Glycosaminoglycan Modulates Growth Factor Signaling (Heparan Sulfate and VEGF)

1. Glycosaminoglycan Modulates Growth Factor Signaling(Heparan Sulfate and VEGF) Glycobiology 2007

2. OutlineI. Brief introduction of GlycosaminoglycansII. Heparan sulfate proteoglycanDistributionStructural diversityFunctional diversityIII. Heparan sulfate and vascular developmentInteraction with vascular endothelial growth factorExperimental approaches

3. I. Glycosaminoglycans (GAGs) Glucoronic acid 3 4 2S, 4S Glucoronic acid 4 4 3 4 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.

4. 6S 6S 6S NS 2S NS 3S NS 2S 6S 6S NS 2S NS NS 2S 6S 6S NS NS NS 2S 6S 6S NS 2S NS NS 2S 6S 6S 6S NS 6S NS 2S NS NS NS 3S 2S NS NS 2S = IdoA Gal = X y l = = G l c A = G l c N A c 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

5. 6S 6S 6S 6S O NS NS 3S 2S NS NS NS 2S NS = IdoA Gal = X y l = = G l c A = G l c N A c II. Heparan Sulfate Structure Linkage region Disaccharide unit Core protein Antithrombin Binding site 6-O-sulfate - - OSO3- O S O O S O 3 3 6 O O O O O 5 - C O O - C O O - 4 O H O S O 1 O O O H O H O H 3 3 2 O O 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

6. II. Specific Ligand Binding Sites Fibroblast growth factor-2 Antithrombin

7. 5 - 1 0 P A P S N S N S N S N S N S N S N S 5 - 1 0 N S N S N S N S N S N S N S 5 - 1 0 P A P S N S 2 S N S 2 S N S 2 S N S N S N S 2 S N S 5 - 1 0 P A P S 6 S 6 S 6 S 6 S 6 S 6 S N S 2 S N S 2 S N S 2 S N S N S N S 2 S N S 5 - 1 0 P A P S 6 S 6 S 6 S 6 S 6 S 6 S N S 2 S N S 2 S N S 2 S N S N S N S 2 S N S 3 S 5 - 1 0 F G F - 1/FGF2 A n t i t h r o m b i n N S - d o m a i n N A / N S - d o m a i n N A - d o m a i n = X y l = G a l = G l c A = G l c N A c = I d o A II. Heparan sulfate biosynthesis Copolymerase EXT1 /EXT2 Chain Polymerization GlcNAcN-deacetylase / N-sulfotransferases (Ndst) GlcA C5 epimerase 2-O-sulfotransferase Chain Modification 6-O-sulfotransferases 3-O-sulfotransferases

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

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

10. II. Functions of Heparan Sulfate Proteoglycan Matrix Proteins Fibronectin Laminin Vitronectin Collagens Fibrillin Tenascin Growth Factors and Morphogens VEGF/VEGFR FGF/FGFR HGF Wnt Hedgehog BMP TGF-b Heparan sulfate proteoglycan Inflammation/Coagulation Antithrombin EC-SOD Heparin cofactor II L- and P-selectins Platelet factor 4 Chemokines, L-8

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.

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

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

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 ofVEGFR 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).

15. III. VEGF/VEGFR2 Signaling Cell surface The expression of VEGFR2 is restricted by hemangioblast (endothelial progenitor cells) and endothelial cells, and plays an essential role in vascular development

16. Vasculature Red cells Blood Leukocytes III. Vasculature

17. FGFs III. Vascular Development: Heparan Sulfate Plays Any Role ? 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

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)

19. 5 - 1 0 P A P S N S N S N S N S N S N S N S 5 - 1 0 N S N S N S N S N S N S N S 5 - 1 0 P A P S N S 2 S N S 2 S N S 2 S N S N S N S 2 S N S 5 - 1 0 P A P S 6 S 6 S 6 S 6 S 6 S 6 S N S 2 S N S 2 S N S 2 S N S N S N S 2 S N S 5 - 1 0 P A P S 6 S 6 S 6 S 6 S 6 S 6 S N S 2 S N S 2 S N S 2 S N S N S N S 2 S N S 3 S 5 - 1 0 F G F - 1/FGF2 A n t i t h r o m b i n N S - d o m a i n N A / N S - d o m a i n N A - d o m a i n = X y l = G a l = G l c A = G l c N A c = I d o A II. Heparan sulfate biosynthesis Copolymerase EXT1 /EXT2 Chain Polymerization GlcNAcN-deacetylase / N-sulfotransferases (Ndst) GlcA C5 epimerase 2-O-sulfotransferase Chain Modification 6-O-sulfotransferases 3-O-sulfotransferases

20. BglII BglII HindIII HindIII Exon 2 Wild Type Ndst1 loxP loxP Floxed Ndst1 BglII BglII HindIII HindIII Cre Deleted Ndst1 BglII HindIII HindIII Ndst1f /f Conditional Gene Knockout: Floxed Ndst1 Mouse Wang L., et al. (2005). Nature Immunology. 6(9): 902-10; MacArthur J., et al. (2007). J Clin Invest. 117(1):153-64; Fuster M, Wang L., et al. (2007). J Cell Biol. (in press).

21. Ndst1f/f X Ndst1f/fTek2Cre+ Ndst1f/fTek2Cre– Mutant Wild type Conditional Ndst1 Mutant Mice TekCre+ • Cre under control of mouse Tek2 promoter • Cre expression only in endothelium & leukocytes Wang L., et al. (2005). Nature Immunology. 6(9): 902-10; Fuster M, Wang L., et al. (2007). J Cell Biol. (in press); Tang N, Wang L., et al (2004). Cancer Cell 6(5) 485-95.

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

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

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

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.