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

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

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

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 (20-100 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

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

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

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

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

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

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 4 Chemokines, L-8 Glycobiology 2007

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

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

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

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

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

III. Vasculature Vasculature Blood Red cells Leukocytes Glycobiology 2007

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

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

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

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): 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). Glycobiology 2007

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): 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. Glycobiology 2007

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

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

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

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