1. Glycosaminoglycan Modulates Growth Factor Signaling (Heparan Sulfate and VEGF)
Glycobiology 2007
Cholrate is an inhibitor of activated sulfate synthesis [31-phosphadenosine 51-phophosulfate (PAPS)], an effective inhibitor of macromolecular sulfation in living cells, inhibite CS and HS sulfation in a concentration-dependent manner.
Glycobiology 2007

2. Outline I. Brief introduction of Glycosaminoglycans II
Outline I. Brief introduction of Glycosaminoglycans II. Heparan sulfate proteoglycan Distribution Structural diversity Functional diversity III. Heparan sulfate and vascular development Interaction with vascular endothelial growth factor Experimental approaches
Glycobiology 2007

3. I. Glycosaminoglycans (GAGs)
Glucoronic acid 3 4
2S, 4S
Glucoronic acid 4 4 3 4
Five classes of GAGs. For better remember, only KS contains Galactose, all other contain glucuronic acid/iduronic acid; DS, CS contain GalNAc; HA, KS, HS contain GlcNAc. HA, DS, CS: beta 1-3; KS, HS: beta 1-4 linkage.
HA has no sulfate; DS: 2S, 4S; CS:4S, 6S.
4S, 6S
GAGs consist of repeating disaccharide units. With the exception of hyaluronan, all other GAGs contain sulfate. Heparan sulfate is similar to heparin except that it contains fewer sulfate groups.
Glycobiology 2007

4. II. Proteoglycan (PG)
Format: Except hyaluronan, all other GAGs covalently link to core proteins to form proteoglycan. e.g. heparan sulfate pretoeglycan (HSPG)
Location: ubiquitous, on cell surface or in the extracellular matrix NS 3S 2S 6S
Heparan sulfate is a long linear sugar chain ( disaccharide units) that is highly negative in charge
It is attached to core proteins on the cell surface and in matrix outside of cells.
Syndecan: cell-membrane spanning core protein, signaling peptide, indicating has own function
Glypican: GPI-anchor linked to cell surface, most study indicating as a coreceptor function. = G l c N A X y
= IdoA
Gal
Glycobiology 2007

5. II. Heparan Sulfate Structure
Disaccharide unit
Linkage region NS 6S 3S 2S
Core
protein
Antithrombin
Binding site
6-O-sulfate O S O -
OSO3- O S O - 3 6 3 O O O O O C O O - 5 C O O - - O O H O H O O S O 4
NO:HS is a linear anionic polysaccharide chains, composed of repeating units of alternating glucosamine linked to a uronic acid. During HS biosynthesis, these disaccharide units undergo modification chiefly through replacement of GlcNAc by sulphate groups, addition of o-sulphate groups, and C-5 epimerization of GlcA to IduA. By virtue of these differential modification possibilities, an HS dsiaccharide can exist in 48 different forms and is thus potentially the most information-dense structure known. Due to the anionic nature of the HS chains, imparted by the sulfation, HSs at the cell surface or in the ECM readilty inteact with diverse ligands, such as VEGF. PDGF is one such HS-binding growth factor. O O H 1 O H O 3 O 3 2 O
NAc O H N H S O - O S O - N H S O - 3 3 3
3-O-sulfate
2-O-sulfate
N-sulfate = G l c N A X y
= IdoA
Gal
Glycobiology 2007

6. II. Specific Ligand Binding Sites Fibroblast growth factor-2
FGF binding site: Biochemical study defined that the binding of HS to bFGF required a minimal length of 5 sugar residues (pentasaccharide) which also require to contain a N- and 2-O-sulfated disaccharide.
Antithrombin site: Biochemical study defined that the binding of HS to AT required a minimal length of 5 sugar residues (pentasaccharide) which also require to contain a all the sulfate groups lighted in Yellow, especially the the 3-O-sulfate group, which is rare.
Antithrombin
Glycobiology 2007

7. II. Heparan sulfate biosynthesis= G l c N A a X y n t i h r o m b F -
1/FGF2 S 2 6 3 I d P S N 5 - 1 d o m a i n A /
Copolymerase
EXT1
/EXT2
Chain
Polymerization
GlcNAcN-deacetylase /
N-sulfotransferases (Ndst)
GlcA C5 epimerase
2-O-sulfotransferase
Chain
Modification
6-O-sulfotransferases
3-O-sulfotransferases
Glycobiology 2007

8. II. Heparan sulfate Structure: Tremendous Diversity
Tremendous structural diversity
1. All the biosynthetic reactions are incomplete
2. Non-template driven, in a substrate-dependent manner
3. In a cluster manner, creating modified region separated by unmodified regions
4. Modifications include epimerization, and N-, 2, 3, 6-O-sulfation, leading a dissacharide can exist in 48 different forms
The expression profile of Heparan sulfate biosynthetic enzymes is regulated in a spatial and temporal manner during development, and has tissue-specificity
Glycobiology 2007

9. Heparan Sulfate Disaccharide Structures
Disaccharide structure: 3 (NS) x 2 (Epi) x 2 (2S) x 2 (6S) x 2 (3S) = 48
Glycobiology 2007

10. II. Functions of Heparan Sulfate Proteoglycan
Growth Factors and Morphogens
VEGF/VEGFR FGF/FGFR HGF Wnt
Hedgehog BMP TGF-b
Matrix Proteins
Fibronectin Laminin
Vitronectin Collagens
Fibrillin Tenascin
Heparan sulfate
proteoglycan
Angiogenesis-related FG, chemokinies and cytokines
Fibroblast growth factors (FGFs) Platelet-derived growth factor (PDGF)
Vascular endothelial growth factor (VEGF) Pleiotrophin
Placental growth factor (PlGF) Platelet factor-4 (PF-4)
Heparin-binding EGF-like growth factor Interleukin-8 (IL-8)
Hepatocyte growth factor (HGF) Macrophage inflammatory protein-1 (MIP-1)
Transforming growth factor-beta (TGF-beta) Interferon-g-inducible protein-10 (IP-10)
Interferon-gamma (IFN-gamma) HIV-Tat transactivating factor
Inflammation/Coagulation
Antithrombin EC-SOD
Heparin cofactor II L- and P-selectins
Platelet factor Chemokines, L-8
Glycobiology 2007

11. III. Heparan Sulfate Modulates Growth Factors
Human VEGF-A Isoforms
The human vascular endothelial growth factor (VEGF) gene, through alternative mRNA splicing of a single VEGF gene, produces six isoforms, which differ by the presence or absence of sequences encoded by exons 6 and 7. Sites of interaction with VEGF receptors (VEGFRs), Neuropilin 1 and HSPGs are indicated on the VEGF206 isoform, as well the enzyme cleavage sites.
Glycobiology 2007

12. III. VEGF Receptors (VEGFRs)
Growth factors and receptors of the VEGF family. The three signaling tyrosine-kinase receptors of the VEGF family (VEGFR-1, VEGFR-2, and VEGFR-3), the accessory isoform specific receptors neuropilin-1 and neuropilin-2, and VEGF binding HSPGs are displayed with their major structural features.
The heparan-sulfate proteoglycans and the neuropilins bind VEGFs, but do not seem to induce biological responses on their own in the absence of the tyrosine-kinase receptors, showing as co-receptors
Glycobiology 2007

13. II. HSPG Functions as Co-receptor
Signaling Event
Mitogenesis
VEGF
Heparan sulfate
Proteoglycan
Endothelial cells
HS structure requirements: minimal 22 saccharide, with N-, and 6-sulfate groups
in VEGF binding site
Glycobiology 2007

14. III. Regulatory Roles at Multiple Points
Proposed roles of HSPGs in VEGF/VEGFR pathway. Locally produced and secreted VEGF, (1) are captured by HS chains and accumulate at the cell surface (2). Interactions with HS support the generation of VEGF gradients (3). HS chains promote stable interactions between VEGF and VEGFR and, modulate the quality of receptor signaling (such as amplitude and kinetics of activation); 4). HSPGs may also regulate the turnover of VEGFR and participate in the internalization of VEGF/VEGFR complexes (5). Shedding of HSPG ectodomains or degradation of HS chains by heparanase may release HS-bound VEGF from the cell surface (6).
Glycobiology 2007

15. III. VEGF/VEGFR2 Signaling
Cell surface
Nitric oxide (NO) is produced by vascular endothelium and smooth muscle, cardiac muscle, and many other cell types. A major function of nitric oxide (NO) in the body is to regulate blood flow. With every heartbeat, a puff of NO is released from the endothelial cells that line our blood vessels. This NO has been assumed to diffuse passively into the surrounding smooth muscle and to react with proteins that cause blood vessels to relax, thereby increasing blood flow.
PGI2
Prostacyclin is mainly synthesized by vascular endothelium and smooth muscle. Effects include:
* inhibitor of platelet aggregation - does not influence platelet degranulation
* relaxation of smooth muscle
* reduction of systemic and pulmonary vascular resistance by direct vasodilation
* natriuresis in kidney
The expression of VEGFR2 is restricted by hemangioblast (endothelial progenitor cells) and endothelial cells, and plays an essential role in vascular development
Glycobiology 2007

16. III. Vasculature Vasculature Blood Red cells Leukocytes
Glycobiology 2007

17. III. Vascular Development: Heparan Sulfate Plays Any Role ?
FGFs
Vasculogenesis: angioblasts differentiate in situ to endothelial cells that connect and form primitive blood vessels
Angiogenesis: the growth and remodeling process of the primitive vascular network into a complex network
Glycobiology 2007

18. Experimental Approaches
Biochemical assays: determine the interaction of heparan sulfate with ligands at defined biochemical conditions (experimental and computational approaches)
Cell based assays: heparan sulfate degradation enzymes, sulfation inhibition, mutant cells (e.g. embryonic stem cell deficient in heparan sulfate biosynthetic enzymes presented in the paper today)
In vivo study: C. elegans, Fly, zebrafish, mouse (systematic or conditional knockout)
Glycobiology 2007

19. II. Heparan sulfate biosynthesis= G l c N A a X y n t i h r o m b F -
1/FGF2 S 2 6 3 I d P S N 5 - 1 d o m a i n A /
Copolymerase
EXT1
/EXT2
Chain
Polymerization
GlcNAcN-deacetylase /
N-sulfotransferases (Ndst)
GlcA C5 epimerase
2-O-sulfotransferase
Chain
Modification
6-O-sulfotransferases
3-O-sulfotransferases
Glycobiology 2007

20. Conditional Gene Knockout: Floxed Ndst1 Mouse
Wild Type
Ndst1
HindIII
BglII
Exon 2
Floxed
Ndst1
BglII
HindIII
loxP
Deleted
Ndst1
BglII
HindIII
Cre
Ndst1f /f
Restriction map of Ndst1 gene
loxP sites flank first exon
Cre deletes the loxP-flanked exon and inactivates the gene
Wang L., et al. (2005). Nature Immunology. 6(9): ; MacArthur J., et al. (2007). J Clin Invest. 117(1):153-64;
Fuster M, Wang L., et al. (2007). J Cell Biol. (in press).
Glycobiology 2007

21. Conditional Ndst1 Mutant Mice
Ndst1f/f X
TekCre+
Cre under control of mouse Tek2 promoter
Cre expression only in endothelium & leukocytes
Ndst1f/fTek2Cre–
Wild type
Ndst1f/fTek2Cre+
Mutant
Using the Cre-loxP system to conditionally knockout Ndst1 in hepatocytes
Cross mice with “floxed” alleles of Ndst1 to transgenic mice expressing Cre-recombinase under control of the rat-albumin promoter
Ndst1 gene is knocked out only in hepatocytes, resulting in less-sulfated liver heparan sulfate
For a control, I studied mice with floxed NDST1 alleles, but without Cre, so the gene will not be deleted
Wang L., et al. (2005). Nature Immunology. 6(9): ; Fuster M, Wang L., et al. (2007). J Cell Biol. (in press);
Tang N, Wang L., et al (2004). Cancer Cell 6(5)
Glycobiology 2007

22. Endothelial Ndst1 mutant mice develop Congenital Diaphragmatic Hernia (CDH)
Right
Left
Dorsal
Ventral
Wild type
Diaphragm
Heart
Sternum
Ventral
Left
Right
Dorsal
Diaphragm
Liver
Heart
Sternum
Sternum
Sternum
Hernia
Rib
Rib
Spine
Spine
Glycobiology 2007

23. Endothelial Heparan Sulfate is essential for vascular development
Mice deficient of Endothelial Ndst1 and Ndst2 are embryonic lethality due to Arrested Vascular Development
Yolk sac
Placenta
Ndst2 mutant
Placenta
Yolk sac
Ndst1/2 mutant
Liver
Liver
Embryo at E12.5
Endothelial Heparan Sulfate is essential for vascular development
Glycobiology 2007

24. NDST1/2 Mutant Endothelial Cells Had
Dramatically Reduced Growth Factor Binding
Ndst2 mutant
Ndst1/2 mutant
Basic FGF
VEGF
Ndst2 mutant
Ndst1/2 mutant
Ndst1/2 deficiency impaired VEGF binding, demonstrating that the interruption of VEGF pathway contributes to the arrested vascular development phenotype
Glycobiology 2007

25. Summary
GASs include hyaluronan, dermatan sulfate, chondroitin sulfate, keratan suflate and heparan sulfate
Heparan sulfate is a sulfated linear polysaccharide, forming HSPG by covalently linked to protein core
Chemical structure of heparan sulfate is highly heterogenous, possessing binding sites for numerous protein ligands involved in diverse biological processes
Heparan sulfate binding site for specific ligand is determined by sulfate pattern and epimerization of uronic acid.
Heparan sulfate regulates vascular development via interaction with growth factors, such as VEGF.
Glycobiology 2007