Download FUCANOMICS & GALACTANOMICS: STRUCTURE vs FUNCTION

Universidade Federal
do
Rio de Janeiro
Fucanomics & Galactanomics: marine
glycans with differential actions in
coagulation and thrombosis
Vitor H. Pomin
Laboratório de Coagulação &
Trombose
September, 2013
INTRODUCTION: “OMICS” studies and “OME” projects
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 such
fields, 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 / - Functional
Genomics / - 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)
GLYCOMICS: overview
CHALLENGES: 1) Structural characterization is difficult. Structural diversity and variety
are both high, probably higher than any other biomacromolecule. Experimentation and
data interpretation are laborious. In addition, previous technology was very limited.
2) Multiple biological functions. For a long time, carbohydrates were considered just a
mere class of energetic and structural molecules. Biological roles have been
discovered and reported just recently. Innumerous functions have been reported.
 Glycomics turned out to be a very extensive project. This is leading to a tendency of
subdivision 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 essentially
of 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-mentioned
tissues
- 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-mentioned
tissues
- 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
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)
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
Algal and invertebrate structures of SGs: heterogeneity vs regularity
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.
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: Medical actions
Therapeutic actions in diverse systems: inflammation; hemostasis and vascular
biology; angiogensis; tumor growth, progression and spreading; oxidative stress;
infections; and nociception.
MOLECULAR INTERACTIONS IN FUCANOMICS AND GALACTANOMICS:
DRUG DEVELOPMENT IN DIFFERENT CLINICAL AREAS
Inflammation
Chemokines
Vascular endothelial
growth factor (VEGF)
P- and L-selectins
Sulfated fucans &
Sulfated galactans
Coagulation
Factors IIa
and Thrombosis
and Xa
Serpins: antithrombin
and heparin cofactor II
VEGF receptor
FGF receptor
basic Fibroblast
Tumor
angiogenesis Growth Factor (FGF2)
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
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
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
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: Chemistry
Regular structures enable accurate and advanced structure-function relationships
O
(i) E. lucunter
O
CH3
O3-SO
HO
(a) L. grisea
n
O
CH3
O
O
O3 SO
HO
O
OSO3O
CH3
HO
CH2OH
O3-SO
O3-SO
HO
O
OH
O
OH
OR4
O
O
(c) S. pallidus
O
CH3
R1O
R3O
n
(m) B. occidentalis (R and R = SO in 66% and 33%, respectively)
(n) G. crinale (R and R = SO in 60% and 15%, respectively)
1
R1-4 = H or SO3-
HO
O
1
O
O
HO
O
O
O
CH3
O
CH3
O
O3-SO
O
HO
O3-SO
O
-
O3 SO
O
R1O
O
CH3
O
O3-SO
O3-SO
O
O
CH3
(d) A. lixula
O
CH3
-
O
CH3
OH
O
3
3
2
n
OH
OH
O
2
(g) S. droebachiensis
O
CH3
HO
HOH2C
O
n
O
CH3
HO
O
O3-SO
n
OH
n
HO
(h) S. franciscanus
O
R1 = H (20%) or SO3 (80%)
-
(e) S. purpuratus I
-
-
O3 SO
O
O
O
CH3
n
O
O
OH
CH2OH
O
R2O
O
O
HO
O
O
O
CH3
n
O3-SO
O3-SO
O3-SO
O
O
O
CH3
O
CH2OH
n
OSO3-
O
CH3
(l) S. plicata
O
CH2OH
(b) L. variegatus
O
O3-SO
O
n
O
CH3
HO
(k) H. monus
HO
O3-SO
-
O3-SO
OSO3O
CH3
O3-SO
HO
n
OH
O
O
CH3
O
O
O
O3-SO
O
HO
HO
CH3
O
O
HO
n
O
O
CH2OH
HO
CH2OH
O3-SO
HO
O
CH3
O
CH2OH
O3-SO
HO
(j) G. crenularis
O
O
O3 SO
(f) S. purpuratus II
CH3
HO
O
O
O3-SO
n
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
Regular structures enable accurate and advanced structure-function relationships
Vitor H. Pomin – Marine Regular Sulfated Homopolysaccharides. Biopolymers 2009, Volume 91, Issue 8:601-609.
(g) S. droebachiensis
FUCANOMICS & GALACTANOMICS: OSTRUCTURE vs FUNCTION
Advanced structure-function relationships:
O
O3 SO
n
OH
O
n
O
HO
O3 SO
HO
-
H3
SUGAR TYPE-DEPENDENT
ANTICOAGULANT ACTION
-
O
O
CH3
O
CH3
(i) E. lucunter
2
O
VS
O
CH2OH
) S. purpuratus
II
O SO
HO
3
(h) S. franciscanus
CH OH
(j) G. crenularis
O
-
n
O
O
CH3
HO
CH2OH O3 SO
HO O
HO
O
O
O
O
n
OH
O
OSO3-
(k) H. monus
O
(l) S. plicata
O
Vitor H. Pomin - Fucanomics and
Current
status in drug discovery, mechanisms of action and role of the well-defined structures.
CHgalactanomics:
2OH
Biochimica et Biophysica Acta (BBA) - General Subjects Volume 1820, Issue 12 2012 1971-9.
FUCANOMICS & GALACTANOMICS: STRUCTURE vs FUNCTION
Advanced structure-function relationships:
SUGAR TYPE-DEPENDENT ANTICOAGULANT ACTION
Vitor H. Pomin – Marine Regular Sulfated Homopolysaccharides. Biopolymers 2009, Volume 91, Issue 8:601-609.
FUCANOMICS & GALACTANOMICS: STRUCTURE vs FUNCTION
Advanced structure-function relationships:
CONFORMATION-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.
Vitor H. Pomin – Marine Regular Sulfated Homopolysaccharides. Biopolymers 2009, Volume 91, Issue 8:601-609.
O
FUCANOMICS
GALACTANOMICS:
CH2OH & O
(l)STRUCTURE
S. plicatavs FUNCTION
HO
O structure-function relationships:
Advanced
O
SULFATION PATTERN-DEPENDENT
ANTICOAGULANT ACTION
n
OSO3-
O
CH2OH
n
O
OSO3
O
-
HOH2C
O
CH2OH
R2O
CH2OH
O
O
R1O
OH
O
OH
R3O
OR4
O
OH
n
(m) B. occidentalis (R and R = SO in ±66% and ±33%, respectively)
(n) G. crinale (R and R = SO in ±60% and ±15%, respectively)
1
R1-4 = H or SO3-
O
1
2
3
2
3
-
-
(g) S. droebachiensis
O
Vitor H. Pomin - Fucanomics and galactanomics: Current status in drug discovery, mechanisms of action and role of the well-defined structures.
O
Biochimica et Biophysica Acta (BBA) - General Subjects Volume 1820, Issue 12 2012 1971-9.
CH3
FUCANOMICS & GALACTANOMICS: STRUCTURE vs FUNCTION
Advanced structure-function relationships:
SULFATION PATTERN-DEPENDENT ANTICOAGULANT ACTION
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
O
(i) E. lucunter
O
CH3
O3-SO
HO
(a) L. grisea
O
O3 SO
OSO3O
CH3
O3-SO
HO
CH2OH
O3-SO
R2O
O
O3-SO
HO
O
OH
OR4
R1O
O
R3O
n
(m) B. occidentalis (R and R = SO in 66% and 33%, respectively)
(n) G. crinale (R and R = SO in 60% and 15%, respectively)
1
R1-4 = H or SO3-
HO
1
O
O
HO
CH3
O
O
O
CH3
O
O3-SO
O
CH3
O
HO
HO
4SO3O
O3-SO
O
O
O
CH3
R1O
4SO3-
O3-SO
O3-SO
O
O
CH3
O
-
O
O
HO
O
O3-SO
n
OH
n
HO
(h) S. franciscanus
O
R1 = H (20%) or SO3 - (80%)
(e) S. purpuratus I
-
O3-SO
O
O3 SO
2SO3-
O
O
CH3
n
2SO3-
O
CH3
CH3
(d) A. lixula
3
3
2
n
OH
2
(g) S. droebachiensis
O
O3-SO
OH
OH
O
CH3
OH
O
O
HO
HOH2C
O
O
(c) S. pallidus
O
CH3
O
O
OH
CH2OH
n
O
CH3
O3-SO
n
O
O
O
[4SO3-]
O
O3-SO
O
CH3
O
CH2OH
n
OSO3-
O
O
[2SO3-] =
HO
O
O
CH3
(l) S. plicata
O
CH2OH
(b) L. variegatus
O
O3-SO
O
n
O
CH3
HO
(k) H. monus
HO
O3-SO
-
O3-SO
OSO3O
CH3
O3-SO
HO
n
OH
O
O
CH3
O
O
O
> 2SO3-
O
HO
O
O3-SO
O
O
HO
n
O
O
CH2OH
HO
CH2OH
O3-SO
HO
O
HO
CH3
O
CH2OH
O3-SO
HO
CH3
> 2SO3-
n
O
CH3
(j) G. crenularis
O
O
(f) S. purpuratus II
CH3
HO
O
O
O3-SO
n
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: STRUCTURE vs FUNCTION
Advanced structure-function relationships:
SULFATION TYPE-DEPENDENT ANTICOAGULANT ACTION
4SO3[2SO3-] = [4SO3-]
2SO3-
Vitor H. Pomin – Marine Regular Sulfated Homopolysaccharides. Biopolymers 2009, Volume 91, Issue 8:601-609.
FUCANOMICS & GALACTANOMICS: 1ST CONCLUSIONS
 SFs and SGs are essentially found in marine organisms such as macroalgae, sea
urchins and sea cucumbers, and ascidians.
 Unlike algal SFs and SGs, the invertebrate molecules have well-defined chemical
structures which allow accurate and advanced structure-function relationships.
 Although SFs and SGs may exhibit many clinical activities, the mechanisms of actions
are 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-sulfated
3-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 on
the anticoagulation, whereas 2-sulfation alone is certainly deleterious. Ongoing NMR
relaxation studies will facilitate further explanations.
 Our dataset strongly support the the fact that the clinical actions of SFs and SGs are
not a mere consequence of sulfation degrees, but intimately correlated to specific
features: anomeric configuration, monosaccharide type, sulfation positions or patterns,
glycosylation position, molecular weights, and so forth.
FUCANOMICS & GALACTANOMICS: STRUCTURE DETERMINATION
NMR structure determination of a new red algal SG (Acanthophora muscoides)
E1/F1
A
CH3
A1
D1
E4
2-O-CH3
5.3
5.3
4.9
4.5
4.1
3.7
3.3
2.9
2.5
2.1
1.7
1.3
4.9
4.5
0.9
1H-chemical
B
G1
B1
shift (ppm)
G1
A1
CH3
b1
F1
2-O-CH3
5.3
5.3
4.9
4.5
4.1
3.7
3.3
2.9
2.5
2.1
1.7
1.3
4.9
4.5
0.9
1H-chemical
C
shift (ppm)
D1 E1/F1
2-O-CH3
C1/c1
CH3
5.3
5.3
4.9
4.5
4.1
3.7
3.3
2.9
2.5
2.1
1.7
1.3
4.9
0.9
1H-chemical
shift (ppm)
4.5
58
62
A
70
F6
C2
C5
74
3.4
2-O-CH3
B6/B6 C6 C6´
E6 E6´ D6/D6´ ´
66
A
6-unsubstituted
6-SO3-
A2-O-CH3
3.6
3.8
F6´
4.0
F6´
78
E6 E6´
C3
82
4.2
B6
F6
4.4
F1
86
E4
4.6
E1
4.8
98
C1
c1
A1
102
58
– chemical shift (ppm)
66
13C
62
94
5.0
E4
D1
B1
E1
5.2
G1
B1
F1
A1
6-unsubstituted
B
B
2-O-CH3
C6 C6´
3.4
3.6
c2-O-CH3
70
F6
78
3.8
F6´
C2
C5
74
4.0
C4
4.2
C3
82
D6
4.4
86
4.6
90
4.8
D1
b1 C1
c1
98
102
58
62
5.0
F1
G1
A1
6unsubstituted
6-SO3-
C
5.4
70
C2
78
C4
C3
82
86
90
94
C1
D1
c1
E1
102
F1
5.3
5.1
4.9
4.7
4.5
1H
4.3
5.0
4.8
4.6
1H
C5
74
5.2
2-O-CH3
C6 C6´
E6 E6´ D6/D6´
66
98
5.2
c1
4.1
– chemical shift (ppm)
3.9
3.7
3.5
3.3
4.4
4.2
4.0
3.8
– chemical shift (ppm)
3.6
3.4
3.2
– chemical shift (ppm)
94
1H
90
FUCANOMICS & GALACTANOMICS: STRUCTURE PROPOSITION
Structures of SGs from Acanthophora muscoides and Botriocladia occidentalis
α-Galp units
β-Galp units
CH2OH
CH2OSO3-
O
HO
O
OH
O
CH2OH
O
O
Unit D (~ 22%) OH
OCH3
β-Galp units
CH2OH
O
O
Unit A (~ 32 %)
α-Galp units
O
VS
R1O
CH2OSO3-
O
-
O
O
OH
O
Unit B (~ 43 %) OH
R1 = H
O
O3SO
O
O
OH
CH2OCH3
HO
O
O
O
OR
Unit C, R = H
(~ 25%)
Unit c, R= CH 3 (minor)
O
O
O
Unit F (~ 22%) OH
CH2OH
HO
OR1
or SO3-
R 2 = H (~ 33%) or SO3- (~ 66%)
R 3 = H (~ 66%) or SO3- (~ 33%)
Unit E (~ 30 %)
O
O
OR2
OR3
CH2OSO3-
O
O
O
Unit G (~ 26%) OH
O
FUCANOMICS & GALACTANOMICS: MW DIFFERENCES
Structures of Acanthophora muscoides and Botriocladia occidentalis
Am
B occ (LMW)
B occ
Refractive Index
1.0
B
A
_
+
Origin
0.8
60
0.6
40
0.4
20
0.2
8
0.0
20
30
40
50
60
Retention Time - min
70
+
FUCANOMICS & GALACTANOMICS: ANTICOAGULATION
SERPIN-DEPENDENT ANTICOAGULANT ACTIVITIES
APTT in serpin-containing plasma
5
125
A
AT/IIa
B
100
T1/T
4
75
3
50
2
25
1
Residual activity
125
0
0
50
100
150
200
250
AT/Xa
C
HCII/IIa
D
100
125
100
75
75
50
50
25
25
0
0
0.001
0.1
mg
10
mL-1
1000
0.001
0.1
10
mg mL -1
1000
Residual activity
Am
UFH
B occ (LMW)
B occ
FUCANOMICS & GALACTANOMICS: ANTITHROMBOSIS
ARTERIAL AND VENOUS MODELS, AND BLEEDING EFFECTS
60
A
50
40
30
20
120
C
80
60
40
10
0.0
Thrombus weight (%)
100
Blood loss - mL
Occlusion time (min)
70
0.4
0.8
1.2
1.6
2.0
2.4
5 10
5 10
5 10
B
80
40
Am
UFH
B occ (LMW)
B occ
0
0.0
0.25
0.50
mg kg-1
0.75
1.00
5 10 (mg kg-1)
FUCANOMICS & GALACTANOMICS: PROTHROMBOTIC EFFECTS
Activation of factor XII
Vmax –mOD min -1
30
25
20
15
10
5
0
1 50
Heparin
1 50
_
1 50
+
B.occidentalis
1 50 (mg mL-1)
A. muscoides
FUCANOMICS & GALACTANOMICS: 2rd CONCLUSIONS
 Unlike the SG from B. occidentalis, the recently characterized SG from A. muscoides is
structurally 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 of
structure-function relationships in anticoagulation and antithrombosis with the natural
low-MW of A. muscoides;
 The native SG from B. occidentalis has high anticoagulant activity due to its significant
activity on serpins (AT and HCII) and proteases (mostly thrombin). It also shows high
serpin-independent anticoagulant activity. The low-MW form lost both activities. SG from
A. 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 the
venous model, however, at only small doses. SG from B. occidentalis is prothrombotic at
higher doses due to its capacity in activate factor XII.
 As opposed to heparin which is highly hemorragic, neither one of the red algal SGs
have shown this side effect.
 Through this comparative study using red algal SGs, we have dissociated the
anticoagulant (both serpin-dependent and serpin-independent), antithrombotic (both
arterial and venous) and hemorragic effects, as compared with the standard heparin.
FUCANOMICS & GALACTANOMICS: ACKNOWLEDGEMENTS
 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
Universidade Federal
do
Rio de Janeiro