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Vitor H. Pomin
Fucanomics & Galactanomics: marine glycans with differential actions in
coagulation and thrombosis
Universidade Federal do
Rio de Janeiro
Laboratório de Coagulação & Trombose
September, 2013
OMICS - English-language neologism that refers to a field of study in biology.
The related suffix-OME is usually used to address the objects of study of suchfields, referring thus to international research projects.
� Genomics (if not the first, one of the first projects to be launched into biology:Human Genome Project at mid-´80s, sheep Dolly from Cloning project at mid-´90s):
- Cognitive Genomics / - Comparative Genomics / - FunctionalGenomics / - Metagenomics / - Nutritional Genomics /- Pharmacogenomics / - Toxicogenomics / - Psychogenomics /- Stem cell genomics / - Epigenomics
� Transcriptomics� Proteomics:
- Immunoproteomics / - Nutriproteomics / - Proteogenomics /- Structural Genomics / - Pharmacoproteomics
� Metabolomics� Metabonomics� Pharmacomicrobiomics� Lipidomics� Glycomics (one of the latest projects to be launched into biology, 21st century,very challenging)
INTRODUCTION: “OMICS” studies and “OME” projects
GLYCOMICS: overview
CHALLENGES: 1) Structural characterization is difficult. Structural diversity and varietyare both high, probably higher than any other biomacromolecule. Experimentation anddata interpretation are laborious. In addition, previous technology was very limited.
2) Multiple biological functions. For a long time, carbohydrates were considered just amere class of energetic and structural molecules. Biological roles have beendiscovered and reported just recently. Innumerous functions have been reported.
� Glycomics turned out to be a very extensive project. This is leading to a tendency ofsubdivision or sectorization to allow the natural flow of the project development. In fact,subprojects, and new terminologies using the suffix-OME are emerging:
FUCANOMICS & GALACTANOMICS: overview
� Sulfated fucans (SFs): marine polysaccharides composed predominantly or essentiallyof α-L-fucopyranosyl units. Usually highly sulfated. Example: Fucoidan
Occurrence:
- cell wall of brown macroalgae- egg jelly coat of sea urchins- body wall of sea cucumber
Function:
- structural component of the extracellular matrices of the above-mentionedtissues- triggers the acrosome reaction in sea urchins through a specie-specific way
� Sulfated galactans (SGs): marine polysaccharides composed essentially of α-L-, α-D-,β-D-galactopyranoses. Usually highly sulfated. Examples: Carrageenans and Agarans
Occurrence:
- cell wall of green/red macroalgae- egg jelly coat of sea urchins- body wall of ascidians
Function:
- structural component of the extracellular matrices of the above-mentionedtissues- triggers the acrosome reaction in sea urchins through a specie-specific way
Laboratories Worldwide: Brazil (CE, RN, RJ, SP, RS), Argentina, USA, Russia, China, Italy, France and Japan.
FUCANOMICS & GALACTANOMICS: Chemistry vs Phylogeny
Heterogeneous and branched structures of brown algal SFs
Pomin V H , and Mourão P A S Glycobiology 2008;18:1016-1027
© The Author 2008. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected]
FUCANOMICS & GALACTANOMICS: Chemistry vs Phylogeny
Regular, homogeneous and well-defined structures of invertebrate SFs
Pomin V H , and Mourão P A S Glycobiology 2008;18:1016-1027
© The Author 2008. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected]
Structures of the repeating units of the α-L-SFs from the cell wall of the sea cucumber (A) and from the egg jelly coats of sea urchins (B-H)
FUCANOMICS & GALACTANOMICS: Chemistry vs Phylogeny
Algal and invertebrate structures of SGs: heterogeneity vs regularity
Pomin V H , and Mourão P A S Glycobiology 2008;18:1016-1027
© The Author 2008. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected]
Structures of the SGs from (A) red algae, (B) green algae, (C) sea grass (marine angiosperm), and marine invertebrates, such as (D) ascidians (also known as tunicates), and (E) sea urchins.
Sulfated fucans &
Sulfated galactans
MOLECULAR INTERACTIONS IN FUCANOMICS AND GALACTANOMICS:
DRUG DEVELOPMENT IN DIFFERENT CLINICAL AREAS
Serpins: antithrombin
and heparin cofactor II
P- and L-selectins
Vascular endothelial
growth factor (VEGF)
Chemokines Factors IIa
and Xa
basic Fibroblast
Growth Factor (FGF2)
VEGF receptor FGF receptor
Inflammation
Coagulation
and Thrombosis
Tumor
angiogenesis
FUCANOMICS & GALACTANOMICS: Medical actions
Therapeutic actions in diverse systems: inflammation; hemostasis and vascular
biology; angiogensis; tumor growth, progression and spreading; oxidative stress;
infections; and nociception.
Vitor H. Pomin - Fucanomics and galactanomics: Current status in drug discovery, mechanisms of action and role of the well-defined structures. Biochimica et Biophysica Acta (BBA) - General Subjects Volume 1820, Issue 12 2012 1971-9.
FUCANOMICS & GALACTANOMICS: Medical actions
Cellular mechanisms in inflammation
Vitor H. Pomin - Fucanomics and galactanomics: Current status in drug discovery, mechanisms of action and role of the well-defined structures. Biochimica et Biophysica Acta (BBA) - General Subjects Volume 1820, Issue 12 2012 1971-9.
FUCANOMICS & GALACTANOMICS: Medical actions
Anti-inflammatory mechanisms of actions of SFs and SGs
Vitor H. Pomin - Fucanomics and galactanomics: Current status in drug discovery, mechanisms of action and role of the well-defined structures. Biochimica et Biophysica Acta (BBA) - General Subjects Volume 1820, Issue 12 2012 1971-9.
Blo
od
flow
TUMOR GROWTH
NEOVASCULARIZATION
METASTASIS
Smooth
muscle cells
Cancer cells
• Angiogenin
• VEGF
• FGF
• TGF-β
CELL DIFFERENTIATION
(mesenchymal-epithelial transition)
+ bFGF+ VEGF
SF or SG
SF or SG SF or SG
SF or SG
Mesodermal cells
XX
X
X
Endothelial
cells
Angioblasts
Basal lamina
FUCANOMICS & GALACTANOMICS: Medical actions
Anti-angiogenic and antitumoral mechanisms of actions of SFs and SGs
Vitor H. Pomin - Fucanomics and galactanomics: Current status in drug discovery, mechanisms of action and role of the well-defined structures. Biochimica et Biophysica Acta (BBA) - General Subjects Volume 1820, Issue 12 2012 1971-9.
Vitor H. Pomin - Fucanomics and galactanomics: Current status in drug discovery, mechanisms of action and role of the well-defined structures. Biochimica et Biophysica Acta (BBA) - General Subjects Volume 1820, Issue 12 2012 1971-9.
FUCANOMICS & GALACTANOMICS: Medical actions
Molecular and cell mechanisms in coagulation/thrombosis
FUCANOMICS & GALACTANOMICS: Medical actions
Molecular mechanisms of actions of SFs and SGs in anticoagulation/antithrombosis
Vitor H. Pomin - Fucanomics and galactanomics: Current status in drug discovery, mechanisms of action and role of the well-defined structures. Biochimica et Biophysica Acta (BBA) - General Subjects Volume 1820, Issue 12 2012 1971-9.
Vitor H. Pomin - Fucanomics and galactanomics: Current status in drug discovery, mechanisms of action and role of the well-defined structures. Biochimica et Biophysica Acta (BBA) - General Subjects Volume 1820, Issue 12 2012 1971-9.
FUCANOMICS & GALACTANOMICS: Medical actions
Molecular mechanisms of actions of SFs and SGs in anticoagulation/antithrombosis
FUCANOMICS & GALACTANOMICS: Chemistry
Regular structures enable accurate and advanced structure-function relationships
Vitor H. Pomin - Fucanomics and galactanomics: Current status in drug discovery, mechanisms of action and role of the well-defined structures. Biochimica et Biophysica Acta (BBA) - General Subjects Volume 1820, Issue 12 2012 1971-9.
O
CH3
O3-SO
O
O3-SO
O
O
CH3
HOHO
O
O
CH3
HOO3
-SO
O
O
CH3
HOO3
-SO
O
(a) L. grisea
n
O
CH3
O3-SO
O
O3-SO
O
O
CH3
HOO3
-SO
O
O
CH3
HOO3
-SO
O
O
CH3
O3-SO
HO
O
(b) L. variegatus
n
O
CH3
O3-SO
O
HO
O
O
CH3
O3-SO
HO
O
O
CH3
HOO3
-SO
O
O
CH3
HOO3
-SO
O
n
(c) S. pallidus
n
(d) A. lixulaO
CH3
HO
O
HO
O
O
CH3
HO
O
OH
O
CH3
O3-SO
O
OH
O
CH3
O3-SO
O
OH
O
CH3
O3-SO
O
O3-SO
O
O
CH3
O3-SO
HO
O
O
CH3
O3-SO
HO
O
n
(f) S. purpuratus IIn
(e) S. purpuratus I
O
CH3
R1O
O
O3-SO
O
R1 = H (20%) or SO3- (80%)
O
CH3
HO
O
O3-SO
O
n
(h) S. franciscanus
O
O
CH3
O3-SO
O
OH n
(g) S. droebachiensis
n
(i) E. lucunter
O
O
CH2OH
O3-SO
O
HO
O
O
CH2OH
HO
O
OSO3- n
(k) H. monus
O
O
CH2OH O
OSO3-
O
HOH2C
OH
O
OH
OH
n
(l) S. plicata
n
R1-4 = H or SO3-
(m) B. occidentalis (R1 and R2 = SO3- in ±66% and ±33%, respectively)
(n) G. crinale (R1 and R2 = SO3- in ±60% and ±15%, respectively)
O
O
CH2OH
OSO3-
OHO
O
CH2OH
OH
OHO
n
(j) G. crenularis
O
CH2OH
R3O
OOR4
O
O
CH2OH
R1O
OR2O
FUCANOMICS & GALACTANOMICS: Medical Actions
Regular structures enable accurate and advanced structure-function relationships
Vitor H. Pomin – Marine Regular Sulfated Homopolysaccharides. Biopolymers 2009, Volume 91, Issue 8:601-609.
FUCANOMICS & GALACTANOMICS: STRUCTURE vs FUNCTION
Advanced structure-function relationships: SUGAR TYPE-DEPENDENT ANTICOAGULANT ACTION
Vitor H. Pomin - Fucanomics and galactanomics: Current status in drug discovery, mechanisms of action and role of the well-defined structures. Biochimica et Biophysica Acta (BBA) - General Subjects Volume 1820, Issue 12 2012 1971-9.
n
(i) E. lucunter
O
O
CH2OH
O3-SO
O
HO
O
CH3
HO
O
O3-SO
O
n
(h) S. franciscanus
VS
Vitor H. Pomin – Marine Regular Sulfated Homopolysaccharides. Biopolymers 2009, Volume 91, Issue 8:601-609.
FUCANOMICS & GALACTANOMICS: STRUCTURE vs FUNCTION
Advanced structure-function relationships: SUGAR TYPE-DEPENDENT ANTICOAGULANT ACTION
Structures of the complexes between different SP (red, yellow) and AT (blue). (A) ternary complex between AT, thrombin (gold) and a heparin derivative (PDB ID 1TB6); (B) AT bonded to the synthetic pentasaccharide (PDB ID 1E03); (C) final structure from a 5 ns MD of AT complexed to a SF decasaccharide with pyranose rings; (D) final structure from a 5 ns MD of AT complexed to a SG decasaccharide with pyranose rings. For (B)–(D), two orientations of the complexes are presented.
FUCANOMICS & GALACTANOMICS: STRUCTURE vs FUNCTION
Advanced structure-function relationships: CONFORMATION-DEPENDENT ANTICOAGULANT ACTION
Vitor H. Pomin – Marine Regular Sulfated Homopolysaccharides. Biopolymers 2009, Volume 91, Issue 8:601-609.
OSO3-
O
HOH2C
OH
O
OH
OH
n
R1-4 = H or SO3-
(m) B. occidentalis (R1 and R2 = SO3- in ±66% and ±33%, respectively)
(n) G. crinale (R1 and R2 = SO3- in ±60% and ±15%, respectively)
O
CH2OH
R3O
O
OR4O
O
CH2OH
R1O
OR2O
FUCANOMICS & GALACTANOMICS: STRUCTURE vs FUNCTION
Advanced structure-function relationships: SULFATION PATTERN-DEPENDENT ANTICOAGULANT ACTION
Vitor H. Pomin - Fucanomics and galactanomics: Current status in drug discovery, mechanisms of action and role of the well-defined structures. Biochimica et Biophysica Acta (BBA) - General Subjects Volume 1820, Issue 12 2012 1971-9.
Vitor H. Pomin – Marine Regular Sulfated Homopolysaccharides. Biopolymers 2009, Volume 91, Issue 8:601-609.
FUCANOMICS & GALACTANOMICS: STRUCTURE vs FUNCTION
Advanced structure-function relationships: SULFATION PATTERN-DEPENDENT ANTICOAGULANT ACTION
FUCANOMICS & GALACTANOMICS: STRUCTURE vs FUNCTION
Advanced structure-function relationships: SULFATION TYPE-DEPENDENT ANTICOAGULANT ACTION
Vitor H. Pomin - Fucanomics and galactanomics: Current status in drug discovery, mechanisms of action and role of the well-defined structures. Biochimica et Biophysica Acta (BBA) - General Subjects Volume 1820, Issue 12 2012 1971-9.
O
CH3
O3-SO
O
O3-SO
O
O
CH3
HOHO
O
O
CH3
HOO3
-SO
O
O
CH3
HOO3
-SO
O
(a) L. grisea
n
O
CH3
O3-SO
O
O3-SO
O
O
CH3
HOO3
-SO
O
O
CH3
HOO3
-SO
O
O
CH3
O3-SO
HO
O
(b) L. variegatus
n
O
CH3
O3-SO
O
HO
O
O
CH3
O3-SO
HO
O
O
CH3
HOO3
-SO
O
O
CH3
HOO3
-SO
O
n
(c) S. pallidus
n
(d) A. lixulaO
CH3
HO
O
HO
O
O
CH3
HO
O
OH
O
CH3
O3-SO
O
OH
O
CH3
O3-SO
O
OH
O
CH3
O3-SO
O
O3-SO
O
O
CH3
O3-SO
HO
O
O
CH3
O3-SO
HO
O
n
(f) S. purpuratus IIn
(e) S. purpuratus I
O
CH3
R1O
O
O3-SO
O
R1 = H (20%) or SO3- (80%)
O
CH3
HO
O
O3-SO
O
n
(h) S. franciscanus
O
O
CH3
O3-SO
O
OH n
(g) S. droebachiensis
n
(i) E. lucunter
O
O
CH2OH
O3-SO
O
HO
O
O
CH2OH
HO
O
OSO3- n
(k) H. monus
O
O
CH2OH O
OSO3-
O
HOH2C
OH
O
OH
OH
n
(l) S. plicata
n
R1-4 = H or SO3-
(m) B. occidentalis (R1 and R2 = SO3- in ±66% and ±33%, respectively)
(n) G. crinale (R1 and R2 = SO3- in ±60% and ±15%, respectively)
O
O
CH2OH
OSO3-
OHO
O
CH2OH
OH
OHO
n
(j) G. crenularis
O
CH2OH
R3O
OOR4
O
O
CH2OH
R1O
OR2O
2SO3-
> 2SO3-
4SO3-
4SO3- 2SO3
-
> 2SO3-
[2SO3-] =
[4SO3-]
Vitor H. Pomin – Marine Regular Sulfated Homopolysaccharides. Biopolymers 2009, Volume 91, Issue 8:601-609.
FUCANOMICS & GALACTANOMICS: STRUCTURE vs FUNCTION
Advanced structure-function relationships: SULFATION TYPE-DEPENDENT ANTICOAGULANT ACTION
4SO3-
[2SO3-] = [4SO3
-]
2SO3-
FUCANOMICS & GALACTANOMICS: 1ST CONCLUSIONS
� SFs and SGs are essentially found in marine organisms such as macroalgae, seaurchins and sea cucumbers, and ascidians.
� Unlike algal SFs and SGs, the invertebrate molecules have well-defined chemicalstructures which allow accurate and advanced structure-function relationships.
� Although SFs and SGs may exhibit many clinical activities, the mechanisms of actionsare known just for inflammation, coagulation/thrombosis and tumoral angiogenesis.
� In inflammation, SGs and SFs can block chemokines, P- and L-selectins. In hemostasis,they block the factors IIa and Xa. In tumoral angiogenesis, they inhibit VEGF and bFGF.
� 2-sulfated 3-linked α-galactan is an anticoagulant polysaccharides, whereas 2-sulfated3-linked α-fucan is not. Their conformational binding preference to serpins are different.
�2,4-di-sulfation and maybe 4-sulfation in 3-linked α-SFs have shown a benefitial effect onthe anticoagulation, whereas 2-sulfation alone is certainly deleterious. Ongoing NMRrelaxation studies will facilitate further explanations.
� Our dataset strongly support the the fact that the clinical actions of SFs and SGs arenot a mere consequence of sulfation degrees, but intimately correlated to specificfeatures: anomeric configuration, monosaccharide type, sulfation positions or patterns,glycosylation position, molecular weights, and so forth.
1H-chemical shift (ppm)
0.91.31.72.12.52.93.33.74.14.54.95.3
A
B
C
CH3
CH3
CH3
2-O-CH3
2-O-CH3
2-O-CH3
4.54.95.3
B1
E4
D1A1
b1
C1/c1
D1
A1
4.54.95.3
4.54.95.3
E1/F1
G1
G1
F1
0.91.31.72.12.52.93.33.74.14.54.95.3
0.91.31.72.12.52.93.33.74.14.54.95.3
1H-chemical shift (ppm)
1H-chemical shift (ppm)
FUCANOMICS & GALACTANOMICS: STRUCTURE DETERMINATION
NMR structure determination of a new red algal SG (Acanthophora muscoides)
E1/F1
3.43.63.84.04.24.44.64.85.05.25.4 3.2
1H – chemical shift (ppm)
3.4
3.6
3.8
4.0
4.2
4.4
4.6
4.8
5.0
5.2c1
D1
A
B
13C
–ch
em
ica
l sh
ift (p
pm
)
102
98
94
90
86
82
78
74
70
66
62
58
102
98
94
90
86
82
78
74
70
66
62
58
A1
B1
C1D1 E1
F1G1
F1G1
C5
C4
C3
C1
A1
b1
C3
C5
C2
C2
6-unsubstituted 6-SO3
-
E4
F6 F6´
C6 C6´
F6 F6´
C6C6´
6-unsubstituted
2-O-CH3
2-O-CH3
c1
c1
3.4
3.6
3.8
4.0
4.2
4.4
4.6
4.8
5.0
5.2
A1H
–ch
em
ica
l sh
ift (pp
m)
B
A1
B1
C
1H – chemical shift (ppm)
3.33.53.73.94.14.34.54.74.95.15.3
102
98
94
90
86
82
78
74
70
66
62
58
C1
C5
C4C3
D1
C2
6-
unsubstituted
2-O-CH3
c1
C6C6´
F1
E1
E6´E6 D6/D6´
D6/D6´E6´E6
B6/B6´
6-SO3-
E1
F1
E4
E6
c2-O-CH3
D6
F6
B6F6´
A2-O-CH3
E6´
FUCANOMICS & GALACTANOMICS: STRUCTURE PROPOSITION
Structures of SGs from Acanthophora muscoides and Botriocladia occidentalis
α-Galp units β-Galp units
O
CH2OH
OCH3
OH
O
OO
CH2OSO3-
OH
HO O
O
O
CH2OSO3-
OH
OH
O
OO
CH2OSO3-
OH
-O3SO O
O
O
OR
O
O
OO
CH2OCH3
OH
HO O
O
O
CH2OH
OH
HO O
O
Unit A (~ 32 %) Unit D (~ 22%)
Unit B (~ 43 %) Unit E (~ 30 %)
Unit C, R = H
Unit c, R= CH3 (minor)(~ 25%)
Unit F (~ 22%)
Unit G (~ 26%)
O
CH2OH
OR1
R1O
O
CH2OH
OR3
OR2
O
OO
α-Galp units β-Galp units
R1 = H or SO3-
R2 = H (~ 33%) or SO3- (~ 66%)
R3 = H (~ 66%) or SO3- (~ 33%)
VS
FUCANOMICS & GALACTANOMICS: MW DIFFERENCES
Structures of Acanthophora muscoides and Botriocladia occidentalis
0 50 100 150 200 250
1
2
3
4
5
T1/T
0
25
50
75
100
125
Resid
ual a
ctiv
ity
0.001 0.1 10 1000
0
25
50
75
100
125
µg mL-1
Resid
ual a
ctivity
µg mL -1
0
25
50
75
100
125
0.001 0.1 10 1000
A B
C D
A mUFHB occ (LMW)B occ
FUCANOMICS & GALACTANOMICS: ANTICOAGULATION
SERPIN-DEPENDENT ANTICOAGULANT ACTIVITIES
AT/IIa
APTT in serpin-containing plasma
HCII/IIaAT/Xa
0.0 0.4 0.8 1.2 1.6 2.0 2.4
10
20
30
40
50
60
70
A
40
60
80
100
Blo
od loss -
µL
5 10 5 10 5 10 5 10 (mg kg-1)
C
Occlu
sio
n tim
e (
min
)
0.0 0.25 0.50 0.75 1.00
0
40
80
120
Thro
mbus w
eig
ht (%
)
mg kg-1
B
FUCANOMICS & GALACTANOMICS: ANTITHROMBOSIS
ARTERIAL AND VENOUS MODELS, AND BLEEDING EFFECTS
A mUFHB occ (LMW)B occ
FUCANOMICS & GALACTANOMICS: PROTHROMBOTIC EFFECTS
Activation of factor XII
0
5
10
15
20
25
30V
ma
x –
mO
D m
in-1
1 50 1 50 1 50 1 50 (µg mL-1)
HeparinB.occidentalis
+_A. muscoides
FUCANOMICS & GALACTANOMICS: 2rd CONCLUSIONS
� Unlike the SG from B. occidentalis, the recently characterized SG from A. muscoides isstructurally very complex and heterogeneous. Besides sulfation at different positions,anhydro sugars and methyl ethers are also observed;
� The MW of B. occidentalis was reduced to allow a comparative study in terms ofstructure-function relationships in anticoagulation and antithrombosis with the naturallow-MW of A. muscoides;
� The native SG from B. occidentalis has high anticoagulant activity due to its significantactivity on serpins (AT and HCII) and proteases (mostly thrombin). It also shows highserpin-independent anticoagulant activity. The low-MW form lost both activities. SG fromA. muscoides has just no anticoagulant activity regardless the plasma type.
� SG from A. muscoides is curiously a potent antithrombotic agent at the arterial model,whereas the one from B. occidentalis is not. Both red algal SGs are antithrombotic at thevenous model, however, at only small doses. SG from B. occidentalis is prothrombotic athigher doses due to its capacity in activate factor XII.
� As opposed to heparin which is highly hemorragic, neither one of the red algal SGshave shown this side effect.
� Through this comparative study using red algal SGs, we have dissociated theanticoagulant (both serpin-dependent and serpin-independent), antithrombotic (botharterial and venous) and hemorragic effects, as compared with the standard heparin.
FUCANOMICS & GALACTANOMICS: ACKNOWLEDGEMENTS
Universidade Federal do
Rio de Janeiro
� Prof. Paulo A.S Mourão
� Prof. Norma Maria Barros Benevides
� Prof. Ana Cristina Vilela-Silva
� Prof. Ana Paula Valente
� Prof. Fabio Ceneviva Almeida
� Stephan Nicollas de Oliveira
� Gustavo Ramalho dos Santos
� Bianca Fernandes Glauser
� Ismael Lino Nilo de Queiroz
� Bruno Pedrosa Fontes
� Ana Luiza Quinderé
� Eros Falcão
� Gabriel Baptista de Sá
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