Biomedical GlycobiologyFrontier of Heparan Sulfate Proteoglycan studyDr. Lianchun Wang [email protected] Sulfate ProteoglycansGrowth Factors and MorphogensVEGF/VEGFR FGF/FGFR HGF WntHedgehog BMP TGF-β Heparan sulfateproteoglycanInflammation/CoagulationAntithrombin EC-SODHeparin cofactor II L- and P-selectinsPlatelet factor 4 Chemokines, ΙL-8 Matrix ProteinsFibronectin LamininVitronectin CollagensFibrillin TenascinProtein Ligands that HSPG interactsFunctions of HSPGs√√√√√√√√Heparan sulfate biosynthesis= GlcNAc= GlcA= Gal= XylAntithrombinFGF-1/FGF2NS NS NSNS NS NSNS2S 2S2S 2S6S 6S 6S 6S 6S6S3S= IdoAPAPSNS NS NSNS NS NSNS2S 2S2S 2SNS NS NSNS NS NSNSNS NS NSNS NS NSNS2S 2S2S2S6S 6S 6S 6S 6S6SPAPSPAPSPAPSNS NS NSNS NS NSNS5-105-105-105-105-105-10NS-domain NA/NS-domain NA-domainCopolymeraseEXT1 / EXT2 GlcNAcN-deacetylase /N-sulfotransferases 1 & 26-O-sulfotransferases 13-O-sulfotransferases 1,2,3a,3b,42-O-sulfotransferaseGlcA C5 epimeraseChainModificationChainPolymerizationHeparan Sulfate DeficienciesWildtypeNS3S6S6S 6S2S6S6SNS NS2S NSNSG= GlcNAc= GlcA= al= Xyl = IdoANdst1-/-6S 6S6SNS2S NSNSExt1-/-Ndst1-/-Ndst2-/-I. Development-related defectsII. Blood CoagulationIII. InflammationIV. Tumor BiologyV. Lipid metabolismOutlineI. Development-related Defects:Embryonic Stem Cells+"LIF‐"LIFSelf‐RenewalPluripo t e ncyGordon Keller Genes Dev. 2005; 19: 1129-1155In vitro Embryonic Stem cell Differentiation mimics earlyin vivo embryonic developmentFGF, Wnt and BMP signaling regulate stem cell fateCreAlter Heparan Sulfate Structure in ES CellsLoxP5(Flo xed)5mou seloxPEXT1loxPCre‐5Ext1'(Ext1+/+)Cre+5Ext1'(Ext1‐/‐)loxPDeficiency of Ext1 leads to defects of early embryonicdevelopment and ES cell self-renewalVascular DevelopmentVasculogenesis:a process wherebyangioblasts differentiate in situ toendothelial cells that connect and formprimitive blood vesselsAngiogenesis:the growth and remodelingprocess of the primitive network into acomplex networkVEGFPDGF, TGFBrachyury+FGFsAll these vascular developmentessential growth factors areheparin-binding proteinsMice Deficient in Endothelial cell (EC) Ndst1 and/or Ndst2Mice Deficient in Smooth Muscle Cell (SMC) Ndst1 andNdst2 (Chronic intestinal pseudo-obstruction ?)II. HSPG and Blood Coagulation Heparin has been widely used asanticoagulant clinically for over 70 years. Heparin does not present in vasculature,instead the similarly structured heparansulfate abundantly exists. Vascular heparan sulfate contains < 0.3%heparin-like structure, can bind antithrombinIII and exhibits anticoagulant activity in vitroand ex vivo, suggesting vascular heparansulfate may function as a naturalanticoagulant. This hypothesis has beenwidely accepted, but has not been proved. Both in vitro and ex vivo conflictingobservations were reported also. Especiallyrecent genetic study (Hs3st1 knockout mice)does not support.Heparan sulfateanticoagulant pathwayHajMohammadi 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 thrombophilic020406080100Ndst1f/fTekCre-Ndst1f/fTekCre+a*Ndst1+/+Ndst1-/-Tail bleedingMice deficient in EC-Ndst1 is thrombophilicCarotid injury 020406080100Ndst1f/fTekCre-Ndst1f/fTekCre+UnstableStableFigure 1. Carotid artery thrombosis induced by FeCl3 treatment.a).Occlusion time; b). Stability of thrombus. .000333666999121212Occlusion Time (min)Occlusion Time (min)OOcccclluussiioonn TTiimmee ((mmiinn))bNdst1f/fTekCre-Ndst1f/fTekCre+P = 0.054n = 14 - 18P < 0.001n = 14 - 18**Stable thrombus (%)Stable thrombus (%)SSttaabbllee tthhrroommbbuuss ((%%))aHeparan Sulfate modulates fibrinolysis ?System controlsblood clot removalIII. HSPG and Inflammation Inflammation is a fundamental mechanism that the body reacts to infection, irritation orother injury. The key feature includes redness, warmth, swelling and pain. The underlying cellular and molecular mechanisms are manifested by the trafficking ofleukocyte from blood circulation into the injured tissues. The leukocyte trafficking into the injured tissue is a multiple-step process, involvingvarious adhesion molecules and chemokines. Heparin inhibits inflammation by blocking L- and P-selectin mediated leukocyte-endothelial cell adhesion. Heparan sulfate modulates inflammatory response ?LeukocyteGPCRNelson 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.Heparan sulfate modulates inflammationRoles: 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, morphogensand 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 growthand metastasis (Based on in vitro cultured tumor cell) (Liu D., et al. Proc Natl Acad SciUSA, 2002). Syndecan and Glypican modulate tumorigenesis (Alexander CM., et al. NatGenet. 2000; Aikawa T. et al. J Clin Invest. 2008). Heparin inhibits tumor metastasis by blocking L- and P-selectin mediated tumorcell adhesion (Borsig L., et al. Proc Natl Acad Sci USA, 2001; Borsig L., et al. Proc NatlAcad Sci USA, 2002) Heparan sulfate chain modulates tumorigenesis and metastasis in vivo ?Tumor growth in vitroDeficiency of heparan sulfate alters tumor cell growth,which can be inhibitory or enhancingTumor angiogenesisHS modulates tumor angiogenesisNdst1+/+Ndst1-/-Ndst1+/+Ndst1-/-Ndst1+/+Ndst1-/-Tumor growthTumor angiogenesisFuster M et al. J Cell Biol. 2007; 177(3): 539-49.Ndst1-/- here means endothelial cell specific Ndst1 knockout miceLipoproteins transport cholesterol, triglycerides and phospholipids throughthe circulationCholesterolPhospholipidsApolipoproteinsNeutral core containingtriglycerides andcholesteryl estersHorton, et al. Principles of Biochemistry. 2002. 16.5.V. HSPG and Lipid metabolism:Lehninger, et al. Principles of Biochemistry. 1993. 675.Major Plasma