Table S1. Molecular and immunological features of patients
P1
Origin
Sex
Age at presentation (mo)
Age at molecular diagnosis (mo)
Mutationa
Effect
Italy
F
24
96
P2
P3
Saudi Arabia Saudi Arabia
M
F
16
8
60
84
P4
P5
Turkey
F
12
12
Turkey
F
25
32
normal
values
c.408A>T
skip exon 5
T136VfsX26
c.247T>C
C83R
c.247T>C
C83R
c.257-2A>T
N86_L87del
+G88R
c.95_97delTAA
I33del
serum IgG (mg/dl)b
180
120
135
<150
<124
184-794 (7-12mo)d
507-1407(12-36mo)d
serum IgA (mg/dl)b
<6,6
<6,6
<6,6
<6,6
<6,6
9-107 (7-12mo)d
18-171 (12-36mo)d
serum IgM (mg/dl)b
81
400
200
80
1990
4-216 (7-12mo)d
18-171 (12-36mo)d
CD3+ (%)b
73
75
76
56
79
53.2-83.1 (4-12mo)e
56.9-83.1 (12-36mo)e
CD19+ (%)b
16
19
20
35
15
7.7-35.3 (4-12mo)e
11.4-43.9 (12-36mo)e
CD27+ IgD+ (%)c
5
10
-
4.7
3.6
3.6-13.4 (12-36mo)e
5.3-22.9 (60-120mo)e
CD27+ IgD- (%)c
0.1
0.5
-
0.0
0.0
0.7-5 (12-36mo)e
2.5-16.3 (60-120mo)e
Immunological features
a listed in CD40base (http://bioinf.uta.fi/CD40base/)
b at presentation
c at molecular diagnosis
d modified from Soldin, S.J., Brugnara, C, & Hicks, J.M., (1999). Pediatric reference ranges (3rd ed.). Washington, DC: AACC Press
e Moratto D., Giliani S., et al. unpubblished observations
Figure S1. Mammalian CD40 proteins alignment
Sequence alignment of CD40 proteins of the indicated species (numbers indicate the starting
amino acid of each aligned box). The positions of the mutations (P2, P4, and P5) are indicated
above the alignment. The conserved amino acids are indicated in reverse type, while other
residues that are also conserved among the sequences are shaded in gray.
Figure S2. Expression analysis of CD40 mutants in primary cells
Surface expression of CD40 in primary CD19+ cells from control (C) and from P1, P2, P4 and
P5 patients, analyzed by flow cytometry with either mAb 5C3-FITC conjugated (upper panels)
or mAb 5C3-PE conjugated (lower panels). Isotype control staining is shown as shaded peaks.
Cells were acquired using a FACSCalibur (BD Biosciences) and analyzed by the FlowJo
software (Tree Star).
Figure S3. Glycosylation analysis
(A) Immunoblot with anti CD40 antibody on control B-LCL and B lymphocytes lysates before
and after deglycosylation with EndoH. A composite pattern, due to partial sensitivity to the
enzyme, likely suggestive of the presence of hybrid sugars, can be observed in wild-type CD40
from a B-LCL. Sensitivity was shown also by CD40 from primary B cells. (B) Cartoon
representing key steps of the N-glycan processing of glycoproteins in the exocytic pathway. Nglycans are transferred on a native protein as high mannose oligosaccarides during protein
synthesis and play a key role in the protein quality-control process in the ER. Indeed, if the
protein folds correctly, it is transported to the Golgi complex where the sugars are further
trimmed, thus acquiring the higher molecular weight complex structure found in the mature
proteins that are targeted to the plasma membrane. If the protein is trapped in the ER, the Nglycans remain in the high-mannose state, and, due to misfolding, it can be delivered to
degradation. Crucial glycosylation steps can be inhibited by drugs as indicated. (C) Pulse-chase
experiment on C and CD40 mutants. Cells were pulsed with 35S Met-Cys (Perkin Elmer) for 1
hour in the absence (-) or presence of 1 mM Castanospermine (Csp). Untreated cells were also
chased for 12 hours in the absence (-) or presence of either 1 mM Deoxymannojirimycin (DMJ)
or 10 µg/ml Brefeldin A (BFA). Cells were lysed at room temperature in RIPA buffer
supplemented with protease inhibitors (Roche) and 10 mM N-ethylmaleimide. CD40 was
immunoprecipitated with mouse anti-CD40 (Lob11, Santa Cruz Biotechnology), and analyzed by
11% SDS-PAGE. Results are representative of two independent experiments.
Figure S4. Localization of CD40 mutants
Intracellular double immunofluorescence staining of CD40 (mouse anti-CD40, lob11, Santa Cruz
Biotechnology, followed by goat anti-mouse AlexaFluor568) with either TGN46, a trans Golgi
network marker (rabbit anti-TGN46, Sigma, followed by goat anti-rabbit AlexaFluor488), or
internalized transferrin localizing in the endosomal recycling compartment (transferrin
AlexaFluor488-conjugate). Analysis was performed by the LSM510META confocal microscope
(Zeiss) with a EC Plan-Neofluar 100x/1.30 oil objective and a scan zoom of 1-1.5x. Digital
images were analyzed with the LSM Image Browser and processed with Adobe Photoshop 6.0.
Co-localization is visible as yellow staining in the merged micrographs. Scale bars, 5 µm.
Figure S5. CD40 expression in HEK-293 transfectants
(A) Flow cytometry analysis of CD40 expression in untransfected HEK-293 cells, a transfected
clone expressing control CD40 (HEK-C), and three selected clones showing decreasing
expression of CD40 mutant P5 (HEK-P5 cl1, cl2, cl3). (B) Immunoblot showing CD40
expression in HEK-293 and HEK transfectants (HEK-C, HEK-P5 cl1, HEK-P5 cl2, HEK-P5
cl3), untreated or treated with PNGaseF or EndoH. Tubulin is shown as loading control.
Figure S6. IκBα degradation and p52 production in P5 mutant
Immunoblots showing IκBα and p52 expression in HEK-293 cells (HEK-293), a clone
expressing control CD40 (HEK-C), and three clones showing decreasing expression of P5
mutant (HEK-P5 cl1, cl2, cl3), non-stimulated (NS) and stimulated with either TNFα or CD40L
for the indicated times. TNFα was used as a stimulation control that activates the “canonical“ but
not the “non-canonical” pathway. Indeed, it induced IκBα degradation but not p52 formation in
both non-transfected and transfected cells. CD40L stimulation induced IκBα degradation and
p52 formation only in CD40 expressing cells depending on CD40 surface expression levels.
Figure S7. Temperature effect on P5 mutant surface expression
Flow cytometry analysis of CD40 expression in B-LCLs from control (C), P1, P2, P4, and P5
patients, after culturing of cells at both 37°C (bold line) and 32°C (dashed line) for 3 days. No
variation in CD40 expression was detectable in cells from C, P1, P2, and P4, while an increase
was evident in P5. Analysis of the geometric mean fluorescence showed a 2.4 folds increment
(from 19.9 at 37°C to 48.1 at 32°C). Staining was performed with the anti CD40 mAb 626.1
followed by a PE-conjugated secondary antibody.
Figure S1
P5
H. sapiens
M. mulatta
E. caballus
S. scrofa
C. lupus
B. taurus
M. musculus
P2 P4
CREKQYLINSQCCSLC
CREKQYLINSQCCSLC
CKGNQYLSGSHCCDLC
CKENQYPTNSRCCNLC
CREKQYLVDSQCCNMC
CGEKQYPVNSLCCDLC
CSDKQYLHDGQCCDLC
CHQHKYCDPNLGLRVQQKG
CHQHKYCDPNLGLRVQQKG
CHQHKYCDPNLGLQVQGTG
CHQHKYCDPNLGLQVQREG
CHQHKYCDPNLGLHVEKEG
CHEHRYCNPNLGLRIQSEG
CHQHRHCEPNQGLRVKKEG
26
77
Figure S2
P1
P4
% of MAX
C
CD40 (mAb 5C3-FITC)
P2
% of MAX
C
CD40 (mAb 5C3-PE)
P5
Figure S3
A
C
EndoH
CD40
-
+
B cells
+
[
actin
B
Degradation
GOLGI
ER
Glu-I
Glu-II
UGGT
(ERMans)
Glu-II
Csp
DMJ
N-acetylglucosamine
mannose
Csp: Castanospermine
C
BFA
glucose
galactose
DMJ: Deoxymannojirimycin
Csp
chase (h)
0
PLASMA MEMBRANE
CYTOSOL
DMJ BFA
12
C
P2
P4
P5
CD40
-
sialic acid
BFA: BrefeldinA
MW
- 46KDa
Figure S4
TGN46
CD40
merge
C
P2
P5
transferrin
C
P2
P5
CD40
merge
B
PNGaseF
EndoH
CD40
[
tubulin -
HEK-P5
cl1
+
HEK-C
HEK-P5 cl1
HEK-P5 cl2
HEK-P5 cl3
HEK-P5 cl2
HEK-P5 cl3
HEK-C
HEK-P5 cl1
HEK-C
HEK-P5 cl3
HEK-293
HEK-P5 cl2
HEK-293
HEK-C
HEK-P5 cl1
% of MAX
Figure S5
A
HEK-P5
cl2
HEK-P5
cl3
CD40 (mAb 626.1)
+
-
Figure S6
TNFα
CD40L
time (h) 0.5 5 NS 0.5 5
TNFα
CD40L
TNFα
CD40L TNFα
CD40L
TNFα
CD40L
0.5 5 NS 0.5 5
0.5 5 NS 0.5 5
0.5 5 NS 0.5 5
0.5 5 NS 0.5 5
HEK-C
HEK-P5 cl1
HEK-P5 cl2
HEK-P5 cl3
IkBα p100 p52 tubulin -
HEK-293
Figure S7
P1
P2
% of MAX
C
CD40 (Mab 626.1)
P4
P5