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

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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

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SLIDE 1

THE MOLECULAR BASIS OF THE ACTIVATION OF FIBROBLAST GROWTH FACTORS BY GLYCOSAMINOGLYCANS

FESTKOLLOQUIUM

  • n the occasion of the 85th Birthday of
  • Prof. Dr. rer. nat. Hans Paulsen, Hamburg 2007
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SLIDE 2
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SLIDE 3
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SLIDE 4
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SLIDE 5
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SLIDE 6
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SLIDE 7
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SLIDE 8

THE MOLECULAR BASIS OF THE ACTIVATION OF FIBROBLAST GROWTH FACTORS BY GLYCOSAMINOGLYCANS

FESTKOLLOQUIUM

  • n the occasion of the 85th Birthday of
  • Prof. Dr. rer. nat. Hans Paulsen, Hamburg 2007
slide-9
SLIDE 9

O O O O COO O OH OSO3 OSO3 OH NHSO3 O O O O COO OH OH OSO3 OSO3 NHSO3 O O O O COO OH OH OSO3 OH NHAc O O O O COO OH OSO3 OSO3 OH NHSO3 O O O O COO OH OSO3 OSO3 OH NHSO3 O O O O OOC OH OH OSO3 OH NHAc O O O O COO OH OSO3 OSO3 OH NHSO3 O O COO OH OH O OSO3 OH NHSO3 OH O O O O COO OH OH OSO3 OH NHAc O O O O COO OH OSO3 OSO3 OH NHSO3 O O COO OH OH OH O OH OH O OSO3

"REGULAR REGION" "REGULAR REGION" DEPOLYMERIZATION

enzymatic chemical

Structure of heparin

slide-10
SLIDE 10

Structure and biology of heparan-sulphate glycosaminoglycans

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SLIDE 11

CHEMICAL STRUCTURE OF HEPARIN-HS

O O O OH OH COO- O O O OH OH COO-

2

  • Sulfate

L-

iduronic N-Sulfate 6

  • Sulfate

N-acetyl 3

  • Sulfate

free am ine D- glucosam ine 2

  • Sulfate

D-

glucuronic Exclusive sequence Major sequence Dom ain NA + NS NA/ NS 2 S-I doA , 3 S-GlcN GlcA / I doA – 6 S- GlcNS NS GlcNH

2

GlcA - GlcNAc NA NA Dom ain NA/ NS Dom ain NS Dom ain AT-binding HEPARI N

Uronic acid (1-4) glucosamine

O O NHA c OH CH

2OH

O O O NH

  • OH

CH2OH O O3S

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SLIDE 12

THREE DIMENSIONAL STRUCTURE OF HS-GAGs

B . Mulloy, et al. Biochem. J. 1 9 9 3 , 239 ,849-858 O O

2 3

1C4 2SO

RO

  • OOC

OSO3

  • OH

O OR

  • OOC

OSO3

  • OH

O OR RO

O OR OH O OR O O S O O O O RO HO O OR O O S O O O

slide-13
SLIDE 13

coagulation factor Xa Xa coagulation factor coagulation factor Xa

SO3

  • SO3
  • SO3
  • SO3

SO3 SO3 SO3

3

SO3

  • SO3
  • heparin

SO3

  • SO3
  • SO3
  • SO3
  • SO-

SO-

heparin pentasaccharide

+ + + +

Arg Lys AT III

+ + + +

Arg Lys AT III Arg

+

The interaction of heparin with AT III

slide-14
SLIDE 14

coagulation factor Xa coagulation factor Xa

SO3

  • SO3
  • SO3
  • SO3
  • SO3
  • +

+ + +

Arg Lys AT III heparin

+

Arg

SO3

  • SO3
  • SO3
  • SO3
  • SO3
  • SO3
  • SO3
  • SO3
  • SO3
  • SO3
  • +

+ + +

Arg Lys AT III heparin

+

Arg pentasaccharide

The interaction of heparin with AT III

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SLIDE 15

Structure of the AT III binding pentasaccharide

O O O OSO3

−

OH NH O O O O COO

−

OH OH OSO3

− / H

OSO3

−

NH O O COO

−

OH OSO3

−

O OSO3

−

OH NH O Ac / SO3

−

SO3

−

SO3

−

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SLIDE 16

Characteristics of selected heparin-binding proteins

Heparin-binding protein Physiological/Pathological role Characteristics of heparin binding Kd 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 IL-8 pro-inflammatory cytokine

  • ca. 6 µ M

18-mer to 20-mer HS/LS/HS promotes SDF-1α 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

anticoagulant activity; influenza and

  • ca. 20 nM

8-mer HS assembles hepatitis B viral entry ApoE lipid transport; AD risk factor

  • ca. 100 nM

8-mer HS localizes pathogen proteins HIV-1 gp120 viral entry 0.3 µ M 10-mer HS inhibits CypA viral localization and entry

  • inhibits

Tat

transactivating factor, primes cells for

  • ca. 70 nM

6-mer HS antagonizes HIV infection HSV gB and gC viral entry into cell

  • inhibits

HSV gD viral entry and fusion

  • GlcNH23S

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 selectins

adhesion, inflammation, and metastasis

µ Μ ≥ 4-mer

HS with GlcNH2 blocks vitronectin cell adhesion and migration

µ Μ

  • removes

fibronectin adhesion and traction

µ Μ

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 AP in amyloid plaque

µ Μ

4-mer HS assembles

slide-17
SLIDE 17

Signal transduction

FGF FGF

GAG Chains

TM Domain TM Domain TM Domain

Kinase Domain

P P

Lipid Bilayer

FGF receptor

FGF FGF

FGF receptor

FGF

Schematic Diagram of a Ternary Complex FGF-GAG-FGFR

slide-18
SLIDE 18

FGF Oligomerization and Ternary Complexes

  • A. D . D iGabrie le , et al, Nature, 1

9 9 8 , 393, 81 2 FGF2 – FGFR-1 L . P e lle grini, Nature, 2 , 407, 1029 FGF I II I I I I I I I I I FGF I I I III FGF FGF I II III J . S chle ssinge r, Mol. Cell, 2 , 6, 743 FGF1 – FGFR-2

FGF FGFR FGFR HS GAG Dimers trans cis Oligomers

slide-19
SLIDE 19

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

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

Heparin-like oligosaccharides

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SLIDE 20

Retrosynthetic Analysis

J.L. de Paz et al. ChemBioChem 2001, 2, 673-685

O O O OBn OP7 O BnO O O OBn O OP6 O BnO O O OBn BnO O BnO R5 MeO2C MeO2C MeO2C OP5 R5 R5 P9O P8O P10O OR4 I ( 2+2 )n P2O OP1 O BnO N3 O CCl3 NH O OH MeO2C OTDS OH OBn P2O O N3 O O P4O MeO2C BnO OP1 OBn OP3 II n = 1,2 n n = 1,2 n III O O O OH OR1 O HO O O OH O OR1 O HO O O OH HO O HO NH NaO2C NaO2C NaO2C OR1 NH NH R2 R2 R1O R1O R1O OR3 R2

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SLIDE 21

R2O BnO O MeO2C PivO OBn O N3 O OR1 O BnO O O2C PivO OBn O N3 O OBz O O O O O

MPEG

OBnO O MeO2C PivO OBn O N3 O O Ph OC(NH)CCl3 BnO BnO O MeO2C BzO OBn O N3 O OAc OC(NH)CCl3 O BnO O MeO2C PivO OBn O N3 O OBz O BnO O O2C PivO OBn O N3 O OBz O O O O O

MPEG

BnO BnO O MeO2C BzO OBn O N3 O OAc R2O BnO O O2C PivO OBn O N3 O OR1 O O O O O

MPEG

O BnO O HO2C HO OBn O N3 O OH O BnO O HO2C HO OBn O N3 O OH O BnO BnO O HO2C HO OBn O N3 O OH O HO O NaO2C NaO3SO OH O NH O OSO3Na O HO O NaO2C NaO3SO OH O NH O OSO3Na O HO HO O NaO2C NaO3SO OH O NH O OSO3Na NaO3S NaO3S NaO3S R1, R2 = Ph-CH- R1, R2 = H R1= Bz, R2= H

+

R1, R2 = Ph-CH- R1, R2 = H R1= Bz, R2= H

+

O OBnO O MeO2C PivO OBn O N3 O O Ph

Solid phase synthesis of regular region hexasaccharide

  • R. Ojeda et al. Chem. Commun. 2003, 2486-2487
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SLIDE 22

NMR Studies of the regular region hexasaccharide Conformation of the iduronate units

G-6 G-4 G-2 I-1 I-3 I-5 1C4: 2SO

72:28 IdoA-5 69:31 IdoA-3 70:30 IdoA-1 Conformational populations

O OR OH OSO3

  • OR
  • O2C

OSO3

  • O2C

RO O RO OH H5 H2 RO O OSO3

  • HO
  • O2C

OR

2SO 4C1 1C4 O OR OH O OR O O S O O O O RO HO O OR O O S O O O

O O O OH OS O3 O HO O O OH O OS O3 O HO O O OH O HO NH OO C OO C OO C OS O3 NH NH SO

3

SO

3

SO

3

OS O

3

OSO3 OS O3 O HO

J.L. de Paz et al. ChemBioChem 2001, 2, 673-685

O O O O O O O O O O O O

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SLIDE 23

Molecular dynamics (PME) of the regular region hexasaccharide

G-6 G-4 G-2 I-1 I-3 I-5

  • 180 -120 -60

60 120 180

  • 180
  • 120
  • 60

60 120 180

Ψ 2 Φ 2

  • 180 -120 -60

60 120 180

  • 180
  • 120
  • 60

60 120 180

Ψ 1 Φ 1

  • 180 -120 -60

60 120 180

  • 180
  • 120
  • 60

60 120 180

Ψ 5 Φ 5

  • 180 -120 -60

60 120 180

  • 180
  • 120
  • 60

60 120 180

Ψ 3 Φ 3

1C4 Conformers

  • 180 -120 -60

60 120 180

  • 180
  • 120
  • 60

60 120 180

Ψ 2 Φ 2

  • 180 -120 -60

60 120 180

  • 180
  • 120
  • 60

60 120 180

Ψ 3 Φ 3

  • 180 -120 -60

60 120 180

  • 180
  • 120
  • 60

60 120 180

Ψ 4 Φ 4

  • 180 -120 -60

60 120 180

  • 180
  • 120
  • 60

60 120 180

Ψ 5 Φ 5

2So Conformers

O O O OH OS O

3

O HO O O OH O OS O

3

O HO O O OH O HO NH OO C OO C OO C OS O

3

NH NH SO

3

SO

3

SO

3

OS O

3

OS O

3

OS O

3

O H O

  • J. Angulo et al.
  • Chem. Commun. 2003, 1512-1513

O O O O O O O O O O O O

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SLIDE 24

Interglicosidic NOEs Anisotropy

G-6 G-4 G-2 I-1 I-3 I-5

NMR Studies of the Regular Region Hexasaccharide

O O O NHSO3

  • HO

O OH

  • O3SO

O

  • OOC

O O NHSO3

  • HO

OSO3

  • OSO3
  • H

H H H H H H H H

H-4 H-2 H-1

109º

700 2.4 H1 – H2 1300 2.5 H2 – H4 τisotropic (ps) Distance (Å) Pair

O O O OH OSO3 O HO O O OH O OSO3 O HO O O OH O HO NH OOC OOC OOC OSO3 NH NH SO

3

SO

3

SO

3

OH OH OH O HO

J.L. de Paz et al. ChemBioChem 2001, 2, 673-685

O O O O O O O O O O O O

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SLIDE 25

Mitogenic activity of the regular region oligosaccharides

LM heparin (3000 Da) HEXASACCHARIDE OCTASACCHARIDE

  • J. L. de Paz, et al. ChemBioChem. 2001, 2, 101-113

n n= 1 , 2

OH HO O O NH OH O OH OSO3

  • O
  • OOC

O O NH HO O OSO3

  • O
  • OOC

O O NH HO O OH OSO3

  • O
  • OOC

OSO3

  • OSO3
  • OSO3
  • SO3
  • SO3
  • SO3
slide-26
SLIDE 26

Mitogenic activity

LW heparin (3000 Da) Regular hexasaccharide Regular octasaccharide Modified hexasaccharide Modified octasaccharide

activator (µG/mL)

Absorbance

Asymmetric octasaccharide Regular hexasaccharide Regular octasaccharide Asymmetric hexasaccharide

  • J. Angulo et al. ChemBioChem. 2004, 5, 55-61

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

slide-27
SLIDE 27

Mitogenic Activity

LM heparin 3000 Asymmetric distribution Regular region 20H 60H

1 10 100 1000 0,2 0,4 0,6 0,8 1,0 1,2 1,4

ABSORBANCE ACTIVATOR (

µ

g/mL)

Regular region Asymmetric distribution 20H 60H

  • J. Angulo et al. ChemBioChem. 2004, 5, 55-61

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

slide-28
SLIDE 28

6.52 6.56 6.60 6.64 0.1 0.2 0.3 0.4

  • 0.01

0.00 0.01 6.52 6.56 6.60 6.64 6.52 6.56 6.60 6.64 6.52 6.56 6.60 6.64

Absorbance RS Radius A: FGF-1 (Mw = 16640 ± 885) B: FGF-1+ LMWH (Mw = 30180 ± 1940) C: FGF-1 + Hexasaccharide (Mw = 18500 ± 540) D: FGF-1 + Octasaccharide (Mw = 19130 ± 790)

Sedimentation equilibrium curves of FGF-1 in the

presence of synthetic oligosaccharides

A B C D

slide-29
SLIDE 29

Asymmetric distribution

I I I NH NH G G G 2 2 6 6 2 NH 6

Regular region

FGF1 activation by synthetic heparin oligosaccharides

1 10 100 1000 0,2 0,4 0,6 0,8 1,0 1,2 1,4

ABSORBANCE ACTIVATOR( µ g/mL)

1 10 100 1000 0,2 0,4 0,6 0,8 1,0 1,2 1,4

ABSORBANCE ACTIVATOR( µ g/mL)

ChemBioChem, 2004, 5, 55-61 O4 O1

G I G I G I G I G I G I

NH NH 2 2 6 NH NH 2 2 6

FGF FGFR

I I I NH NH NH I I I NH NH NH G G G 6 6 6 G G G 2 2 2

20H 60H

I I I NH NH NH I I I NH NH NH G G G 6 6 6 G G G 2 2 2

20H 60H LM heparin 3000 Assymmetric distribution Regular region 20H 60H LM heparin 3000 Asymmetric distribution Regular region 20H 60H

slide-30
SLIDE 30

O O HO O O2C O3SO OH O NH O OSO3 O HO O O2C O3SO OH O NH O OSO3 HO HO O O2C O3SO OH O NH O OSO3 O3S O3S O3S O O HO O O2C O3SO OH O NH O OH O HO O O2C HO OH O NH O OSO3 HO HO O O2C O3SO OH O NH O OH O3SO Ac SO3 O O HO O O2C OSO3 OH O NH O OH O HO O O2C OSO3 O O NH O OH HO HO O O2C OSO3 OH O NH O OH SO3 SO3 SO3 O O HO O O2C HO OH O NH O OSO3 O HO O O2C HO OH O NH O OSO3 HO HO O O2C HO OH O NH O OSO3 SO3 SO3 SO3 O O HO O O2C O3SO OH O NH O OH O HO O O2C HO OH O NH O OSO3 HO O2C O3SO OH O O3SO SO3 HO O NH O OSO3 Ac 1 2 3 4 5

slide-31
SLIDE 31

O4 O1 O1 O4

O O HO O O2C O3SO OH O NH O OH O HO O O2C HO OH O NH O OSO3 HOHO O O2C O3SO OH O NH O OH O3SO Ac SO3 O O HO O O2C O3SO OH O NH O OH O HO O O2C HO OH O NH O OSO3 HO O2C O3SO OH O O3SO SO3 HO O NH O OSO3 Ac

FGF-1 Activator FGF-2 Inhibitor

slide-32
SLIDE 32

O O O O O O O O O O O O O O O O O

O O O

L ow B inding H igh B inding

slide-33
SLIDE 33

SPR: FGF-1 Binding Constants

P.M. Nieto, 2006

slide-34
SLIDE 34

77-84

77 84 77 84

HSQC-NOESY 15N-1H HNCA ASSIGNMENT of 3D-experiments

1H 13C 1H 1H

7 Hz 11 Hz

C C N N C H C H H C O C H H H H H O

  • A. Canales et al. PEBS J., 2006, 273, 4716-4727
slide-35
SLIDE 35

Superposition of 20 structures rmsd=0.94 Å 1432 NOEs : 260 sequencial 667 medium range 505 long range

Stereoview of the best fit superpositions of the structure of asymmetric hexasaccharide-bound FGF-1

  • A. Canales et al. PEBS J., 2006, 273, 4716-4727
slide-36
SLIDE 36

BINDING SITE MAPPING

Binding site

15N-1H-HSQC

  • bound state
  • free protein
  • A. Canales et al. PEBS J., 2006, 273, 4716-4727
slide-37
SLIDE 37

40 60 80 100 120 140

  • 1

1

S

2 complex

  • S

2 free G129 K132 T137 L147 G124 H107 G134 I144 Y139 S152 G124 G129 H107 H116

Element ∆S2 LOOP 1 0.16 SHEET 4 0.16 TURN 3 0.26 SHEET 5 0.14 SHEET 6 0.13 LOOP 2 0.28 TURN 6

  • 0.16

SHEET 10 0.18 TURN 7 0.34 SHEET 11 0.20 LOOP 4 0.12 SHEET 12 0.14

Conformational entropy loss amounts to ca. 56 kJ/mol

  • A. Canales, 2004

40 60 80 100 120 140 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5

Chem shift

The flexibility of the complex

40 60 80 100 120 140 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5

Chem shift

  • A. Canales J. Biomol. NMR, 2006, 35, 225-239
slide-38
SLIDE 38

The structure of the bound hexasaccharide

13C-filtered- NOESY

CHAIR-BOAT EQUILIBRIUM IN THE BOUND STATE

  • A. Canales et al. J. Am. Chem. Soc. 2005, 127, 5778-5779

5.00 5.10 5.20 5.30 5.40 5.00 5.10 5.20 5.30 5.40 3.00 3.50 4.00 4.50

H1I5 H5 H5 H2 H2 H1I1 H3,4 H3 H4 H1G6 ↓ H1G2

↓

H1G4 H1I3 H2 H4 H3 H5 H2 H4 H2,3 H4 H3 H3,4 H2,5 H1G2

↓

H1G6 ↓ H1I3 H1G4 H1I5

HO O O NH HO O OH

  • O3SO

O

  • OOC

O O NH HO OH OSO3- H H H H H H H H H O OH HO O

  • OOC

O O NH HO OH H H H H H H H H H O OH

  • O3SO

O

  • OOC

H H H H H

iPr

SO3- Ac SO3-

GlcN-6 IdoA-5 GlcN-4 IdoA-3 GlcN-2 IdoA-1 H1I5-H6G4

A B O O OR OH OSO3

  • OR
  • OOC
  • OOC

OSO3- HO OR RO 5H 2H 5H 2H

1C4 2SO

0.0 2.5 5.0 7.5 10 .0 0.0 2.5 5.0 7.5 10.0
slide-39
SLIDE 39

O OR OH O OR O O S O O- O NH O N H4

+

O OR HO O OR

  • O

O S O O O- N H4

+

NH O

Dynamics of the hexasaccharide within the Complex

slide-40
SLIDE 40

AUTODOCK

First docking run: Global search for the binding site Second step: local search of the binding site (rigid ligand) Third step: local search of the binding site (flexible ligand)

The Complex: NOE INFORMATION

SER 130 ARG 133 LYS 127 LYS 142

  • A. Canales, J. Angulo, 2004
slide-41
SLIDE 41

ACKNOWLEDGMENTS

Synthesis (IIQ) José L. de Paz Rafael Ojeda Ricardo Lucas Niels-Christian Reichardt Olimpia Terentí Structure (IIQ, CIB) Pedro M. Nieto Jesús Angulo Jesús Jiménez-Barbero Angeles Canales Biological Activity (CIB) Guillermo Giménez-Gallego Rosa M. Lozano Financial support DGCYT (PB96-0820) DGCYT (BQU02-03734) Fundación Ramón Areces Fundación Francisco Cobos