SupplementaryInformation
Index
1.
FigS1.HPLCchromatogramsofGGHGandDTH-NH2
2.
FigureS2MassspectraofGGHGandDTH-NH2
3.
FigureS3HPLCChromatogramK21(Cl2CF)-Hepcidin
4.
FigureS4MALDI-TOFMSofK21(Cl2CF)-Hepcidin
5.
FigS5.ExtrapolationoflogconditionalCuIIaffinities
6
FigS6 SpectrophotometrictitrationspectraofDTHFPIAIF-NH2withCuII
7
TableS1ExtrapolationoftheLogconditionalCuIIaffinitiesforDTHFPIAIF-NH2
8
FigureS7Analysis of K21(Cl2CF)-Hepcidin-CuII complex by mass spectroscopy
9.
Ferroportininternalisationassayandhepcidininternalisationassay
mA U
A
1200
3.15
1000
800
600
400
200
0
0
5
10
15
20
R etentiontime(min)
B
Fig S1. HPLC chromatograms of GGHG and DTH-NH2
Using a monitoring wavelength of 220 nm, GGHG shows a retention time of 3.15 min. (A) and
DTH-NH2 a retention time of 3.05 min. (B)
2000
1800
3 .0 5
1600
1400
mA U
1200
1000
800
600
400
200
0
0
5
10
R e te ntiontim e (m in)
15
20
A
C:\DECA XP DATA\RH-newPC\M17607
22/08/2012 10:51:17
PP05 MW:375 10 uL/mL in 0.1% FA in 50%
NL: 2.73E8
M17607#51-374 RT:
0.48-3.57 AV: 324 F:
+ p ESI Full ms
[50.00-500.00]
327.00
100
90
80
Relative Abundance
70
60
50
40
30
164.00
20
10
0
100
270.00
255.00
312.07
184.07
133.00
74.00
384.00
370.87 395.07
445.00
485.07
NL: 1.09E8
M17607#647-741 RT:
6.22-7.06 AV: 95 F: +
p ESI Full ms2
[email protected]
[90.00-500.00]
308.93
90
80
70
60
213.00
50
251.93
40
30
292.00
20
10
195.07
110.07 137.80
270.00
0
100
150
200
327.00
250
344.33 378.07 420.47 442.60 480.27
300
350
400
450
500
m/z
B
C:\DECA XP DATA\RH-newPC\M17715
19/10/2012 14:55:43
PP14 MW:370 20uL/mL in 0.1% FA in 50% M
NL: 3.12E8
M17715#12-132 RT:
0.19-2.09 AV: 121 T: +
p ESI Full ms
[50.00-1000.00]
371.1
100
90
Relative Abundance
80
70
60
50
740.9
40
30
20
10
200.0
110.1
458.1
256.1 354.1
0
100
534.1
621.1
670.0
762.9
855.8 903.8 970.9
NL: 2.16E7
M17715#175-211 RT:
3.08-3.74 AV: 37 F: +
p ESI Full ms2
[email protected]
[100.00-1000.00]
353.9
90
80
70
256.0
60
50
155.1
40
336.0
30
292.0
20
10
110.1
239.0
371.1
221.1
0
100
200
300
400
483.9
500
m/z
606.5
600
936.5
700
800
900
1000
Figure S2 Mass spectra of GGHG and DTH-NH2
(A) Mass spectrum of GGHG, 10 µl/ 1 ml 0.1% FA in 50% methanol, ESI, MS (-ve) molecular
weight of GGHG is 327. (B) Mass spectrum of DTH-NH2, 20 µl/ 1 ml 0.1% FA in 50%
methanol, ESI, MS (-ve) molecular weight of DTH-NH2 is 371.
21
FigureS3HPLCChromatogramK (Cl2CF)-Hepcidin
FigureS4MALDI-TOFMSofK21(Cl2CF)-Hepcidin
Fig S5. Extrapolation of log conditional CuII affinities
Extrapolation of log conditional CuII affinities of K21(Cl2CF)-Hepcidin determined in different
percentages of acetonitrile (MOPS, pH 7.4). y = -0.0058x + 7.22
FigureS6SpectrophotometrictitrationspectraofDTHFPIAIF-NH2withCuII
(A)TitrationspectraofDTHFPIAIF-NH2withCuIIinaacetonitrile:0.1MKClsolution
(50:50).[DTHFPIAIF-NH2]=1.18mmol/Land[CuII]=0.29mmol/L,ratioofL:M=4.1,
initially15.28mLof0.1MKClat25°C,pHfrom2.04to6.60.
(B)TitrationspectraofDTHFPIAIF-NH2withCuIIinaacetonitrile:0.1MKClsolution(1:2).
[DTHFPIAIF-NH2]=2.54mmol/Land[CuII]=0.61mmol/L,ratioofL:M=4.2,initially15.60
mLof0.1MKClat25°C,pHfrom1.82to6.69.
TableS1ExtrapolationoflogconditionalCuIIaffinitiesforDTHFPIAIF-NH2
PercentageAcetonitrilein0.1M 50
33
0a
KClsolution
LogK7.4b
8.7
8.4
7.8
pCuII7.4c
9.7
9.4
8.8
aExtrapolationfromthe50%and33%stabilityconstants
blogconditionalCuIIaffinitiesatpH7.4,recalculatedfrompH-independentstability
constants
cpCuII
-5
II
-6
7.4undertheconditions[ligand]Total=10 M,[Cu ]Total=10 M,pH=7.4
Figure S7. Analysis of Hepcidin-CuII complex using MALDI-TOF MS in the linear
mode.
Hepcidin and CuII are present in equimolar amounts. (A), 2 nmol/L at pH 3; (B), 15.6
nmol/L at pH 7.0; (C), 500 nmol/L at pH 7.0; (D), 600 at pH 7.0 nmol/L
FerroportinInternalizatioAssay
A stable cell line (Madin-Darby Canine Kidney, MDCK) was generated, which constitutively
expressed human ferroportin (Fpn) fused at its C-terminus to the HaloTag protein (Promega
Corp.). The internalization of Fpn was traced by labeling these cells with the fluorescent
HaloTag-TMR (tetramethylrhodamine) ligand, which covalently attached to the HaloTag
protein. Fpn-HaloTag MDCK cells were seeded in 100 µL DMEM medium (Dulbecco's
Modified Eagle Medium with 10% fetal bovine serum (FBS) containing 1% Penicillin, 1%
Streptomycin and 450 µg/ml G-418) per well of 96 well microplates (MicroClear, Greiner).
After overnight incubation at 37°C/5% CO2, the HaloTag-TMR ligand was added to the cells
at 2 µmol/L final concentration and incubated for 15 min. at 37°C/5% CO2. Cells were washed
with Phosphate Buffered Saline (PBS), 100 µl DMEM medium was added and the cells were
incubated for 30 min. at 37°C/5% CO2. The medium was replaced with 50 µl fresh DMEM
medium and 50 µl of the test compound was added diluted in DMEM medium. Hepcidin doseresponse experiments were carried out with either 8 or 11 concentration dilution series of CFhepcidin analogues and reference hepcidin (Bachem, Cat. No H-5926) spanning
concentration ranges of 4 µmol/L to 0.2 nmol/L for labelled-hepcidin analogues and 2 µmol/L
to 0.03 nmol/L for the reference hepcidin. Dose-response experiments were carried out with
either 3 wells or 4 wells per peptide concentration for 8 and 11 concentration dilution series,
respectively. After overnight incubation at 37°C/5% CO2, 25 µL of Draq5 (Biostatus, Cat.
No DR51000) was added to 2.5 µmol/L final concentration and incubated for 10 min. at
37°C/5% CO2 to stain cell nuclei. After washing the cells with 200 µl of DMEM
medium without phenol-red (GIBCO, Cat. No 11880), cells were fixed in 100 µl of 4%
Paraformaldehyde (PFA, Electron Microscopy Sciences, Cat. No 15710-S) in PBS for 15
minutes at room temperature. The PFA solution was removed and cells were washed with
PBS leaving 100 µl per well and plates were sealed with foil plate seal. TMR (520-550 nm
excitation / 560-630 nm emission / 1000 ms
exposure time) and Draq5 (620-640 nm excitation / 650-760 nm emission / 200 ms
exposure time) fluorescence images were acquired using the Operetta high
content screening system (Perkin Elmer) using a 20x long working-distance
objective. Four pictures were acquired per well and fluorescence channel covering
ca. 2000 cells per well. The acquired image data was imported into the Columbus
image data storage and analysis system (Perkin Elmer) for analysis. Image
analysis included detection of nuclei (Draq5 fluorescence), definition of
cytoplasmic region followed by application of the Ridge SER algorithm to analyze
the texture of the TMR fluorescence in the cytoplasmic region as a quantitative
measure for subcellular localization of ferroportin. High TMR SER Ridge values
correlated with Fpn localized to the cell surface and low TMR SER Ridge values
with the absence of Fpn from the cell surface. Relative values for Fpn
internalization in percent were calculated as follows: Fpn Internalization (%) =
(1 – ((SER Ridge – Average of SER Ridge of 1000 nmol/L hepcidin)/(Average
of SER Ridge of 0 nmol/L hepcidin – Average of SER Ridge of 1000 nM
hepcidin))) * 100. EC50 values were calculated with the relative Fpn internalization
data using “log(agonist) vs. response” curve fitting of Prism 5 software (GraphPad
Software Inc., version 5.02). For each data set the fit of the “log(agonist) vs.
response (three parameters)” model was compared to the fit of the “log(agonist) vs.
response – Variable slope (four parameters)” model and the EC50 data of the
preferred model was used.