smalp conference, september 18thsmalp conference, september 18th. alexandra desrames . inserm u1134...

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


Lux, Blood 2016

Band 3 : major protein









-> Study of RBCs protein complexes using SMA

Lux, Blood 2016

Band 3 : major protein

RhD/RhCE : Rh blood group Ag







4

A. Desrames – SMALP conference – Sept 18th

Hypotonic lysis

Methods

Removal of spectrin network Spectrin network is preserved

RBC


Hypotonic lysis

RBC

Methods


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



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



8

A. D

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con

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t 18

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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 Ghosts (LIS)

SMA

LIS-SMALP

5 mM NaPO4 pH8

Pellet/supernatant

Ultracentr.

0.91

1.25

0.99

1.10


Superdex 200 increase 3.2/300 Flow rate 0.08 ml/min

Gel filtration


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 Ghosts (LIS)

SMA

LIS-SMALP

5 mM NaPO4 pH8

Pellet/supernatant

Ultracentr.

0.91

1.25

0.99

1.10



Negative staining electron microscopy, 30 000X

(JM Verbavatz, ImagoSeine platform, Jacques Monod Institute)

Gel filtration


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 Ghosts (LIS)

SMA

LIS-SMALP

5 mM NaPO4 pH8

Pellet/supernatant

Ultracentr.

0.91

1.25

0.99

1.10


-> What about protein conformation ?


Gel filtration


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)


(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)



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



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


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 :



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


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


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


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


RhD + fractions loaded on GF column :

Superdex 200 increase 10/300 Flow rate 0.4 ml/min



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


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

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con

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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


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


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

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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)


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 !


smalp conference, september 18thsmalp conference, september 18th. alexandra desrames . inserm u1134...

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