Supplementary Information
Supplementary Methods
Tryptic Digestion of Protein Mixture
The dialyzed protein mixtures were each suspended in 20 l of 0.1% RapiGest
(Waters Co., Milford, MA) in 50 mM ammonium bicarbonate, and then vortexed.
The resulting solutions were reduced, alkylated and digested with TPCK-trypsin as
reported previously (Motoyama, 2006).
Trifluoroacetic acid was added to the
mixture at 0.5% final concentration to halt the digestion and to inactivate RapiGest
surfactant.
ldentification of proteins using liquid chromatography coupled with electrospray
tandem mass spectrometry (LC/MS/MS)
Digested protein mixtures were subjected to LC/MS/MS-based protein identification
analysis as described previously (Motoyama, 2007).
Briefly, the mixtures were
loaded by an autosampler (SI-2 semi-micro HPLC system, Shiseido Co., Ltd., Tokyo,
Japan) onto a fused-silica trapping column (100 m i.d. x 1 cm, JupiterProteo C14,
10 m, Phenomenex, Torrance, CA).
The trapping column was desalted with
starting buffer (0.1% formic acid, 5% acetonitrile/purified water) for approximately
30 min, then directly connected to a fused-silica analytical capillary column (100 m
i.d. x 12 cm, JupiterProteo C14, 4 m, Phenomenex) by changing the position of a
two-way switching valve.
Peptides in the digested mixtures were separated with a
90-min gradient of 5-75% acetonitrile.
The column flow-rate was set to 300~400
nl/min by adjusting the length of a split resistance capillary (50 m i.d. x 50~200
mm).
Peptides eluted from the column were directly electrosprayed into a hybrid
mass spectrometer (LTQ-Orbitrap, Thermo Fisher Scientific, Waltham, MA). The
mass spectrometer was operated in an automatic parallel data-dependent MS/MS
mode.
After one accurate-mass survey scan by the Orbitrap at 60,000 mass
resolution, MS/MS spectra for the largest 5 precursor ions in the survey scan were
automatically acquired by LTQ under the control of the Xcalibur data system
(Thermo Fisher Scientific, Waltham, MA).
Collected MS/MS spectra were
searched to identify proteins with the SEQUEST algorithm running on Bioworks
software (Thermo Fisher Scientific, Waltham, MA).
A non-redundant human
protein database (NCBI, 2010) was used for the identification of proteins.
Stringent search criteria were used to minimize false identification of proteins (Sf
1
score > 0.85, Peptide probability > 0.001, Number of top matches: > 1, Precursor
mass tolerance: <10 ppm, Minimum number of peptides to identify proteins: 1,
Enzyme specificity: half-tryptic or fully tryptic peptides only).
Time lapse study
Keratinocytes were plated onto 35 mm glass-based dishes and incubated at 37oC
until 80% or 100% confluency.
In the latter case, further differentiation
stimulation was applied by exposure to air and addition of 2 mM calcium.
The
active caspase-14 expression vectors were transfected into these keratinocytes
using FuGENE (Roche) during the growth phase.
Morphological changes of
keratinocytes expressing active caspase-14 were observed by means of confocal
microscopy (LSM5 PASCAL, Carl Zeiss).
The lens was a Plan APOCHROMAT 20x
magnification and Keima-Red or Azami-Green was detected using an argon laser.
Effect of KLK5 on mesotrypsinogen activation
In order to examine whether KLK5 is able to activate mesotrypsinogen, 10 l of
active KLK5 (0.25 g/ml) was incubated with 25 l of mesotrypsinogen (0.5 g/ml)
in 55 l of serine protease assay buffer (0.1 M TrisHCl (pH 8.0) + 1 mM CaCl 2 +
0.1% Tween 20) for 30 min at 37oC.
After addition of 10 l of 100 M
Boc-Gln-Ala-Arg-methylcoumarin (MCA) (Peptide Institute, Osaka, Japan), the
mixture was incubated for 30 min at 37oC.
The activity of mesotrypsin was
measured using an ARVOTM X3 (Perkin Elmer, Inc., Waltham, MA) with excitation
at 355 nm and emission at 460 nm, and the mean values of duplicate assays were
taken as relative intensity.
Differences in increment of MCA liberation were
calculated.
Comparison of TUNEL-positive cells in revC14- and mesotrypsin-transfected cells.
Keratinocytes
in
the
growth
phase
were
transfected
with
pInv-HA-revC14,
pInv-HA-active mesotrypsin or pInv-HA-revC14 and pInv-HA-active mesotrypsin.
After reaching confluency, cultures were air-exposed for 15 min and 1.5 mM
calcium-containing medium was added. Culture was continued for a further two days.
After fixation with cold methanol, keratinocytes were subjected anti-HA antibody
staining and TUNEL staining.
Construction of an expression vector for enzyme-dead revC14 (revC14mut)
Active site cysteine (228Cys) was changed to Ala using PrimeSTAR® Mutagenesis
2
Basal Kit (Takara Bio Inc. , Otsu, Japan) according to the Manufacturer’s instruction.
Mutagensis
primers
used
5’-TACAGGCCGCTCGAGGAGAACAAAGGGA-3’,
5’-TCCTCGATCGGCCTGTATGATGTACAC-3’.
PCR
were
forward:
and
reverse:
was
carried
out
using
pCMV-HA-revC14 as a template.
Preparation of recombinant FLG-N and FLGmut.
Recombinant FLG-N proteins with or without mutation in the linker region were
prepared as follows. Briefly, pCMV-HA/FLG-1.5-KR and pCMV-HA/FLGmut-KR were
cut with Sph I and Sal I, and inserted into the pQE30 vector. Recombinant FLG-N and
FLGmut were purified with Ni-Agrose chromatography.
Effect of mesotrysin on recombinant FLG-N and FLGmut.
Using recombinant FLG-N and FLGmut proteins as substrates, cleavage of the linker
region by mesotrypsin was investigated. Briefly, recombinant FLG-N or FLGmut (0.2
mg/ml each, 5 l) was incubated with 5 l of mesotrypsin (0.01 or 0.1 g/ml) in 20 l of
serine protease assay buffer and incubated at 37oC for 30min.
Reaction was
terminated by adding 10 l of 4x SDS PAGE sample buffer and subjected to SDS-PAGE
followed by Western blot.
References
Motoyama A, Venable JD, Ruse CI and Yates JR, 3rd (2006) Automated ultra-high-pressure
multidimensional protein identification technology (UHP-MudPIT) for improved
peptide identification of proteomic samples. Anal Chem 78: 5109-5118
Motoyama A, Xu T, Ruse CI, Wohlschlegel JA and Yates JR, 3rd (2007) Anion and cation
mixed-bed ion exchange for enhanced multidimensional separations of peptides and
phosphopeptides. Anal Chem 79: 3623-3634
3
Supplementary figure legends
Fig. S1.
An N-terminal fragment of profilaggrin including A, B and truncated
filaggrin domains induces DNA degradation. (A) Western blot analysis of FLG-N
expressed in keratinocytes.
FLG-N was recognized with anti-N-terminal antibody
as well as anti-filaggrin mAb.
The latter showed an additional band, which is
considered to be a degradation product. (B) Time lapse study for FLG-N-transfected
keratinocytes. An N-terminal fragment containing A-domain, B-domain and
truncated filaggrin repeat (FLG-N) was fused with a fluorescent protein,
Keima-Red, and expressed in growing keratinocytes.
Keratinocytes were
maintained under an atmosphere of 5% CO2 in air at 37oC and time lapse study was
carried out using PASCAL confocal microscopy. Micrographs were taken at every 15
min starting from 2 hrs after transfection and continuing up to 30 hrs.
Light
micrographs and fluorescence images of Keima-Red-fused FLG-N were merged and
serial images every 1 h are shown.
Fig. S2.
Expression of various constructs in keratinocytes.
hydrolytic activity between purified caspase-14 and revC14.
(A) Comparison of
Active caspase-14
was purified from cornified extracts. revC14 was isolated with Ni-Agarose and
further purified by means of Mono Q chromatography.
Enzymatic activities were
measured using Ac-WEHD-MCA as a substrate. (B) Expression of transfected
proteases in keratinocytes. Since all the transfected proteases possess HA-tag,
expression of these proteases was confirmed by Western blotting with anti-HA
antibody. Lane 1; keratinocyte extracts transfected with involucrin promoter-driven
HA vector (pInv-HA),
lane 2; pCMV-HA-vector, lane 3; pInv-HA-revC14
(constitutively
caspase-14),
active
4;
pInv-HA-active
mesotrypsin,
5;
pCMV-HA-revC14 + pInv-HA-active mesotrypsin, lane 6; pInv-HA-active KLK5,
lane 7; pInv-HA-active KLK5 + pCMV-HA-revC14, lane 8; pInv-HA-KLK7. Yellow
arrowheads: revC14, red arrowheads: mesotrypsin, white arrowheads: KLK5, blue
arrowheads: KLK7. (C) Co-localization of active mesotrypsin-expressing cells and
TUNEL-positive cells in differentiated keratinocytes. Mesotrypsin expression was
confirmed with anti-HA staining. TUNEL assay was performed simultaneously.
An enlarged image of the boxed area is also shown to the right.
Scale bars: 100 m.
(D) Mesotrypsin in growing keratinocytes does not induce DNA degradation.
Active
mesotrypsin
was
expressed
using
CMV
promoter
(pCMV-HA-mesotrypsin), and did not cause any detectable change.
4
vector
In contrast,
expression of FLG-N induced DNA degradation as shown in the right panels.
Anti-HA staining and TUNEL assay were carried out.
Fig. S3.
(A)
Scale bars: 50 m.
Expression of various constructs in keratinocytes.
Effect
of
KLK5
on
mesotrypsinogen.
KLK5
was
incubated
with
mesotrypsinogen and the change of mesotrypsin activity was measured after 30 min.
(B) The ratio of TUNEL-positive cells to HA-positive cells was evaluated. Most of the
revC14-expressing
cells
were
also
TUNEL-positive
(95%).
Active
mesotrypsin-expressing cells showed lower TUNEL-positivity (72%). Co-expression of
both proteases resulted in 95% TUNEL-positivity. (C) Effect of enzyme-dead revC14
(revC14mut) on TUNEL positivity. Active site cysteine (originally 228Cys) of revC14
was changed to Ala and expressed in keratinocytes using the pCMV-HA vector and
stained with anti-HA Ab (red) and TUNEL (green).
pCMV-HA vector and
pCMV-HA-revC14 were also expressed and presented for comparison.
(D) Using
recombinant FLG-N protein (55 kD) and FLGmut protein (four Arg residues in the
linker region were changed to Leu residues, 55 kDa) as substrates, cleavage of the
linker region by mesotrypsin was investigated. Detection was done using anti-FLG-N
(H300).
Lane 1, 2, 3 (mesotrypsin control, including 0, 0.01, and 0.1 g/ml
mesotrypsin), Lane 5 (FLG-N control), 6 (FLG-N + 0.01 g/ml mesotrypsin), 7 (FLG-N +
0.1 g/ml mesotrypsin), Lane 9 (FLGmut control), 10 (FLGmut + 0.01 g/ml
mesotrypsin), 11 (FLGmut + 0.1 g/ml mesotrypsin), Lane 4, 8, 12 (molecular weight
markers).
Fig. S4. Time lapse study confirmed that caspase-14-induced changes took place
only in differentiated keratinocytes.
For this study, a fluorescent protein,
Azami-Green, was fused with revC14. (A) Images from the time lapse study after
transfection of pCMV-HA-AG-revC14 in the growth phase.
Micrographs were
taken at every 5 min starting from 2 hrs after transfection and continuing up to 26
hrs. Serial images every 50 min are shown. Larger images obtained at 6 h and 22
h are also shown in the right panel.
Arrows indicate divided cells. Note that all
the revC14-expressing cells maintained their fluorescence. (B) Time lapse study
after transfection of pInv-HA-AG-revC14 in the differentiated state.
The
involucrin promoter-driven revC14 vector (pInv-HA-AG-revC14) was transfected in
the growth phase and culture was continued after confluence.
5
Two days after
confluence, 2 mM calcium was applied and time lapse study was started 48 hrs later.
Merged images obtained every 1.2 h are shown.
Colored arrowheads indicate
three examples of revC14-expressing cells that disappeared within a relatively
short observation period.
Enlarged images obtained at time points 0 (starting
time) and 24 h are also shown.
Note that most of the revC14-expressing cells
showed morphological changes, and their fluorescence faded (circles).
6