PA-IIL/Fuc - quonia.cz

Transkript

Application Potential of Lectins
– Sugar Binding Proteins
Michaela Wimmerová
Wimmerová
National Centre for Biomolecular Research
&
Department of Biochemistry
Faculty of Science, Masaryk University, Brno,
Brno, Czech Republic
Masaryk University, Brno, Czech Republic
National Centre for Biomolecular Research & Department of Biochemistry
Protein/carbohydrate recognition at the surface of cells
(and glyco-conjugates)
Immunity
Cell
Pathogens
Development
Immunity &
Development
Signalling
Bacteria
Virus
Toxins
Antibodies
Diferentiation
Morphogenesis
Cancerogenesis
Pathogenesis
Hormones
…
Science, 2003
Lectins
- Sugar binding proteins
- Ubiquitous
- Generally week affinity
towards monosaccharides
- Avidity effect through multivalency
-Tandem repeat
-Oligomerisation
-Surface presentation
ABH(O) blood group system
OH CH OH
2
Gal
GalNAc
O
HO
Gal
OH CH2OH
O
OH
NAc
O
HO
CH2OH
O
HO
O
AcN
O
O
OH
O
CH3
OH
GlcNAc
Fuc
Blood group A
B
H (0)
Type 1 core
Bacterial strategies for
exploiting host glycans
AB
Fimbrial
Soluble
lectins
adhesins, flagellins, cap flagelar proteins
5 toxins
FimH, CupB,…
Cyanovirin,
Choleratoxin,
Choleratoxin
calcium
, enterotoxins,…
enterotoxins
dependent
,… lectins
Pseudomonas aeruginosa
Gram-negative bacterium
Opportunistic human pathogen
Colonization – cystic fibrosis patients (90% mortality)
Production of two soluble lectins - PA-IL and PA-IIL
Protein
gene
size
specificity
affinity
PA-IL
lecA
121 aa
D-Gal
medium (mM)
PA-IIL
lecB
114 aa
L-Fuc >>> D-Ara>D-Man
strong (µ
µM)
Virulence of bacterium –
adhesion to host cell surface
biofilm formation
secretion of hydrolytic enzymes
and toxic compounds
In vivo study on mice
Pulmonary lesions from
P. aeruginosa
Chemani et al., Infect Immun , 77, 2065 (2009)
(coll. B. Guery, K. Faure – CHU Lille)
Bacterial load in lungs
Inoculum = 5×
×106 UFC/smouse
n = 10/group. * p < 0.05, *** p < 0.001 vs PAO1
In vivo study on mice
(coll. B. Guery, K. Faure – CHU Lille)
Experimental model of acute pneumoniae in mice
Co-administration endotracheal PA + sugars (15mM)
Survival (days)
BalB/c
Inoculum = 5×
×107 UFC/mouse
n = 20/group * p < 0.05 vs PAO1
Chemani et al., Infect Immun , 77, 2065 (2009)
Experimental methods to study
biomolecular interactions
Molecular biology and Biochemistry
Gene
Protein
Structure
Specificity
Crystallography
ELLA
SPR
Glycochips
Mosquito, Minstrel UV
BiaCore 3000
Mutagenesis
Affinity
Isothermal titration
microcalorimetry
SPR
VP-ITC, VP-DSC, ITC200
Lectin PA-IIL from Pseudomonas aeruginosa
Wimmerova et al., J Mol Model ,15, 673 (2009)
Kd ~ 10-6 M
PA-IIL tetrameric arrangement
Mitchell et al., Nature Struct. Biol. 9, 918 (2002),
Mitchell et al., Proteins, 58, 735(2005)
PA-IIL binding site with L-fucose (1 Ǻ)
In vitro testing of more then 60 fucose-based synthetic
competitors (synthesised in René Roy laboratory (Montreal, Canada))
25
OH
O
N N
N
NHAc
O HO
O
20
OH
O HO
O
15 HOHO
COOMe
O HO
N N
N
NHAc
OH
N
O
Dendrimers of Fuc14GN
NHAc
O
OH
O
N N
O
O
OH
O
O
Isoxazole-(R)
OH
21
HO
OH
L-Fuc(α
α1-4)GlcNac
O
O
OH
OH
HO
N AcHN
N
N
O
HO
HO
HO
Triazole-(R)
O
HO
Natural ligands
22
addition at C5
O
OH
AcHN
other additions at C1
H
O
10
5
N
N
N
N
N
H
N
NHAc
OH
O
O
HO
OH
HN
23
N
N
NHAc
N
HO
O
0
OH
28
27
30
41
43
59
56
57
55
10
9
1
4
5
52
51
50
is a
fu c
Me
o se
t -a
-L -F
uc
48
49
12
O
OHO
H
O
L ew
15
14
16
13
21
18
19
22
20
HO
24
O
OH
O
O HO
N
N
26
O HO
23
inhibition potency
HO
OH
O
OH
O
inhibitors
National Centre for Biomolecular Research
& Department
of Biochemistry
Marrot
et al.
ChemMedChem
2007
PA--IIL like lectins from other pathogens
PA
Pseudomonas
aeruginosa
Ralstonia
solanacearum
Chromobacterium
violaceum
Specificity loop: amino acids 22-23-24
PA-IIL: L-Fuc >>> D-Man
MeFuc
Lewis A
KD ~ 0.43 µM
KD ~ 0.21 µM
RS-IIL: D-Man >>> L-Fuc
MeMan
KD ~ 0.23 µM
MeFuc
2-FucLac
3-FucLac
MeMan
KD ~ 1.7 µM
KD ~ 0.13 µM
KD ~ 0.08 µM
KD ~ 2.7 µM
CV-IIL: L-Fuc > D-Man
BclA: D-Man
Burkholderia
Ce(noce)pacia
Burkholderia
ambifaria
Burkholderia
dolosa, …
Interaction between fucose and calcium in lectins
MBP-C/Fuc (1RDI)
MBP-A/Fuc (1KWW)
P-selectin/SLex (1G1T)
PA-IIL/Fuc (1GZT)
Ralstonia solanacearum
• aggressive soil phytopathogen
• may infect animals and humans
RSL – fucose-binding lectin
(Sudakevitz et al. (2002) J. Biochem. 132, 353-358,
Kostlánová et al. (2005) J. Biol. Chem. 280, 27839-27849 )
Xyloglucan :
Fuc
Not calcium-dependent !!!
a1,2
Gal
b1,2
Xyl
KD for MeMe-α-Fuc = 7.3 10-7 M
a1,6
Xyl
a1,6
Xyl
a1,6
Glc b1,4 Glc b1,4 Glc b1,4 Glc
The high affinity can be attained
with very different binding sites
Plant lectins
KD ~ 10-3-10-4
RSL/MeFuc
PA-IIL/Fuc
BC2L-C lectin from B. cenocepacia
N-terminal part
Related to TNF-like domains
.
C-terminal part
Ca-dependent lectin domain
homologous to lectins from PAIIL superfamily.
N-terminal domain
1aa
C-terminal domain
129aa
157aa
272aa
Glycan array with 377 human related glycans
(Consorcium for functional glycomics,
glycomics, Core H)
Fuc
Man
Gal
GlcNAc
Carbohydrate recognition
Immunity
Development
Signalling
Diferentiation
Morphogenesis
Cancerogenesis
Pathogenesis
…
L
E
C
T
I
N
S
Histochemistry
Disease-related
glycoconjugate alteration
Blood typing
Cell separation
Bone marrow separation
Mitogenic stimulation
Therapeutic targetting
…
Carbohydrate recognition
mainly used:
Plant lectins
Histochemistry
L
Disease-related
E
glycoconjugate alteration
Generally week affinity
C
towards monosaccharides
Blood typing
Lectins
pathogens
are promising
Increasing
through from
multivalency
T
Cell separation
-Tandem repeat
targets
for
further
protein
engineering
I
-Oligomerisation
Bone marrow separation
-Multivalent ligands
N Mitogenic stimulation
S
engineered lectins,
Therapeutic targetting
artificial lectins
…
Outcomes
Drug design
- antiadhesive
compounds – alternative way for solving
antibiotics resistence
~ glycomimetics inhibitors
Protein engineering of lectins
for bioanalysis and biotechnology
- design
of the highhigh-affinity lectins with desired
properties
mutagenesis in silico & in vitro
Co
Co--operations on lectin projects
CERMAV-CNRS,
CERMAVCNRS, Grenoble
Anne Imberty
Annabelle Varrot
Serge Pérez
ESRF, Grenoble
Edward P. Mitchell
Gianluca Cioci
University of Ontario
Miguel Valvano
Glycobiochemistry
Martina Pokorná
Peter Kysel
Nikola Kostlánová
Jana Mrázková
Marie Pokorná
Petra Rozumová
Ondřej Šulák
Gita Jančaříková
Jan Komárek
Lenka Malinovská
Michal Ďurech
Josef Houser
Lucie Olšová
Lenka Brůnová
Lucia Slepánková
Eva Dejmková
Andrea Fleková
University of Vienna
Computational chemistry
Paul Kosma
Jaroslav Koča
Jan Adam
Zdeněk Kříž
$$$
Martin Prokop
Czech Ministry of Education
Sushil K. Mishra
Czech Science Foundation
Mizutani Foundation
for Glycosciences
French Ministry of Science
French Cystic Fibrosis foundation
Affinity of bacterial lectins towards ligands (ITC)
Soluble lectins
PA-IIL/fucose
PA-IIL/MeFuc
CV-IIL/MeFuc
RS-IIL/MeMan
RSL / MeFuc
2.9 10-6 M
4.3 10-7 M
1.7 10-6 M
2.3 10-7 M
7.3 10-7 M
PA-IIL/Lewis a
CV-IIL / 2’-fucosyllactose
CV-IIL / 3’-fucosyllactose
RSL / 2’-fucosyllactose
2.1 10-7 M
1.3 10-7 M
8.0 10-8 M
2.5 10-7 M
Adhesins
E. coli fimbrial lectins
FimH /mannose 2.3 10-6 M
Cholera AB5 toxin
CTB/GM1os
4.3
10-8
AB5 toxins
M
Tetanus toxin
NeNT/GD1b
NeNT/GT1b
4.5 10-8 M
5.5 10-8 M
Perret S et al, Biochem J 2005; Kostlánová N et al, J Biol Chem 2005; Pokorná M et al, Biochemistry 2006;
Bouckaert J et al, Mol Microbiol 2005; Turnbull WB et al, J Am Chem Soc 2004; Krell T et al., Biotechnol Appl
Biochem 2003
Isothermal titration microcalorimetry
Ka (x104)
∆G
∆H
[M-1]
[kJ/mol]
[kJ/mol]
PA-IILwt
Adam et al, BMC Struct Biol 2008
-T∆S
[kJ/mol]
Mutant
S22A
reverse specificity
-41.3
4.9
order to
-17.8
-5.9
monosaccharides !
Me-α
α-Fuc
235
-36.4
Me-α
α-Man
1.4
-23.7
Me-α
α-Fuc
26.4
-30.9
-37.1
6.2
Me-α
α-Man
36.0
-31.7
-24.3
-7.4
Me-α
α-Fuc
418
-37.8
-33.9
-3.8
Me-α
α-Man
1.9
-24.4
-16.7
-7.7
Me-α
α-Fuc
524.3
-38.4
-40.1
1.8
Me-α
α-Man
2.3
-24.9
-27.1
2.1
S22A
S23A
G24N
mutants S23A, G24N:
The same specificity order
to monosaccharides
Mutagenesis in silico - Program TRITON
MODELLER
modelling of mutant structures from a source protein
TRITON
graphic interface – interactive specification of input data
and visualisation of outputs
MOPAC / DRIVER
AutoDock
calculation of the reaction
pathway
Calculation of the binding
energies and binding modes
Prokop et al, Bioinformatics 2008
http://ncbr.chemi.muni.cz/triton
Experimental methods to study
biomolecular interactions
Molecular biology and Biochemistry
Gene
Protein
Structure
Specificity
Crystallography
ELLA
SPR
Glycochips
Mosquito, Minstrel UV
BiaCore 3000
Mutagenesis
Affinity
Isothermal titration
microcalorimetry
SPR
VP-ITC, VP-DSC, ITC200

PA-IIL/Fuc - quonia.cz

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