A dedicated receptor for dead cell-associated antigen cross

Immunity, Volume 36
Supplemental Information
F-Actin Is an Evolutionarily Conserved
Damage-Associated Molecular Pattern Recognized
by DNGR-1, a Receptor for Dead Cells
Susan Ahrens, Santiago Zelenay, David Sancho, Pavel Hanč, Svend Kjær, Christoph Feest, Georgina
Fletcher, Charlotte Durkin, Antonio Postigo, Mark Skehel, Facundo Batista, Barry Thompson, Michael
Way, Caetano Reis e Sousa, and Oliver Schulz
Inventory of Supplemental Information
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Supplemental figures
o Fig. S1 related to Fig. 1
o Fig. S2 related to Fig. 2
o Fig. S3 related to Fig. 3
o Fig. S4 related to Fig. 6
o Fig. S5 related to Fig. 7
o Fig. S6 related to Fig. 5
Supplemental experimental procedures
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Figure S1: mDNGR-1 ECD and hDNGR-1-Fc bind to dead cells. UV-treated HeLa cells
were stained with (A) mDNGR-1 CTLD or mDNGR-1 ECD or (B) hDNGR-1-Fc and
analyzed by flow cytometry. (A) Dot plots showing staining of dead (TO-PRO 3+) but not live
cells with FLAG-mDNGR-1-CTLD (middle panel) or FLAG-mDNGR-1 ECD (right panel).
No double positive cells were found in samples stained without the DNGR-1 reagents (left
panel). (B) Dot plots showing binding of hDNGR-1Fc (right panel) but not the control hDCSIGN-Fc (left panel) to dead cells. For A and B, one out of three representative experiments is
shown.
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Figure S2: Biochemical characterization of DNGR-1 ligand. (A) HeLa cell lysate was
treated for 1h at 37°C with different concentrations of papain or trypsin or left untreated as
indicated. Samples were analyzed by dot blot (left panel) for the presence of DNGR-1 ligand
or SDS-PAGE and SYPRO Ruby staining (right panel) to assess proteolysis. One
representative of two experiments is shown. (B) HeLa cell lysate was either boiled or not and
treated with 5% SDS or transfer buffer containing 20% methanol as indicated. Samples were
analyzed by dot blot with FLAG-mDNGR-1 ECD. One out of two independent experiments
is shown. (C) HeLa cell lysates were prepared by Triton X-100 (lane 1) or Digitonin in DDM
(lane 2) lysis, subjected to native PAGE and blotted under native conditions onto
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nitrocellulose membranes. Immobilized proteins were detected using FLAG-mDNGR-1 ECD.
The latter was also used in the native PAGE as control for the detection of FLAG-tagged
protein (lane 3). The arrow indicates the bottom of the gel pocket. Data are representative of 5
independent experiments. (D) HeLa cell lysate (0.5ml) was fractionated by size exclusion
chromatography. Absorbance profile (280nm) of eluted proteins (left panel) and dot blot
analysis for DNGR-1 ligand of selected fractions (right panel) from one out of two
experiments. Elution volumes of molecular weight standards are marked by arrows.
Figure S3: Phalloidin does not inhibit binding of DNGR-1 to its ligand. Fixed and
permeabilized HeLa cells were incubated with the indicated concentrations of unlabeled
phalloidin before staining with Acti-stain 670 phalloidin and mDNGR-1 ECD and analysis by
flow cytometry. Upper panels show the dot plot profiles and lower graph shows quantitation
of the data as mean fluorescence intensity in each channel normalized to the signal in the
absence (zero control) of unlabelled phalloidin. One of two representative experiments is
shown.
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Figure S4: hDNGR-1-Fc binds directly to F-actin. Muscle F-actin was stained with either
hDNGR-1-Fc or control hDC-SIGN-Fc. A488-phalloidin was added as a counterstain to
reveal actin filaments. Data were acquired as monochrome images and converted into color
images with ImageJ. Composite images are shown on the right. Graphs depict pixel intensity
of the two channels along the white line indicated on the micrograph. Data shown are from
one of three experiments with similar results.
Figure S5: The hemITAM motif of DNGR-1
mediates signaling in response to F-actin. (A) The
indicated amount of non-muscle F-actin with or
without phalloidin was added to B3Z-Syk-mDNGR1 cells F-buffer and complete RPMI medium served
as controls. (B) The indicated amount of nonmuscle F-actin with or without phalloidin or freezethawed MEFs pre-treated with jasplakinolide or
latrunculin B were added to B3Z-Syk-mDNGR-1
Y7F mutant cells at a ratio of 1:5. PMA plus
ionomycin served as a positive control. Histograms
in A and B show absorbance after addition of βgalactosidase substrate to lysed cells. Bars represent
the average of duplicate wells. One out of two
experiments is shown.
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Figure S6: Bundling of highly purified actin by α-actinin increases detection by DNGR1. (A) 100µg of non-muscle actin was separated by SDS-PAGE and the gel was stained with
Coomassie Blue. Prominent band represents actin (molecular weight approx. 43 kDa) (B)
Table showing number of unique identified peptides for non-actin proteins present in
preparations of purified human non-muscle, rabbit muscle and S. cerevisiae actin as detected
by mass spectrometry. Note that this number provides a qualitative score indicating the
presence of a given protein rather than its abundance. Shaded rows represent proteins that
have no known ortholog in S. cerevisiae. (C) 0.6µM of G- or F-actin (human non-muscle),
0.13µM α-actinin or a mixture of F-actin and α-actinin were incubated for 10 min before 2fold serial dilution (indicated by the wedge) and transfer onto nitrocellulose membranes.
Membrane was probed with FLAG-mDNGR-1 ECD. One representative out of three
experiments is shown. (D) Co-sedimentation of DNGR-1 and F-actin in a pelleting assay. 1µg
non-muscle F-actin was incubated alone (– α-actinin) or with 250ng of α-actinin (+ α-actinin)
and subsequently incubated with a 2-fold dilution series of hDNGR-1-Fc starting at 2µg.
After ultracentrifugation, samples were analyzed for the presence of hDNGR-1-Fc and actin
in the supernatant and pellet fractions.
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Supplemental Experimental Procedures
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Mass spectrometry analysis of DNGR-1 pull downs
FLAG-peptide eluted material from DNGR-1 ECD and control pull-downs was
separated by SDS-PAGE. Gel lanes were manually cut from top to bottom into 1mm thick
slices and the gel pieces were placed in a 96-well microtiter plate and destained with 50% v/v
acetonitrile and 50 mM ammonium bicarbonate, reduced with 10 mM DTT, and alkylated
with 55 mM iodoacetamide. After alkylation, proteins were digested with 6 ng/µL Trypsin
(Promega, UK) overnight at 37 °C. The resulting peptides were extracted in 2% v/v formic
acid, 2% v/v acetonitrile. The digest was analyzed by nano-scale capillary LC-MS/MS using a
nanoAcquity UPLC (Waters, UK) to deliver a flow of approximately 300 nL/min. A C18
Symmetry 5 µm, 180 µm x 20 mm µ-Precolumn (Waters, UK), trapped the peptides prior to
separation on a C18 BEH130 1.7 µm, 75 µm x 250 mm analytical UPLC column (Waters,
UK). Peptides were eluted with a gradient of acetonitrile. The analytical column outlet was
directly interfaced via a modified nano-flow electrospray ionisation source, with a hybrid
linear quadrupole fourier transform mass spectrometer (LTQ Orbitrap XL/ETD,
ThermoScientific, San Jose, USA). Data dependent analysis was carried out, using a
resolution of 30,000 for the full MS spectrum, followed by eight MS/MS spectra in the linear
ion trap. MS spectra were collected with an automatic target gain control of 5x105 and a
maximum injection fill time of 100 ms over a m/z range of 300–2000. MS/MS scans were
collected using an automatic gain control value of 4x104 and a threshold energy of 35 for
collision induced dissociation. LC-MS/MS data were then searched against a protein database
(UniProt KB) using the Mascot search engine programme (Matrix Science, UK). Database
search parameters were set with a precursor tolerance of 5 ppm and a fragment ion mass
tolerance of 0.8 Da. One missed enzyme cleavage was allowed and variable modifications for
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oxidized methionine, carbamidomethyl cysteine, pyroglutamic acid, phosphorylated serine,
8
threonine and tyrosine were included. MS/MS data were validated using the Scaffold
programme (Proteome Software Inc., USA). All data were additionally interrogated manually.
Reporter cell lines and DNGR-1 ligand and agonist reporter assays
BWZ cells stably expressing the extracellular domain of mouse DNGR-1 fused to the
transmembrane region from NKRP1B and the intracellular tail of CD3ζ (BWZ-mDNGR-1-ζ
cells; (Sancho et al., 2009)) were grown in RPMI 1640 containing 10% FCS, 2mM glutamine,
50 μM 2-mercaptoethanol, 100 units/ml penicillin, 100 μg/ml streptomycin (complete RPMI
medium). Mouse embryonic fibroblasts transformed with SV40 large T antigen (MEFs;
(Sancho et al., 2009)), as well as B3Z cells stably co-expressing Syk and either a wildtype
mDNGR-1 or a signalling-deficient mutant of mDNGR-1 (Y7F) (Sancho et al., 2009), were
also grown in complete RPMI medium.
To assess the presence of the DNGR-1-ligand, BWZ-mDNGR-1-ζ cells were plated in
96 well plates (1x105 cells/well) in the presence of the indicated stimuli. After overnight
culture, cells were washed once in PBS and LacZ activity was measured by lysing cells in
CPRG (Roche)-containing buffer. 1-4 hours later O.D. 595 was measured using O.D. 655 as a
reference. To assess Syk-dependent signaling through DNGR-1 in response to ligand
(DNGR-1 agonist assay), B3Z-Syk-mDNGR-1 wildtype or B3Z-Syk-mDNGR-1 Y7F mutant
cells were plated in 96 well plates (1x105 cells/well) in the presence of either F-actin at
indicated concentrations or live or freeze-thawed MEFs. MEFs were either left untreated or
were pre-treated for 2h with 100nM Jasplakinolide or for 1h with 5µM Latrunculin B. To
induce necrosis, MEFs were pelleted by centrifugation and shock frozen in liquid nitrogen for
30 seconds, thawed, resuspended in medium, counted and added to reporter cells at 2x104
corpses/well.
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Confocal microscopy
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HeLa cells were cultured on fibronectin-coated coverslips overnight at 37°C, fixed in
4% paraformaldehyde in PBS for 15 min and washed and permeabilized in 0.1% Triton-X100
in PBS for 4 min. After washing with PBS, cells were left in blocking buffer (1% BSA + 2%
FCS in PBS) overnight and stained with FLAG-mDNGR-1 ECD, followed by 7H11 mAb and
A546-conjugated goat-anti-rat antibody (Invitrogen), as well as the DNA dye DRAQ5
(Biostatus Ltd). For vaccinia virus infection, HeLa cells cultured on fibronectin-coated glass
coverslips were infected with the wild type Western Reserve strain or a mutant strain (A36RYdF; (Rietdorf et al., 2001)) unable to induce actin tails at a multiplicity of infection of 2 in
serum-free MEM, as described (Arakawa et al., 2007). One hour post infection (hpi), the
medium was replaced with medium containing 10% FCS. Infected cells were fixed with 4%
paraformaldehyde at 8 hpi. In some experiments, A488-phalloidin (1:400) was added to stain
the actin cytoskeleton. For analysis of dead cells, HeLa cell UV-irradiated and stained with
mDNGR-1 ECD and Cy3-conjugated mouse-anti-FLAG antibody were counterstained with
A488-phalloidin and allowed to settle on poly-L-lysine-coated coverslips. Coverslips were
mounted using Fluoromount-G (Southern Biotech) and images were collected using a laser
scanning confocal microscope (LSM 510; Zeiss).
For confocal imaging of Drosophila tissues, ovaries and wing imaginal discs were isolated
from female flies and larvae, respectively. Ovaries were dissected in PBS, fixed for 20 mins
in 4% paraformaldehyde + PBS, washed for 30 minutes in PBS in 0.1% Triton X-100 and
blocked for 15 minutes in 5% normal goat serum in PBS in 0.1% Triton X-100. FLAGmDNGR-1 ECD was added to the samples and incubated overnight at 4°C. Imaginal discs
were processed as above except that they were dissected on ice, fixed in 4%
paraformaldehyde + PBS for 30 min, and blocked using 0.1% BSA (Sigma). All samples
were further stained with Cy3-conjugated mouse-anti-FLAG antibody (1:200) and 1 μg/ml
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DAPI (Invitrogen) for 2 hrs at room temperature before mounting in Vectashield (Vector
labs). In some experiments, A488-phalloidin (1:400) was also added to stain the actin
cytoskeleton. Samples were imaged on a Leica SP5 confocal microscope.
Native PAGE, gel filtration chromatography and Coomassie staining
For native PAGE, proteins within the lysate were separated on a 4-20% Tris-Glycine
gel (Invitrogen) and transferred to nitrocellulose membranes using methanol-free transfer
buffer. Blotted membranes were blocked overnight and probed for DNGR-1 ligand using
FLAG-mDNGR-1 ECD and HRP-anti-FLAG antibody as described for dot blotting.
For gel filtration, 0.5ml cell lysate was injected onto a Superose 6 column, which had
been equilibrated in PBS, and proteins were separated by fast protein liquid chromatography
at a flow rate of 500µl/min using the Akta system (GE Healthcare). Eluted proteins were
collected in 0.5ml fractions and analyzed by dot blot for the presence of DNGR-1 ligand.
For Coomassie protein staining of actin preparations, 100µg of non-muscle actin was
separated by SDS-PAGE on a 4-20% polyacrylamide gel and stained with Coomassie Blue
(Bio-Rad).
Supplemental Reference
Arakawa, Y., Cordeiro, J.V., Schleich, S., Newsome, T.P., and Way, M. (2007). The release of vaccinia virus
from infected cells requires RhoA-mDia modulation of cortical actin. Cell Host Microbe 1, 227–240.

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