Biomedical Glycobiology Frontier of Heparan Sulfate Proteoglycan study Dr Lianchun Wang Lwang ccrc uga edu Heparan Sulfate Proteoglycans Protein Ligands that HSPG interacts Growth Factors and Morphogens Matrix Proteins VEGF VEGFR FGF FGFR HGF Wnt Hedgehog BMP TGF Fibronectin Laminin Vitronectin Collagens Fibrillin Tenascin Heparan sulfate proteoglycan Inflammation Coagulation Antithrombin EC SOD Heparin cofactor II L and P selectins Platelet factor 4 Chemokines L 8 Functions of HSPGs Heparan sulfate biosynthesis Copolymerase EXT1 EXT2 Chain Polymerization GlcNAcN deacetylase N sulfotransferases 1 2 NS NS NS NS 5 10 PAPS NS NS NS NS NS NS 5 10 GlcA C5 epimerase NS NS NS NS 2 O sulfotransferase NS 2S NS 2S NS 2S NS NS 6S 6S 6S 6S NS 2S NS 2S NS 2S NS FGF 1 FGF2 NS domain Xyl Gal 6S NS 6S NS 2S NS 3 O sulfotransferases 1 2 3a 3b 4 6S 5 10 PAPS 6S NS 2S NS 2S NS 2S NS Chain Modification NS 2S NS 6 O sulfotransferases 1 6S 5 10 PAPS 5 10 PAPS 6S NS 6S 6S NS 2S NS 3S 5 10 Antithrombin NA NS domain GlcA NA domain GlcNAc IdoA Heparan Sulfate Deficiencies 6S 6S 6S 6S 6S NS NS 2S NS 2S NS 3S NS Wildtype Ext1 Ndst1 Ndst2 6S NS Xyl 6S 2S NS Gal 6S Ndst1 NS GlcA GlcNAc IdoA Outline I Development related defects II Blood Coagulation III Inflammation IV Tumor Biology V Lipid metabolism I Development related Defects Embryonic Stem Cells Self Renewal LIF Pluripotency LIF In vitro Embryonic Stem cell Differentiation mimics early in vivo embryonic development Gordon Keller Genes Dev 2005 19 1129 1155 FGF Wnt and BMP signaling regulate stem cell fate Alter Heparan Sulfate Structure in ES Cells LoxP Floxed mouse loxP EXT1 Cre loxP Cre Ext1 Ext1 loxP Cre Ext1 Ext1 Deficiency of Ext1 leads to defects of early embryonic development and ES cell self renewal Vascular Development FGFs Brachyury Vasculogenesis a process whereby angioblasts differentiate in situ to endothelial cells that connect and form primitive blood vessels Angiogenesis the growth and remodeling process of the primitive network into a complex network VEGF All these vascular development essential growth factors are heparin binding proteins PDGF TGF Mice Deficient in Endothelial cell EC Ndst1 and or Ndst2 Mice Deficient in Smooth Muscle Cell SMC Ndst1 and Ndst2 Chronic intestinal pseudo obstruction II HSPG and Blood Coagulation Heparin has been widely used as anticoagulant clinically for over 70 years Heparin does not present in vasculature instead the similarly structured heparan sulfate abundantly exists Vascular heparan sulfate contains 0 3 heparin like structure can bind antithrombin III and exhibits anticoagulant activity in vitro and ex vivo suggesting vascular heparan sulfate may function as a natural anticoagulant This hypothesis has been widely accepted but has not been proved Both in vitro and ex vivo conflicting observations were reported also Especially recent genetic study Hs3st1 knockout mice does not support Heparan sulfate anticoagulant pathway HajMohammadi S et al J Clin Invest 2003 111 7 989 99 Weitz JI J Clin Invest 2003 111 7 952 4 News and Views Mice deficient in EC Ndst1 is thrombophilic Tail bleeding 100 a 80 60 40 20 0 f fTekCre f fTekCre Ndst1 Ndst1 Ndst1 Ndst1 Mice deficient in EC Ndst1 is thrombophilic Carotid injury 12 Occlusion Time min a P 0 054 n 14 18 n im e m iT69 n io s u lc c Occlusion O Time min 36 912 0 100 b Unstable Stable 80 P 0 001 s n 14 18 60 u b m o rth 40 le b a tS Stable thrombus Stable thrombus 20 0 03 Ndst1f fTekCre Ndst1f fTekCre Ndst1f fTekCre Ndst1f fTekCre Figure 1 Carotid artery thrombosis induced by FeCl3 treatment a Occlusion time b Stability of thrombus Heparan Sulfate modulates fibrinolysis System controls blood clot removal III HSPG and Inflammation Inflammation is a fundamental mechanism that the body reacts to infection irritation or other injury The key feature includes redness warmth swelling and pain The underlying cellular and molecular mechanisms are manifested by the trafficking of leukocyte from blood circulation into the injured tissues The leukocyte trafficking into the injured tissue is a multiple step process involving various adhesion molecules and chemokines Heparin inhibits inflammation by blocking L and P selectin mediated leukocyteendothelial cell adhesion Heparan sulfate modulates inflammatory response Leukocyte Nelson Rm et al Blood 1993 82 11 3253 8 Xie X et al J Biol Chem 2000 275 44 34818 25 Wang L et al J Clin Invest 2002 110 1 127 36 GPCR Heparan sulfate modulates inflammation Roles 1 Interacts with L selectin to facilitate leukocyte endothelial cell adhesion 2 Immobilize and present chemokine s facilitating leukocyte firm adhesion 3 Particitates chemokines abluminal to luminal cell surface transcytosis Wang L et al Nat Immunol 2005 6 9 902 10 Parish CR Nat Immunol 2005 6 9 861 2 News and Views IV HSPG and tumor biology Heparan sulfate has been supposed to interact with growth factors morphogens and adhesion molecules to modulate tumor biology Heparan sulfate is required for tumorigenesis Esko JD et al Science 1988 Tumor cell surface heparan sulfate can both promote and inhibit tumor growth and metastasis Based on in vitro cultured tumor cell Liu D et al Proc Natl Acad Sci USA 2002 Syndecan and Glypican modulate tumorigenesis Alexander CM et al Nat Genet 2000 Aikawa T et al J Clin Invest 2008 Heparin inhibits tumor metastasis by blocking L and P selectin mediated tumor cell adhesion Borsig L et al Proc Natl Acad Sci USA 2001 Borsig L et al Proc Natl Acad Sci USA 2002 Heparan sulfate chain modulates tumorigenesis and metastasis in vivo Tumor growth in vitro Deficiency of heparan sulfate alters tumor cell growth which can be inhibitory or enhancing Tumor angiogenesis HS modulates tumor angiogenesis Tumor growth Tumor angiogenesis Ndst1 Ndst1 Ndst1 Ndst1 Ndst1 Ndst1 Ndst1 here means endothelial cell specific Ndst1 knockout mice Fuster M et al J Cell Biol 2007 177 3 539 49 V HSPG and Lipid metabolism Lipoproteins transport cholesterol triglycerides and phospholipids through the circulation Phospholipids Cholesterol Neutral core containing triglycerides and cholesteryl esters Apolipoproteins Horton et al Principles of Biochemistry 2002 16 5 Major Plasma Lipoproteins Chylomicrons VLDL LDL From intestine From liver Cholesterol rich Triglyceride rich Triglyceride rich ApoB100 ApoB48 ApoB100 Bad cholesterol ApoE ApoE HDL Protein rich Phospholipid rich ApoAI ApoAII Good cholesterol Scale bar 0 1 m Lehninger et al Principles of Biochemistry 1993 675 Lipoprotein