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SMALP conference, September 18th
Alexandra Desrames
INSERM U1134 / National Institute for Blood Transfusion
Detergent-free isolation of native red blood cell membrane complexes
1
RBC membrane organization
Lux, Blood 2016
- Human Red Blood Cell (RBC) membrane :
-> Proteins carrying blood group antigens (ABO, Rh …)
-> Protein network involved in cell shape and mechanical properties
- Previous biochemical studies performed in presence of detergent :
-> Loss of complexes
-> Large number of models
2 A. Desrames – SMALP conference – Sept 18th
RBC membrane organization
Lux, Blood 2016
Band 3 : major protein
- Human Red Blood Cell (RBC) membrane :
-> Proteins carrying blood group antigens (ABO, Rh …)
-> Protein network involved in cell shape and mechanical properties
- Previous biochemical studies performed in presence of detergent :
-> Loss of complexes
-> Large number of models
3 A. Desrames – SMALP conference – Sept 18th
RBC membrane organization
-> Study of RBCs protein complexes using SMA
Lux, Blood 2016
Band 3 : major protein
RhD/RhCE : Rh blood group Ag
- Human Red Blood Cell (RBC) membrane :
-> Proteins carrying blood group antigens (ABO, Rh …)
-> Protein network involved in cell shape and mechanical properties
- Previous biochemical studies performed in presence of detergent :
-> Loss of complexes
-> Large number of models
4
A. Desrames – SMALP conference – Sept 18th
Hypotonic lysis
Methods
Removal of spectrin network Spectrin network is preserved
RBC
5 A. Desrames – SMALP conference – Sept 18th
Hypotonic lysis
RBC
Methods
6 A. Desrames – SMALP conference – Sept 18th
RBC membrane solubilization by SMA
RBC Ghosts (LIS)
SMA
LIS-SMALP
5 mM NaPO4 pH8
Pellet/supernatant
Ultracentr.
Same migration profile between ghosts and SMALPs -> Proteins are soluble G3PD is lost during washing step with buffer 300 mM NaCl, 50 mM NaPO4 pH 8
Instant Blue staining
SDS-PAGE and Coomassie staining
7 A. Desrames – SMALP conference – Sept 18th
RBC membrane solubilization by SMA
RBC Ghosts (LIS)
SMA
LIS-SMALP
5 mM NaPO4 pH8
Pellet/supernatant
Ultracentr.
Same migration profile between ghosts and SMALPs -> Proteins are soluble Spectrins, adducins, 4.1 and actin are removed by VLIS treatment G3PD is maintained in SMALPs
Ghosts (VLIS) VLIS-SMALP Pellet/supernatant
0.3 mM NaPO4 pH8
Instant Blue staining -> Further analysis by gel filtration
SDS-PAGE and Coomassie staining
SDS-PAGE and Coomassie staining
8
A. D
esrames – SM
ALP
con
ference – Sep
t 18
th
Band 3
4.1
Ankyrin
4.2
p55 Actin G3PD
Spectrin
Stomatin 25
70
55
35
100
250
130
0 .0 0 .5 1 .0 1 .5 2 .0 2 .5
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
S M A J D
V e (m l)
mA
U 2
80
nm
1 2 3 4 5 9 12
1 2 3 4 5 9 12
RBC membrane solubilization by SMA
RBC Ghosts (LIS)
SMA
LIS-SMALP
5 mM NaPO4 pH8
Pellet/supernatant
Ultracentr.
0.91
1.25
0.99
1.10
Instant Blue staining
Superdex 200 increase 3.2/300 Flow rate 0.08 ml/min
Gel filtration
9 A. Desrames – SMALP conference – Sept 18th
Band 3
4.1
Ankyrin
4.2
p55 Actin G3PD
Spectrin
Stomatin 25
70
55
35
100
250
130
0 .0 0 .5 1 .0 1 .5 2 .0 2 .5
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
S M A J D
V e (m l)
mA
U 2
80
nm
1 2 3 4 5 9 12
1 2 3 4 5 9 12
Fraction 1 : no protein Excessive SMA ? Pieces of membranes ?
RBC membrane solubilization by SMA
RBC Ghosts (LIS)
SMA
LIS-SMALP
5 mM NaPO4 pH8
Pellet/supernatant
Ultracentr.
0.91
1.25
0.99
1.10
Instant Blue staining
Superdex 200 increase 3.2/300 Flow rate 0.08 ml/min
Negative staining electron microscopy, 30 000X
(JM Verbavatz, ImagoSeine platform, Jacques Monod Institute)
Gel filtration
10 A. Desrames – SMALP conference – Sept 18th
Band 3
4.1
Ankyrin
4.2
p55 Actin G3PD
Spectrin
Stomatin 25
70
55
35
100
250
130
0 .0 0 .5 1 .0 1 .5 2 .0 2 .5
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
S M A J D
V e (m l)
mA
U 2
80
nm
1 2 3 4 5 9 12
1 2 3 4 5 9 12
Fraction 1 : no protein Fractions 2-3 enriched in spectrin and Band 3 Fractions 3-4-5 enriched in proteins 4.1 and 4.2 Same profile with VLIS-SMALPs
RBC membrane solubilization by SMA
RBC Ghosts (LIS)
SMA
LIS-SMALP
5 mM NaPO4 pH8
Pellet/supernatant
Ultracentr.
0.91
1.25
0.99
1.10
Instant Blue staining
-> What about protein conformation ?
Superdex 200 increase 3.2/300 Flow rate 0.08 ml/min
Gel filtration
11 A. Desrames – SMALP conference – Sept 18th
SMALPs-RhD interaction with anti-RhD antibodies
Rh blood group system : 54 antigens carried by protein RhD or RhCE
D is the Rh antigen most involved in allo-immunisation (pregnancy, transfusion)
-> Several anti-D antibodies available in the lab
12 TM
RhD protein
RhCE protein
RHD
gene
RHCE
gene
1p34-36
RhD-positive RhD-negative
RhCE protein
RhD+/RhD- genotypes and phenotypes
No RhD protein
(Deleted RHD gene)
12 A. Desrames – SMALP conference – Sept 18th
(Mouro et.al, Nat Genetics, 1993)
1/ SMALP-RhD immunopurification
RhD -
Free SMA -
A1 A2 A3 A4 A5 A1 A2 A3 A4 A5 130
100
70
55
35
25
15
Anti-RhD LOR15C9
Ab LOR15C9 reacts non specifically with free SMA
0 .0 0 .5 1 .0 1 .5 2 .0 2 .5
0
2 0 0
4 0 0
R h p o s
V e (m l)
mA
U 2
80
nm
RhD+ SMALPs
RhD detection in gel filtration fractions :
- RhD is mainly detected in fraction 4 -> selected for IP
- Gel filtration profile from RhD- is similar -> fraction 4 taken as negative control for IP
4
13
(Apoil et.al, Br J Haematol. 1997)
A. Desrames – SMALP conference – Sept 18th
1/ SMALP-RhD immunopurification
Immunopurification with monoclonal Ab F5, conformation-dependent (Goossens et.al, JI methods, 1987)
Ab bound on protein A sepharose then pre-cleared SMALPs are added
Washes in SMA Buffer
Elution with Laemmli buffer
Detection on WB with Ab LOR15C9, conformation-independent
E W4 FT FT W4 E
anti-RhD (F5)
Rh+ Rh-
35 RhD
RhD is partially retained by and eluted from the F5-resin
14 A. Desrames – SMALP conference – Sept 18th
1/ SMALP-RhD immunopurification
Immunopurification with monoclonal Ab F5, conformation-dependent
Ab bound on protein A sepharose then pre-cleared SMALPs are added
Washes in SMA Buffer
Elution with Laemmli buffer
Detection on WB with Ab LOR15C9, conformation-independent
E W4 FT E W4 FT FT W4 E
buffer anti-RhD (F5)
Rh+ Rh-
35 RhD
RhD is partially retained by and eluted from the F5-resin RhD is not detected in eluate from resin alone
RhD conformation is conserved in SMALPs 15
A. Desrames – SMALP conference – Sept 18th
2/ Microscale Thermophoresis
Protein-ligand interaction method, based on complexes mobility in a thermic gradient
Size of complexes (sample in presence or absence of ligand) impacts on their mobility
Samples (analyte or ligand) must be fluorescent
Method recently used with SMALPs (Bada-Juarez et.al, BBA 2020)
What about RBCs-SMALPs ? 16 A. Desrames – SMALP conference – Sept 18th
2/ Microscale Thermophoresis
Samples must be homogenous for MST (advice from MST platform members)
Ghosts -> VLIS-SMALPs -> GF -> RhD+ fraction :
Negative staining electron microscopy, 30 000X
(JM Verbavatz, ImagoSeine platform, Jacques Monod Institute)
Heterogenous material in samples : - Liposomes (L) - Nanodiscs of various sizes (10-50 nm diameter)
L
L
-> First isolation step using sucrose gradient ultracentrifugation 17
A. Desrames – SMALP conference – Sept 18th – unpublished data
2/ Microscale Thermophoresis – Sample preparation
Western blot anti RhD :
S 1
250
130
100
70
55
35
25
Spectrin
Band 3
4.2 4.1
G
Stomatin
Ankyrin
2 3 4 5 6 7 8 9 10 12 11
Pon
ceau
stain
ing
W
B RhD
35
25
SMALPs loaded on a sucrose gradient (2,5 – 5 – 10 – 30%)
Sucrose
(B Raynal, Molecular Biophysics Platform, Pasteur Institute)
RhD is mostly detected in fractions 8-9 Band 3, 4.1, 4.2 and stomatin are also present in these fractions
-> Second isolation step using gel filtration 18
A. Desrames – SMALP conference – Sept 18th – unpublished data
5 1 0 1 5 2 0 2 5
0
2 5
5 0
7 5
1 0 0
R h p o s F 8
m l
mA
U 2
80
nm
2/ Microscale Thermophoresis – Sample preparation
RhD + fractions loaded on GF column :
Superdex 200 increase 10/300 Flow rate 0.4 ml/min
RhD+
35
25
250
130
100
70
55
35
25
15
RhD+ GF fraction also contains Band 3, 4.1 and 4.2 proteins
Co
om
assie sta
inin
g
WB (Anti-D)
19
A. Desrames – SMALP conference – Sept 18th – unpublished data
5 1 0 1 5 2 0 2 5
0
2 5
5 0
7 5
1 0 0
R h p o s F 8
m l
mA
U 2
80
nm
2/ Microscale Thermophoresis – Sample preparation
RhD + fractions loaded on GF column :
Superdex 200 increase 10/300 Flow rate 0.4 ml/min
Negative staining electron microscopy, 23 000X
(JM Verbavatz, ImagoSeine platform, Jacques Monod Institute)
RhD+
Monodisperse nanodiscs, mean diameter = 12.7 nm (ImageJ software)
-> Sample quality is acceptable for MST
35
25
250
130
100
70
55
35
25
15
WB (Anti-D) C
oo
ma
ssie stain
ing
20
A. Desrames – SMALP conference – Sept 18th – unpublished data
3/ Microscale Thermophoresis – Results
SMALPs fluorescent labelling : eosin-5-maleimide (EMA), lexc = 524 nm
EMA binds to Band 3 and RhD proteins (same complex)
RhD-SMALPs incubated with EMA then washed by Amicon
Monoclonal Ab MS26 : epitope D9
21 Cartron JP, Rouillac C, Le Van Kim C, Mouro I, Colin Y. Transfus Clin Biol. . 1996
A. D
esrames – SM
ALP
con
ference – Sep
t 18
th
3/ Microscale Thermophoresis – Results
SMALPs fluorescent labelling : eosin-5-maleimide (EMA), lexc = 524 nm
EMA binds to Band 3 and RhD proteins (same complex)
-> MST is not suited for RhD-SMALPs analysis
(B Baron, Molecular Biophysics Platform, Pasteur Institute)
22
SMALPs RhD+
SMALPs RhD-
SMALPs + buffer
SMALPs + Ab MS26
SMALPs do not diffuse in the thermic gradient No difference between RhD+ and RhD- samples A. Desrames – SMALP conference – Sept 18th – unpublished data
4/ ELISA assays – Preliminary results
First ELISA assays with SMALPs described very recently (Ayub et. al, BBA July 2020)
SMALPs prepared for MST were used for ELISA
First trials with anti-Band 3 Ab (most abundant protein)
-> Bric155, linear epitope
1000
2000
4000
5000
8000
10000
16000
20000
32000
0 .0
0 .5
1 .0
1 .5
2 .0
D a ta 1
S M A L P s d ilu tio n
OD
49
0 n
m
B ric 1 5 5Bric155 + anti-mouse-HRP
- Ctrl- : no SMALP, Ab background - Strong dose-dependant signal
C 911
N 1
360
P854L E658K
V557M
T552I K551N
P548L
Bric155 Band 3
23 A. Desrames – SMALP conference – Sept 18th – unpublished data
SMALPs prepared for MST were used for ELISA
First trials with anti-Band 3 Ab (most abundant protein)
-> Bric155, linear epitope -> Bric6, conformation-dependent
500
1000
2000
4000
5000
8000
10000
16000
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
B r ic 6
S M A L P s d ilu tio n
OD
45
0 n
m
O D 4 5 0 n m
- High Ab background - Weak dose-dependant signal
1000
2000
4000
5000
8000
10000
16000
20000
32000
0 .0
0 .5
1 .0
1 .5
2 .0
D a ta 1
S M A L P s d ilu tio n
OD
49
0 n
m
B ric 1 5 5Bric155 + anti-mouse-HRP
- Ctrl- : no SMALP, Ab background - Strong dose-dependant signal
Bric6 + anti-mouse-HRP
First ELISA assays with SMALPs described very recently (Ayub et. al, BBA July 2020)
4/ ELISA assays – Preliminary results
Band 3
24
A. D
esrames – SM
ALP
con
ference – Sep
t 18
th
Conclusions
Perspectives
SMALPs can be generated from RBC membrane
Most proteins observed in ghosts are detected in SMALPs
RhD can be purified by IP with conformation-dependent antibody
Semi-purification by sucrose gradient followed by GF leads to homogenous nanodisc samples
RBC-SMALPs can be used in ELISA assays but not in MST
Small scale preparation (200 µl RBCs): study of pathological RBCs (genetic or infectious diseases)
25
Mass spectrometry on each GF fraction (ongoing)
ELISA to be continued Large scale preparation (> 1 ml RBCs)
A. Desrames – SMALP conference – Sept 18th
INSERM U1134/INTS/Université de Paris, Paris, France
Sandrine Genetet Païline Delcourt Dominique Goossens Isabelle Mouro-Chanteloup
Molecular biophysics platform, Pasteur Institute, Paris, France
Patrick England Bertrand Raynal Bruno Baron
ImagoSeine platform, Jacques Monod Institute, Paris, France
Jean-Marc Verbavatz
Thank you !
26 A. Desrames – SMALP conference – Sept 18th
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