activity

-->chain elongation

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N-deacetylase/N-sulfotransferase
(NDST)

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bifunctional enzyme -->
GlcNAc ---> GlcNS

4 isoforms (diff expression)

PAPS = S donor

creates small clusters of consecutive N-sulphated disaccharides

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GlcNS

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substrate for biosynthetic enzymes

---> sulphated domains

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GlcA C5 epimerase

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GlcA --> IdoA
alters HS conformation

1 isoform

*requires adjacent GlcNS

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2-O-sulfotransferase (2OST)

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addition of S to 2-O of IdoA (& rarely GlcA)

1 isoform

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6-O-sulfotransferase (2OST)

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adds S to 6-O of GlcNAc/GlcNS

3 isoforms

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3-O-sulphotrasferase

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adds S to 3-O GlcNAc/S
**** V.rare

7 isoforms

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6-O endosulfatase (sulf)

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removes 6-O-S from HS chains

@ cell surface or secreted into ECM

2 isoforms

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different HS biosynthetic enzyme isoforms have different ______ ______

THEREFORE HS structure depends on _______ expressed

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substrate specificities

isoforms

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HS domains

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Sulphated domains

Non-sulphated domains

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HS domain structure determined by ...

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No of IdoA and S additions

Patterning of S additions

Length of sulphated/nonsulphated domains

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HS domain structure is ______ specific AND ________ between species

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tissue
conserved

HIGHLY REGULATED

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Heparin

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Highly sulphated analogue of HS

1.8-2.4 S/diasaccaride

higher GlcNS %
higher IdoA %

only synthesised in MAST CELLS

Core protein = Serglycin

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Most HSPGs function via ________ with ______

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interactions
proteins

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HSPG:protein interactions

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via HS side chains***

via extracellular protein core
via cytoplasmic domain of core protein

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4 broad functions of HSPG:protein interactions

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1) Coagulation/Fibrinolysis
- Antithrombin III, Heparin cofactor II, thrombin etc.

2) Growth, Signalling and Inflammation
-FGF & FGFRs, HGF, VEGF, TGFb, BMP, Wnts, L & P selectins etc.

3)ECM molecules
-laminin, fibronectin, thrombospondin, type I III & IV collagen etc.

4)Lipolysis
-lipoprotein lipase, hepatic lipase, LDL etc

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HS binding motif

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conserved sequence in some HS binding proteins

in others
HS-interaction domain formed after folding

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GAGs are v.______ charged due to ______ and _ groups

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negatively
carboxyl
S

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SAS domain

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2 short S-domains seperated by N-acetylated regions

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Single HS domain binding to a single protein

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e.g ATIII

enzyme involved in blood clotting
protease inhibitor --/ THROMBIN

binds specific sequence:
GlcNAc/S(6S)-GlcA-GlcNS(3,6S)-IdoA(2S)-GlcNS(6S)

binding --> conformational change ---> increased activity ~1000

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SAS domains bind 2 distinct proteins

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e.g ATII and Thrombin

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SAS domains span 2 binding sites in a single protein

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e.g CXCL12/SDF1 (Cytokine)

HS:CXCL12 complex increase t1/2 and increases chance of receptor binding

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SAS domains bind a protein homodimer

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e.g VEGF

regulates diffusion, t1/2 and affinity for signalling Rs

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Single domain/SAS stabilises a hetero-oligomeric complex

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e.g FGF & FGFR

Herparin/HS = coreceptor

necessary for Ternary Signalling Complex

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Generally for SAS binding
S domain _______ & distance between S domains is _______

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composition
important

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Sulfation patterning is important for protein binding in proteins that ______ a ____ component e.g _-_

---> all or nothing

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require
rare
3-O

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3-O found in
___ of heparin chains
___ of HS chains

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30%
0.3%

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Proteins can recognise _____ _____ alone

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charge density

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Proteins can recognise charge density and ______ ______

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sequence specificity

e.g FGF1

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consequences of HS:protein binding

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Regulation of enzyme activity
Binding to signalling receptor (FGF)
Protection of ligand against degredation
Immobilisation of proteins at sites of production --> GRADIENTS
Provision of a reservoir of ligands for future mobilisation

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Mice as a model for PG / HS biosynthetic enzyme function

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genes v.similar in mouse & human

similar no. of isoforms

provide model for human disease

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3 major types of HSPGs

expressed at distinct times, in discrete locations and at varying amounts

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Syndecan (4)
Glypican (6)
Perlecan (1)

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Syndecans

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Intracellular + TM + (variable) extracellular domain

can be cleaved

isoforms differentially expressed - >1/cell

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Syndecan 1

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major form in epithelial

KO - viable and fertile, some defects in WOUND HEALING & Wnt SIGNALLING

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Syndecan 3

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major form in neuronal cell types

KO - viable and fertile, decreased food intake/body weight due to SIGNALLING IN HYPOTHALAMUS

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Syndecan 4

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widely expressed at low levels

KO- viable fertile, delayed WOUND HEALING

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Glypican function

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GPI anchored
variable globular structure
3-4 HS/core
all isoforms in most adult tissues

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Glypican 1 KO

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viable and fertile

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Glypican 2 KO

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viable and fertile

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Glypican 3 KO

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developmental overgrowth
perinatal death
cystic kidneys
abnormal lung development

* similar to Simpson-Golabi-Behmel syndrome

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Simpson-Golabi-Behmel syndrome

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pre/post-natal overgrowth
distinct facial apperance
cleft palate
syndactyly
polydactyly
cystic & dysplastic kidneys
congenital heart defects
rib & vertebral abnormalities

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Perlecan function

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ubiquitous expression in basement membranes

multiple roles in
cell growth & differentiation
tissue organisation

essential roles in
neuromuscular function
cartilage formation

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Perlecan KO

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die at E10-12 due to rupture of basement membranes around heart

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Perlecan _______ found in human diseases Dyssegmental Dysplasia & Schwartz-Jampel syndrome

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mutation

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EXT1 / EXT2 KO

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usually ubiquitous

lethal at E6.5- 8.5
fail to undergo gastrulation

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Multiple Osteochondroma (MO)

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-> exostoses - cartilage-capped benign outgrowths

higher risk of subsequent tumours

caused by heterozygous Ext1/Ext2
*HAPLOINSUFFICIENT

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Ext2 +/- mice

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-MO

HS reduced by 50%
alterations in distribution of paracrine signalling molecules

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NDST1 KO

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lethal shortly after birth

critical defects in craniofacial & brain development
abnormal lung development

HS - V.reduced N-sulphation (and therefore O-sulphation and epimerisation

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NDST2 KO

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viable and fertile

do NOT produce heparin
HS unaffected

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NDST1 & NDST2 KO

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embryos die prior to gastrulation

HS completely lacks N-sulphation and further modifications (except a few GlcNAc 6S)

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Sulphated HS

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essential during gastrulation to direct cell fate

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Epimerase KO and 2OST KO have similar phenotypes

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Die @ birth
Skeletal defects
Lack Kidneys

Epimerase KO
Lung defects
HS with no IdoA but increased N-sulphated domains

2OST KO
HS - increased level of N and 6-O sulphation

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6OST

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differential expression
different substrate specificities

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6OST1 KO

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late embryonic lethality (E15.5 - perinatal stage)

major reduction in 6S in most tissues

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6OST2 KO

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viable & fertile

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6OST1 6OST2 KO

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embryonic lethal at slightly earlier stage

HS - no 6S but compensatory increase in 2S

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3OST1

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thought to be major isoform creating ATIII binding site

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3OST1 KO

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NO pro-coagulant phenotype
redundacy?

eye lesions
male infertility
altered response to cardiac shock

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Sulf1 or Sulf2 KO

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viable and fertile

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Sulf1 & Sulf2 KO

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low survival rates
complex variable phenotypes

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Labelling HS chain

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1)Pre-isolation labelling
-H3-glucosamine

2)Label reducing end of diasaccharide chain using FLUORESCENT TAG e.g AMAC

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Purifying HS

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ion exchange chromatography

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Chain composition

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% of each type of diasaccharide in chain

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Domain organisation

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length of sulphated/non-sulphated domains

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Breakdown chain for analysis via ...

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1)Enzymes (3)
2)Chemical breakdown
-low pH nitrous acid

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Hearinase I

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cleaves GlcNS(+-6S)-*IdoA(2S)*

*essential

determine spacing between S-domains - (cleaves only S-domains)
position of 2S residues

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Heparinase II

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cleaves GlcNAc/S(+-6S)-GlcA/IdoA(+-2S)

broad activity

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Heparinase III

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cleaves GlcNAc/S-GlcA

determine size of S-domains
(cleaves only non-sulphated domains)

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pH1.5 nitrous acid

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deamination cleavage at GlcNS
--->SO4 release
GlcNS--> anydromannose

can distinguish between IdoA & GlcA

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epitope specific Abs

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allows patterns of sulphation to be fingerprinted

can be fluorescently tagged ---> flow cytometry
microscopy

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anti-heparan sulphate stub AB

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3G10

can be fluorescently tagged
-->
can sse number of HS chains/cell

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HS-protein interactions

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use fluorescently tagged proteins as probes
(e.g. ATIII, FGF)