THE MOLECULAR BASIS OF THE ACTIVATION OF FIBROBLAST GROWTH FACTORS - PowerPoint PPT Presentation

THE MOLECULAR BASIS OF THE ACTIVATION OF FIBROBLAST GROWTH FACTORS BY GLYCOSAMINOGLYCANS FESTKOLLOQUIUM on the occasion of the 85th Birthday of Prof. Dr. rer. nat. Hans Paulsen, Hamburg 2007

THE MOLECULAR BASIS OF THE ACTIVATION OF FIBROBLAST GROWTH FACTORS BY GLYCOSAMINOGLYCANS FESTKOLLOQUIUM on the occasion of the 85th Birthday of Prof. Dr. rer. nat. Hans Paulsen, Hamburg 2007

Structure of heparin OSO 3 OSO 3 OSO 3 COO O OSO 3 O O OSO 3 O O O COO O O O OH O COO O OH OH O O O OH OH COO O O OH OSO 3 COO O O OH OH O OH O NHSO 3 OSO 3 O NHSO 3 OSO 3 NHAc NHSO 3 OH OH NHSO 3 OSO 3 "REGULAR REGION" "REGULAR REGION" OSO 3 OSO 3 OOC OSO 3 O O O O O O COO OH O OH enzymatic COO O OH OH O OH OH O OH O NHSO 3 OH NHSO 3 NHAc OSO 3 OH DEPOLYMERIZATION OSO 3 OSO 3 OSO 3 COO O O O O O chemical O COO O O OH COO O OH OH O OH OH OH O O OH OH OH NHSO 3 OSO 3 NHAc OH

Structure and biology of heparan-sulphate glycosaminoglycans

CHEMICAL STRUCTURE OF HEPARIN-HS CH 2 OH Uronic acid (1-4) glucosamine COO - O O O OH OH O O O D - glucuronic 2 - Sulfate N-Sulfate NHA c OH 6 - Sulfate D- N -acetyl CH 2 OH glucosam ine L - iduronic 2 - Sulfate O O O 3 - Sulfate COO - OH OH free am ine O O O - OH O 3 S NH Dom ain Major sequence Exclusive sequence GlcNH NA GlcA - GlcNAc 2 NS GlcA / I doA – 6 S- GlcNS 2 S-I doA , 3 S-GlcN NA/ NS NA + NS NS Dom ain NA/ NS Dom ain NA Dom ain AT-binding HEPARI N

THREE DIMENSIONAL STRUCTURE OF HS-GAGs 1 C 4 2 S O OH RO RO - OSO 3 - OOC - OOC OH O O OR OR - OSO 3 O O S O OR OR 2 O O O OH O O O O O 3 O HO RO OR O O S O O B . Mulloy, et al. Biochem. J. 1 9 9 3 , 239 ,849-858

The interaction of heparin with AT III pentasaccharide heparin heparin - - - SO 3 SO 3 SO 3 - - - SO - SO - - SO 3 SO 3 SO 3 - - - - SO 3 SO 3 SO 3 SO 3 SO 3 SO 3 SO 3 3 coagulation coagulation coagulation factor factor factor + + + Arg Arg + + + + + + Lys Lys Arg AT III AT III Xa Xa Xa

The interaction of heparin with AT III pentasaccharide heparin heparin - - - SO 3 SO 3 SO 3 coagulation coagulation - - - - - - - - - SO 3 SO 3 SO 3 factor factor - - - SO 3 SO 3 SO 3 SO 3 SO 3 SO 3 SO 3 SO 3 SO 3 + + Arg Arg + + + + + + + + Lys Lys Arg Arg AT III AT III Xa Xa

Structure of the AT III binding pentasaccharide − OSO 3 − / H OSO 3 − − OSO 3 O COO O O O O − − COO OH O OSO 3 O OH OH OH O O O − NH O OSO 3 NH OH NH − SO 3 − SO 3 − Ac / SO 3

Characteristics of selected heparin-binding proteins Heparin-binding protein Physiological/Pathological role Characteristics of heparin binding K d Oligosaccharide size Sequence features Function proteases/esterases AT III coagulation cascade serpin ca. 20 nM 5-mer GlcNS6S3S enhances SLPI inhibits eslastase and cathepsin G ca. 6 nM 12-mer to 14-mer IS enhances C1 INH inhibits C1 esterase ca. 100 nM - HS enhances VCP protects host cell from complement nM - unclear growth factors FGF-1 cell proliferation, differentiation, nM 4-mer to 6-mer IdoA2S-GlcNS6S activates signal morphogenesis, and angiogenesis transduction FGF-2 (same as FGF-1) nM 4-mer to 6-mer IdoA2S-GlcNS (same as FGF-1) chemokines PF-4 inflammation and wound healing nM 12-mer HS/LS/HS inactivates heparin ca. 6 µ M IL-8 pro-inflammatory cytokine 18-mer to 20-mer HS/LS/HS promotes SDF-1 α pro-inflammatory mediator ca. 20 nM 12-mer to 14-mer HS localizes lipid-binding proteins Annexin II receptor for TPA and plasminogen, ca. 30 nM 4-mer to 5-mer HS unclear CMV and tenascin C Annexin V ca. 20 nM 8-mer HS assembles anticoagulant activity; influenza and hepatitis B viral entry ApoE lipid transport; AD risk factor ca. 100 nM 8-mer HS localizes pathogen proteins 0.3 µ M HIV-1 gp120 viral entry 10-mer HS inhibits CypA viral localization and entry - - - inhibits Tat ca. 70 nM 6-mer HS antagonizes transactivating factor, primes cells for HIV infection HSV gB and gC viral entry into cell - - - inhibits HSV gD viral entry and fusion - - GlcNH 2 3S inhibits Dengue virus envelope viral localization ca. 15 nM 10-mer HS inhibits protein Malaria CS protein sporozoite attachment to hepatocytes ca. 40 nM 10-mer HS inhibits adhesion proteins ≥ 4-mer selectins µ Μ HS with GlcNH 2 blocks adhesion, inflammation, and metastasis µ Μ vitronectin cell adhesion and migration - - removes µ Μ fibronectin adhesion and traction 8-mer to 14-mer HS with GlcNS reorganizes HB-GAM neurite outgrowth in development ca. 10 nM 16-mer to 18-mer HS mediates µ Μ AP in amyloid plaque 4-mer HS assembles

Schematic Diagram of a Ternary Complex FGF-GAG-FGFR FGF GAG Chains FGF FGF FGF FGF receptor receptor FGF FGF TM Domain TM Domain TM Domain Lipid Bilayer Kinase P P Domain Signal transduction

FGF Oligomerization and Ternary Complexes FGF2 – FGF1 – FGFR-1 FGFR-2 J . S chle ssinge r, Mol. Cell , 2 0 0 0 , L . P e lle grini, Nature , 2 0 0 0 , 407, 6, 743 1029 A. D . D iGabrie le , et al , Nature, 1 9 9 8 , 393 , 81 2 I I I Dimers I Oligomers trans cis I II I I II I FGF FGF FGF FGF FGF I I III III I I I I FGFR FGFR HS GAG

Heparin-like oligosaccharides F E D C B A O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Retrosynthetic Analysis OR 1 OH NaO 2 C OR 1 OR 3 O O OR 1 O OH NaO 2 C O HO NH O OH O R 2 NaO 2 C O O R 1 O HO NH O HO O R 2 HO NH R 1 O O R 2 III R 1 O n = 1,2 n OP 7 OBn MeO 2 C OP 6 O OR 4 O OP 5 O OBn R 5 O BnO MeO 2 C O OBn O MeO 2 C O O P 10 O R 5 BnO O BnO O R 5 BnO P 9 O O P 8 O II n n = 1,2 ( 2+2 ) n OP 1 OBn O MeO 2 C P 2 O O OP 3 BnO I N 3 O P 4 O OP 1 OBn MeO 2 C O NH OTDS P 2 O O BnO O CCl 3 OH OH N 3 J.L. de Paz et al. ChemBioChem 2001 , 2 , 673-685

Solid phase synthesis of regular region hexasaccharide O MPEG O O O OR 1 R 1 , R 2 = Ph-CH- O O O O MeO 2 C OBn MeO 2 C OBn O O Ph Ph O 2 C OBn O OBnO O OBnO O R 2 O OC(NH)CCl 3 O R 1 , R 2 = H O O N 3 N 3 BnO O N 3 PivO PivO + R 1 = Bz, R 2 = H PivO O O MPEG O O OBz OR 1 R 1 , R 2 = Ph-CH- O O 2 C OBn OAc O O MeO 2 C OBn O O O MeO 2 C OBn R 2 O R 1 , R 2 = H O BnO O N 3 BnO BnO O OC(NH)CCl 3 N 3 O BnO O N 3 PivO R 1 = Bz, R 2 = H PivO + BzO O O MPEG O O OBz OBz O O 2 C OBn OAc O O MeO 2 C OBn O O O MeO 2 C OBn O O BnO N 3 O BnO BnO O O N 3 BnO N 3 O PivO PivO BzO OH OH O HO 2 C OBn OH O O HO 2 C OBn O O O HO 2 C OBn O O BnO N 3 O BnO BnO O N 3 O BnO N 3 O HO HO HO OSO 3 Na OSO 3 Na O OSO 3 Na NaO 2 C OH O O NaO 2 C OH O O O NaO 2 C OH O O HO NH O HO HO O NH O HO NaO 3 S NH O NaO 3 SO NaO 3 S NaO 3 SO NaO 3 S R. Ojeda et al. Chem. Commun . 2003 , 2486-2487 NaO 3 SO

NMR Studies of the regular region hexasaccharide Conformation of the iduronate units OS O 3 OH OO C O OS O 3 O O O OH OS O 3 O OO C HO NH 1 C 4 O OH O OO C O OR O SO OS O 3 HO 3 NH O O HO SO HO OH 3 NH O OS O - O 2 C 3 SO O 3 OSO 3 G-6 I-5 G-4 I-3 G-2 I-1 OR - OSO 3 O O O O O O O O 2 S O O O O O H 2 H 5 O O - RO OSO 3 - O 2 C OH S O Conformational populations O RO OR OR O O O OH 1 C 4 : 2 S O O 4 C 1 O O O IdoA-1 O 70:30 HO RO HO OR RO IdoA-3 OR O 69:31 - O 2 C O S - OSO 3 O IdoA-5 72:28 O J.L. de Paz et al. ChemBioChem 2001 , 2 , 673-685

Molecular dynamics (PME) of the regular region hexasaccharide OS O 3 OH OO C O O OS O 3 O O OH OS O O 3 OO C HO NH O OH OO C O O O SO HO OS O 3 3 NH O O H O SO HO 3 NH O OS O 3 SO 3 OS O 3 G-6 I-5 G-4 I-3 G-2 I-1 O O O O O O J. Angulo et al. O O O O O O Chem. Commun . 2003 , 1512-1513 1 C 4 Conformers 180 180 180 180 120 120 120 120 60 60 60 60 0 0 0 0 Ψ 1 Ψ 2 Ψ 3 Ψ 5 -60 -60 -60 -60 -120 -120 -120 -120 -180 -180 -180 -180 -180 -120 -60 0 60 120 180 -180 -120 -60 0 60 120 180 -180 -120 -60 0 60 120 180 -180 -120 -60 0 60 120 180 Φ 1 Φ 2 Φ 3 Φ 5 2 So Conformers 180 180 180 180 120 120 120 120 60 60 60 60 0 0 0 0 Ψ 2 Ψ 3 Ψ 4 Ψ 5 -60 -60 -60 -60 -120 -120 -120 -120 -180 -180 -180 -180 -180 -120 -60 0 60 120 180 -180 -120 -60 0 60 120 180 -180 -120 -60 0 60 120 180 -180 -120 -60 0 60 120 180 Φ 2 Φ 3 Φ 4 Φ 5

NMR Studies of the Regular Region Hexasaccharide OSO 3 OH OOC O O OSO 3 O O OH OSO 3 O OOC HO NH O OH O OOC O O SO HO OH 3 NH O O HO SO HO 3 NH O OH SO 3 OH G-6 I-5 G-4 I-3 G-2 I-1 Anisotropy O O O O O O O O O O O O H-4 Interglicosidic NOEs 109º H-2 H-1 H - OSO 3 - OOC H H H O OH HO O O O H H - NHSO 3 - NHSO 3 Distance O τ isotropic (ps) H Pair H HO (Å) H O - O 3 SO O - OSO 3 H1 – H2 2.4 700 H2 – H4 2.5 1300 J.L. de Paz et al. ChemBioChem 2001 , 2 , 673-685