Characterization of HEK293 Cells Stably Transfected with EPCR ...

Characterization of HEK293 cells stably transfected
with EPCR cDNA
Study of binding of recombinant mutated protein C on
endothelial cell protein C receptor
B. Saposnik Ph.D.
V. Vermillard
M. Aiach Ph.D.
S. Gandrille Ph.D.
U. 765
C. Esmon Ph.D.
Cardiovascular Biology Department
Head: J. Emmerich, M.D., Ph.D.
Head: C. Esmon Ph.D.

Binding of PC
to its receptor
EPCR
Protein C
EPCR
Mosnier et al, Blood, 2007, 109, 3161-3172

Domaine sérine protéase
Serine protease domain
Preparation by site-directed mutagenesis
of a series of recombinant protein C
bearing mutations in the RGDS sequence
A A A
177 182
*
R R G D S P
A
P
A
K
A
Y
A
E
A
N
T
A
Vers la membrane
To the Gla domain
Dahlbäck et Villoutreix (JTH 2003)

Evaluation of PC/EPCR interaction by mean of flow cytometry analysis
PC binding revelation by incubation
1/ with anti-PC mAb (mouse IgG)
2/ with anti-mouse IgG antibody
phycoerythrin ( )-labelled
Cell over-expressing
EPCR
rWt PC or rMutated PC

Preparation of cells (HEK293) over expressing EPCR
Stable transfection of HEK 293 cells
Selection of clones with a high level of EPCR expression
using flow cytometry analysis
Selection by anti-EPCR mAb binding
Selection by rWT PC binding
Anti-EPCR antibody
Cell over-expressing
EPCR

Selection of transfected HEK 293 cells
20
15
Counts
10
5
0
1 10 1 10 2 10 3 10 4
Counts
Counts
30
25
20
15
10
5
0
HEK 293 EA.hy 926
(EPCR " - ") (EPCR+)
1 10 1 10 2 10 3 10 4
Fluorescence

Kd of PC / EPCR interaction = 30 nM (Fukudome K and Esmon CT J Biol Chem, 1994)
Binding of increasing rWT PC concentrations
200
10 20
40
60
100
0
The Kdapp observed (35 nM) is consistent with a binding of PC on EPCR

Binding of four mutated recombinant PC to transfected HEK cells
Mean fluorescence
300
200
100
0
25
50
Protein C concentration (nM)
PC WT
RGD178-180AAA
R178A
G179A
D180A

Comparison of EPCR detection methods
Counts
Counts
30
25
20
15
10
5
0
30
25
20
15
10
5
0
HEK 293
1 10 1 10 2 10 3 10 4
EA.hy 926
1 10 1 10 2 10 3 10 4
Fluorescence
Cells + PC + anti-PC antibody
Cells + anti EPCR antibody (JRK 1494)

Correlation between the two detection methods
Ratio
Mean fluorescence of HEK transfected cells
Mean fluorescence of HEK non transfected cells
50
JRK 1494 + anti-mAb-PE
40
30
20
10
R2= 0.70
0
0 10 20 30 40 50
PC + anti-PC + anti-mAb-PE

This validates the cell clones we used to study mutated recombinant PC
binding on EPCR
Remains to be performed :
Inhibition of PC binding on cells incubated with anti-EPCR antibody