Báo cáo khoa học: An orphan dermaseptin from frog skin reversibly assembles to amyloid-like aggregates in a pH-dependent fashion pptx

An orphan dermaseptin from frog skin reversibly assembles
to amyloid-like aggregates in a pH-dependent fashion
Ruth Go
¨
ßler-Scho
¨
fberger
1
,Gu
¨
nter Hesser
2
, Martin Muik
3
, Christian Wechselberger
4
and
Alexander Jilek
1
1 Institute of Organic Chemistry, Johannes Kepler University Linz, Austria
2 CSNA Center for Surface- and Nanoanalytics, Johannes Kepler University Linz, Austria
3 Institute of Biophysics, Johannes Kepler University Linz, Austria
4 Center for Biomedical Nanotechnology, Upper Austrian Research, Linz, Austria
Keywords
amphibian skin; amyloid; bioactive peptide;
cytotoxicity; self-assembly
Correspondence
A. Jilek, Institute of Organic Chemistry,
Johannes Kepler University Linz,
Altenbergerstrasse 69, A-4040 Linz, Austria
Fax: +43 732 2468 8747

Tel: +43 732 2468 9498
E-mail:
(Received 6 April 2008, revised 5 August
2009, accepted 7 August 2009)
doi:10.1111/j.1742-4658.2009.07266.x
Dermaseptin PD-3-7 (aDrs) from frog skin contains three aspartic acid resi-
dues resulting in a negative net charge at neutral pH, as opposed to numer-
ous other dermaseptins which are cationic helical antimicrobial peptides.
Still, this peptide can be fitted into an amphipathic a helix by an Edmundson
wheel projection. However, folding to the proposed helix was induced to
only a low extent by zwitterionic vesicles or even detergents. Furthermore,
no evidence of antibacterial or cytotoxic activity from soluble aDrs could be
obtained. The peptide has an inherent propensity to an extended conforma-
tion in aqueous solution and self-assembles into amyloid fibrils in a reversible
pH-controlled fashion, which was studied in some detail; above pH 5, the
amyloid fibrils disassemble in a cooperative manner. This is probably caused
by deprotonation of both side chain and terminal carboxyl groups, which
results in intermolecular electrostatic repulsion. At neutral pH, this process
proceeds instantaneously to the soluble form. Within the transition interval
(pH 5–6.5), however, ‘backward’ granular aggregates, 10–500 nm in size, are
formed. Such metastable amorphous aggregates, which are quickly released
from an amyloid depot by a shift in pH, can mediate a strong cytotoxic
effect. This activity does not involve lysis or interference with the cellular
redox status, but apparently acts via an as yet unidentified mechanism. In
this study, we present a new member of an emerging class of self-assembling
frog skin peptides with extraordinary self-aggregation properties, which may
potentially be relevant for biological processes.
Structured digital abstract
l
MINT-7256467: Dermaseptin (uniprotkb:O93455) and Dermaseptin (uniprotkb:O93455) bind

(
MI:0407)bycircular dichroism (MI:0016)
l
MINT-7255686: Dermaseptin (uniprotkb:O93455) and Dermaseptin (uniprotkb:O93455) bind
(
MI:0407)bybiophysical (MI:0013)
l
MINT-7256439: Dermaseptin (uniprotkb:O93455) and Dermaseptin (uniprotkb:O93455) bind
(
MI:0407)byfluorescence microscopy (MI:0416)
l
MINT-7256449: Dermaseptin (uniprotkb:O93455) and Dermaseptin (uniprotkb:O93455) bind
(
MI:0407)byelectron microscopy (MI:0040)
l
MINT-7256430: Dermaseptin (uniprotkb:O93455) and Dermaseptin (uniprotkb:O93455) bind
(
MI:0407)byfluorescence technologies (MI:0051)
Abbreviations
aDrs, dermaseptin PD-3-7; LDH, lactate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; POPC,
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; SUV, small unilamellar vesicles; TEM, transmission electron microscopy; ThT, thioflavin T.
FEBS Journal 276 (2009) 5849–5859 ª 2009 The Authors Journal compilation ª 2009 FEBS 5849
Introduction
Amphibian skin glands are known to contain a vast
variety of biologically active peptides, such as neuro-
peptides, peptide hormones, opioid peptides and pep-
tide antibiotics [1,2]. Many of these may help the
individuals to interact with their environment, i.e. they
are targeted exogenously [3]. In the skin of Pachymedu-
sa dacnicolor, a tree frog from southern Mexico, the

opioid peptides [Ile2]-deltorphin and dermorphin [4],
tryptophyllins [5] and five heterogenous defence-related
peptides have been detected [6]. The latter group are
termed dermaseptins, because their members share a
conserved pre-pro-region, whereas the mature peptides
can have very different structural characteristics [7]. The
majority of these dermaseptins are cationic linear anti-
microbial peptides able to adopt an amphipathic a helix
[8]. These properties enable the peptides to intercalate
into phospholipid bilayers in a detergent-like manner
[9,10]. However, some skin peptides derived from pre-
cursors with the characteristic pre-pro-region do not
share these structural features. For example, the derma-
septin PD-3-7 (DMS5_PACDA) of this species, which
in principle could form an amphipathic a helix, differs
from classical cationic antimicrobial peptides because it
has a negative net charge [6]. Because of this we hence-
forth call this peptide ‘anionic dermaseptin’ (aDrs).
In this study, we tested whether aDrs has any anti-
bacterial or cytotoxic activity. We discovered that aDrs
can form amyloid-like aggregates at acidic pH. We
tested the antibacterial or cytotoxic activity of these
aggregates with the aim of obtaining insight into the
functional relevance of self-assembly and their mode of
action. Furthermore, we also investigated the aggrega-
tion behaviour of aDrs homologues with a neutral or
positive net charge.
Results
Secondary structure induction by vesicles and
detergents

Induction of secondary structure, an amphipathic helix
in particular, by phospholipid bilayers is a good indica-
tor of whether a peptide is membrane active. We used
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)
unilamellar vesicles and dodecylphosphocholine deter-
gent micelles to mimic the outer leaflet of eukaryotic cell
membranes, which is rich in phospholipids with the
zwitterionic phosphocholine headgroup. By contrast,
SDS micelles favour an initial interaction of cationic
antibacterial peptides by electrostatic attraction in a
similar manner to negatively charged bacterial cell
membranes [9,11]. The CD spectrum of soluble aDrs in
buffer is that of a random coil, with 30% extended
conformation and 5% turn structures independent of
pH (Fig. S1). Addition of POPC small unilamellar vesi-
cles (SUVs) resulted in an increase in the helical struc-
ture up to 2% (7% at pH 8), although the b-sheet
fraction remained largely unchanged. The presence of
SDS or dodecylphosphocholine may have led to an over-
estimated value for the helical fraction of up to 20%
(Fig. 1).
Antibacterial and cytotoxic activity of soluble aDrs
We tested the antibacterial activity of aDrs against the
gram-positive Bacillus subtilis and the gram-negative
Escherichia coli using an inhibition zone assay. Mono-
meric aDrs displayed no antibiotic action in the entire
concentration range up to 32 lm. Cytotoxic activity
against eukaryotic Spodoptera frugiperda (Sf9) insect
cells and NIH-3T3 mouse fibroblast cells was investigated
by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazo-

lium bromide (MTT) assay, which is sensitive to the
redox potential of the cells. We detected an insignificantly
low effect of aDrs on these cell lines (< 10% reduction
compared with control at 16 lm; data not shown).
Characterization of aDrs aggregates
aDrs aggregates after prolonged incubation at acidic
pH. The amyloid-like (in a strict sense) nature of
Fig. 1. Induction of secondary structure in soluble aDrs. CD spectra
of 100 l
M aDrs (solid line) and after addition of POPC SUVs
(dashed line) and dodecylphosphocholine detergent micelles (dotted
line). [h]
MRW
, mean residue molar ellipticity.
Self-assembly of anionic dermaseptin R. Go
¨
ßler-Scho
¨
fberger et al.
5850 FEBS Journal 276 (2009) 5849–5859 ª 2009 The Authors Journal compilation ª 2009 FEBS
these aggregates grown in vitro at pH 2 was probed
using several well-established methods [12] (Fig. 2).
First, we tested the binding of the amyloid-specific
dyes Congo Red and thioflavin T (ThT), which are
commonly used in amyloid diagnostics. Staining the
aggregates with Congo Red yielded green birefrin-
gence, whereas incubation with ThT gave green fluor-
escence. Second, a high degree of b-sheet secondary
structure was detected using FTIR spectroscopy.
Investigation of the second derivative of the amide I¢

region demonstrated that maxima at 1619 and
1635 cm
)1
were most likely. These bands can be
attributed to b-sheet structures. A band at
 1685 cm
)1
, which is often observed in b-sheet-rich
proteins, is almost absent (< 1%) [13,14]. Bands at
1609 and 1659 cm
)1
were most likely to be contribu-
tions from side chain amides [15]. Other maxima are
present at 1648 and 1671 cm
)1
, which can probably
be assigned to random coils and sterically restricted
amide bonds such as those present in turns, respec-
tively. Fitting the Gauss peaks onto the band posi-
tions revealed a total contribution of b-sheet
structures of  80%. Third, transmission electron
microscopy (TEM) investigations revealed long
(> 1 lm) fibre bundles. Single fibres (protofilaments)
were found to have a uniform thickness of 5 nm.
Fibril formation is a reversible process dependent
on pH
When exposed to pH 8, the fibrils instantaneously
underwent a phase transition to the predominantly
monomeric random coil form of the peptide because
this could pass freely through the membrane (3.5 kDa

cut-off) during equilibrium dialysis. We henceforth
refer to this state as the ‘soluble’ form of the peptide.
However, this process was found to be reversible.
After a change to pH 2, an amyloidous, pH-responsive
gel was again formed after a lag phase of 45 h (Fig. 3).
We exposed aDrs fibrils to various pH values in
order to investigate their pH-dependent stability. The
resulting profile revealed a sharp transition at pH 5
and a sigmoidal decrease in ThT fluorescence in the
transition interval towards higher pH values (Fig. 3).
Structural rearrangement of aggregates
During fibril breakdown at pH 6, we observed the for-
mation of metastable granular aggregates of different
size and morphology (Fig. 4). Aggregate size ranged
from 10 to 500 nm. These aggregates retained some
ThT-binding ability and caused the sigmoidal shape of
the transition profile in Fig. 3, which was measured
A
B
C
D
Fig. 2. Amyloid-like characteristics of aDrs
aggregates. (A) Light micrograph of Congo
Red bound to aDrs reveals ‘apple-green’
birefringence between crossed polarisators.
Scale bar, 50 lm. (B) Fluorescence micro-
scopy image of ThT bound to aDrs reveals
green fluorescence. Scale bar, 50 lm. (C)
FTIR spectrum of the amide I’ region and
second derivative. (D) Negative stain elec-

tron-micrographs of aDrs fibrils formed at
acidic pH. Scale bar, 200 nm.
R. Go
¨
ßler-Scho
¨
fberger et al. Self-assembly of anionic dermaseptin
FEBS Journal 276 (2009) 5849–5859 ª 2009 The Authors Journal compilation ª 2009 FEBS 5851
1 h after the addition of buffer. This may indicate that,
in a pH interval close to the transition point, a certain
degree of stacked b-sheet structure is temporarily
preserved.
After incubation at pH 6, TEM inspection of
aliquots of the sample revealed that the number of
amorphous aggregates was drastically reduced after
5 days. After 14 days, such aggregates were apparently
undetectable (data not shown). However, equilibrium
dialysis, independent of time, yielded 20 lm of soluble
aDrs (12–14 kDa membrane cut-off). Thus, only a
A
B
Fig. 3. Phase behaviour of aDrs as a function of pH. (A) Response
of aDrs to repeated pH shifts (arrows). The kinetics of 200 l
M
aDrs was measured by recording the change in fluorescence of
ThT at 482 nm over time. After a lag period of 45 h, fluorescence
increased linearly upon a decrease in pH to pH 2. (B) Effect of pH
on aDrs amyloid-like aggregates. Fluorescence of ThT bound to
aDrs aggregates derived from fibrils recorded at 482 nm as a func-
tion of pH after incubation for 1 h.

A
B
C
D
Fig. 4. Characterization of metastable amorphous aggregates of
aDrs. (A) Negative stain electron micrographs of aggregates formed
from fibrils at pH 6.0 show large metastable amorphous aggregates
after 30 min. Scale bar, 100 nm. (B) Time dependency of the disas-
sembly of aDrs fibrils after a pH increase to pH 6.0 followed by ThT
fluorescence. (C) CD spectra of fibrils (dashed line), aggregates
formed from fibrils at pH 6.0 after 5 min (solid line) and soluble aDrs
(dotted line). [h]
MRW
, mean residue molar ellipticity. (D) FTIR spectra
of the amide I’ region of fibrils (dashed line), aggregates formed
from fibrils at pH 6.0 after 5 min (solid line) and soluble aDrs (dotted
line). All spectra contain a contribution of  20% from a band at
1635 cm
)1
(thin line) which probably stems from b sheets.
Self-assembly of anionic dermaseptin R. Go
¨
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¨
fberger et al.
5852 FEBS Journal 276 (2009) 5849–5859 ª 2009 The Authors Journal compilation ª 2009 FEBS
small fraction of total aDrs had passed through the
membrane. We therefore suggest that, at pH 6, at the
final state stable oligomers (n ‡ 5) are formed, which
could not be characterized in detail because of the

detection limits of negative-stain TEM and light scat-
tering. Nevertheless, the putatively aggregated character
was found not to hinder retransition to the amyloidous
state upon a decrease in pH (data not shown).
In the FTIR spectra recorded 5 min after the pH-
shift to 6.0, a minor peak ( 10%) at 1621 cm
)1
was
clearly seen (Fig. 4). This suggested that a significant
number of interchain hydrogen bonds are still present
in the aggregates. Interestingly, this fraction remained
unchanged over the following hour, whereas ThT
fluorescence decreased further by  40% over this time
(Fig. 4). Within this interval, fibrils are no longer
detectable by TEM. In FTIR spectra of the soluble
peptide, the 1621 cm
)1
band finally disappeared in
favour of band contributions at 1645 cm
)1
(random
coil) and  1658 cm
)1
. The latter band is within the
range of contributions from a helices. CD spectra,
however, do not support an a-helical content. These
spectra are virtually indistinguishable from those of
the soluble form.
Antibacterial and cytotoxic activity of
disassembling fibrils

Next, we were interested to see whether aggregated
forms of aDrs would be biologically active. Because
the inhibition zone assay is not applicable for nondif-
fusable aggregates, we preincubated bacteria with aDrs
fibrils (up to 16 lm equivalent) at pH 5.0 in a liquid
growth inhibition assay. This yielded colony numbers
equal to the control experiment. Furthermore, mea-
surement of D indicated that cells not had been lysed
(data not shown).
By contrast, when aDrs fibrils were co-incubated
with insect cells at pH 6.0, their ability to reduce
MTT was significantly reduced compared with
controls (Fig. 5). We suspected that these aggregates
might have a transient character, therefore we also
tested the effect of fibrils preincubated in culture med-
ium at pH 6 (‘backward’ aggregates) for different
time spans, as well as potential ‘forward’ oligomers
which might be formed from soluble aDrs at pH 6
(incubated for 7 days) (Fig. 5). In these experiments,
the toxicity of aDrs ‘backward’ aggregates decreased
with incubation time, with an apparent typical half-
life for the toxic species of 5 days. By contrast, the
soluble form did not develop any noteworthy toxic
activity upon incubation in culture medium at pH 6
(Fig. 5).
Cellular effects on Sf9 cells
Localization studies may provide a clue to the poten-
tial target organelle and thus the mode of cytotoxic
action. A fluorophor was attached via a thioether link-
age to an additional Cys residue which was appended

to the C-terminus. BODIPY C1–Fl was chosen for this
purpose because of its moderate hydrophobicity and
its suitability for laser techniques. The labelled peptide
(aDrs*) was shown to have similar aggregation and
cytotoxic properties as unlabelled aDrs (Fig. S2). Con-
focal fluorescence microscopy revealed that both aggre-
gated and soluble aDrs* were found at the plasma
membrane in the early stages (1 h) and after 12 h were
intracellularly distributed within Sf9 target cells, but
did not enter the nucleus (Fig. S3). With the setup
used, we observed no autofluorescence of the cells
(data not shown). Leakage of cytoplasmic material
(blobs) was not observed at any time.
In order to investigate the progression of the poison-
ing effect of aDrs aggregates (12 lm) we also varied
A
B
Fig. 5. Cytotoxic effect of disassembling fibrillar aDrs aggregates
on Sf9 insect cells investigated by the MTT assay. (A) Cells were
treated with lyophilized fibrils resuspended in medium at pH 6.0 at
the indicated peptide concentration for 24 h. (B) Cells were treated
with ‘backward’ aggregates (15 l
M) after preincubation in medium
at pH 6.0 for the indicated intervals. ‘F’, soluble aDrs (15 l
M) was
incubated in medium at pH 6.0 for 1 week. S, soluble aDrs (15 l
M).
(A,B) Reported values are relative to control cells without peptide.
R. Go
¨

ßler-Scho
¨
fberger et al. Self-assembly of anionic dermaseptin
FEBS Journal 276 (2009) 5849–5859 ª 2009 The Authors Journal compilation ª 2009 FEBS 5853
the incubation time with the cells. MTT reduction
levels were detectably affected after as early as 3 h of
incubation (Fig. S3). Furthermore, replacement of the
culture medium 5 min after the application of aggre-
gates did not reduce the toxic effect, which indicated a
rapid interaction with the cells (data not shown).
The release of intracellular material can be shown by
determining the cytoplasmic enzyme lactate dehydro-
genase (LDH) in the culture medium. Within the first
12 h after application of the aggregates (12 lm), only a
minute fraction of the total LDH levels present in the
cells could be detected in the supernatant (Fig. S3).
We also investigated the redox status in cells treated
with aggregates at 15 lm. The results show no detect-
able increase in reactive oxygen species following expo-
sure for 3 h compared with the control (Fig. S4).
Variants and pH-stable aggregates
aDrs variants were designed to confirm the role of the
carboxyl side chains in the pH effect (Table 1). There-
fore, in peptide N4 we replaced all three aspartic acids
by asparagines. Peptide N4 formed fibrils at pH 2 with
a delayed kinetics. However, when the solution was
brought to pH 6.0, the fibrils rearranged to form stable
amorphous aggregates with some ThT-binding capabil-
ity, similar to the metastable aggregates of aDrs. Inter-
estingly, the same behaviour was observed for a

C-terminally amidated aDrs (aDrsa) (data not shown).
However, C-terminal amidation of peptide N4 (N4a)
fully stabilized the fibrils at neutral pH (Fig. S5). Dis-
assembly of amyloid-like aDrs is thus likely caused by
a cumulative repulsive effect of negative charges. Nota-
bly, the modifications in N4a also confer a potent
cytotoxic activity (R. Go
¨
ßler-Scho
¨
fberger and A. Jilek,
unpublished observations) which was not characterized
in detail, to the monomeric form.
Discussion
Most cationic antimicrobial peptides are unstructured
in solution. Folding into the active amphipathic con-
formation is triggered by binding to the target mem-
brane. In this study, we used vesicles and micelles to
see whether aDrs would also fold to the proposed
amphipathic helix. The CD spectra indicated a high
propensity for an extended conformation in solution
and only a low increase in helical secondary structure
upon addition of zwitterionic vesicles or even deter-
gents. This may not suffice for a lytic activity
(Fig. 1). Indeed, we could not detect any antibacterial
or significant cytotoxic activity of the monomer, sug-
gesting a function divergent from classical peptide
antibiotics.
Reversible self-assembly of aDrs is controlled
by pH

At acidic pH, after a lag period, aDrs forms amyloid-
like b-sheet aggregates (Fig. 2). Such delayed kinetics
are frequently observed during the formation of amy-
loid and result from the obligatory formation of an
energetically unfavourable intermediate, which acts as
a nucleus for fibre growth [16,17]. Interestingly, the
peptide does not contain aromatic residues, which in
other cases are thought to contribute significantly to
the propensity of a polypeptide to form amyloid
[18,19]. Three aspartic acids as well as the unprotected
C-terminus were shown to be responsible for the disso-
ciation of the b-sheet stacks above pH 5.0, when the
side chain and terminal carboxyl groups are deproto-
nated (Fig. 3). Such a highly cooperative reversible
unfolding transition in a narrow pH interval is known
for small globular proteins and has recently also been
described for small model peptides, which underwent
sharp pH-dependent ‘melting’-like phase transitions.
[20]. The high speed of the fibril-to-monomer transi-
tion compared with the kinetics of their formation,
however, may be indicative of different pathways. The
refolding of crucial parts of the polypeptide dependent
on pH changes in organelles or the pH gradients in
glands may control its function in many biological
processes. The pH therefore serves as an activity
switch or positional marker. In frog skin, the granular
glands, in which the secretory peptides are formed,
undergo various morphological changes during their
de novo formation or regeneration which most likely
also include a decrease in pH [21]. Just like mamma-

lian skin, which has a pH of 4.5–5.5, frog skin also
has a pH which may be essential for several key
defence functions [22]. As a consequence, maturation
of the secretory granules might be accompanied by
aggregation of aDrs. In turn, environmental pH would
trigger the dissociation of these aggregates after release
of the gland contents.
Table 1. Peptide sequences of aDrs and variants in single letter
code. C* denotes fluorophor coupled to cysteine via a thioether
linkage.
5101520
aDrs LLGDLLGQTSKLVNDLTDTVGSIV-OH
aDrs* C*-OH
aDrs a NH
2
N4 N N N
N4a N N N NH
2
Self-assembly of anionic dermaseptin R. Go
¨
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¨
fberger et al.
5854 FEBS Journal 276 (2009) 5849–5859 ª 2009 The Authors Journal compilation ª 2009 FEBS
Metastable granular aggregates are formed
during disassembly
Within the transition interval (pH 5–6.5), metastable
amorphous ‘backward’ aggregates were transiently
formed during melting of the amyloid-like fibrils
(Fig. 4). The clearance or rearrangement of these gran-

ules apparently comprised two observable overlapping
processes which reflect their dynamic character: (a) dis-
ruption of the remaining longitudinal ridges which are
formed by the aligned b sheets and are thought to con-
stitute the ThT-binding sites (Fig. 4) [23]; and, with a
different kinetics, (b) breaking of residual interchain
hydrogen bonds which are still present in the granules,
yet to a lesser extent when compared with the amyloid
form of the peptide (Fig. 4). Otherwise, the structure
of the aggregated peptide seems to resemble that of
the soluble form because, according to CD and FTIR
spectra, it consists of comparable contributions from b
hairpin and randomly ordered segments. The half-life
of these aggregates, which were found to be cytotoxic
to cultured insect cells, was  5 days at pH 6 (Fig. 5).
Presumably, within the transition interval the final
state is nontoxic oligomeric within the range
5–25 mers, which could not be visualized by TEM.
Thus a critical oligomer size or their metastable char-
acter may be required for cytotoxicity. By contrast, at
pH 8, a predominantly monomeric state is instanta-
neously formed.
Granular aggregates cause neither cytolysis nor
generation of reactive oxygen species
It is widely accepted that the aggregation state of
antimicrobial peptides in solution is an important
parameter determining their activity against eukaryo-
tic cells [24,25]. Some antimicrobial peptides such as
dermaseptin S4 aggregate to cytotoxic amorphous
structures [24,26]. Cytolytic activity, however, is

mediated by the folded amphipathic monomer, which
is localized at the plasma membrane, where high
effective concentrations can be reached at the sites of
impact of the aggregates. We were interested in deli-
neating the fundamental biochemical mechanism
through which aDrs granular intermediates are cyto-
toxic to eukaryotic cells. Apparently, the peptide
quickly interacted with the cells and penetrated into
the cytoplasm independent of the aggregation state
(Fig. S3). Cellular damage, however, was induced only
by the aggregated form and became detectable after
3 h. During these stages, the integrity of the cell
membrane remained largely intact because no LDH
(M
r
, 140 kDa; diameter,  8 nm) was released from
the cells [27]. These results rule out a cytolytic
mechanism.
In some instances, antimicrobial peptides such as
apidaecin [28] are directed against intracellular targets
rather than against the cell membrane. However,
protofibrils, the prefibrillar ‘forward’ oligomers, are
suspected of being the effectors of cellular damage
associated with numerous protein deposition diseases
[29], including neurodegenerative disorders such as
Alzheimer’s or Parkinson’s disease [30], and amyloi-
doses [31,32]. The inherent cytotoxicity of protofibrils
is characterized by the intracellular localization of the
polypeptides, the intactness of the cell membrane, the
onset of apoptosis and mitochondrial damage [33,34].

Although the action of aDrs fulfils the first two
criteria, we did not detect the generation of reactive
oxygen species, which appear during apoptosis and
accompany protofibril action. Therefore, our results
indicate that aDrs aggregates act via an independent,
as yet unidentified mechanism.
In contrast to protofibrils, amyloid fibrils are not
inherently cytotoxic. Nevertheless, the cytotoxicity of
certain Ab-amyloid species may depend on structural
parameters [34,35]. Furthermore, cytotoxicity can be
induced by binding secondary components such as
GM1 ganglioside [36].
A correlation of amyloid with innate immune
defence
A few cases of diverse functional roles for amyloid in
biology have been shown to date [37]. Whenever amy-
loid participates in physiological processes, its forma-
tion appears to be tightly regulated or the prolonged
presence of toxic polymerization intermediates is
avoided. In strong contrast, it has been suggested that
protofibrils and oligomeric states of antimicrobial pep-
tides share common features of cytotoxic action [38,39].
This hypothesis may hold for aDrs. Further studies are
needed in order to fully understand the processes lead-
ing to membrane penetration and ultimately to cellular
damage. Very recently, another dermaseptin, derma-
septin S9, has been found to form amyloid in vitro.
Interestingly, soluble, weakly self-associated forms of
dermaseptin S9 were shown to mediate the chemotaxis
of human leukocytes, whereas spherical aggregates of

the same peptide exerted antimicrobial activity [40].
In conclusion, there is now emerging evidence that
amyloid-like aggregates and their intermediate states
could be an integrative component of the innate
immune system, for which amphibian skin is an
approved model system [41–43]. Remarkably, the
noncytotoxic aDrs from this source exhibits a reversible
R. Go
¨
ßler-Scho
¨
fberger et al. Self-assembly of anionic dermaseptin
FEBS Journal 276 (2009) 5849–5859 ª 2009 The Authors Journal compilation ª 2009 FEBS 5855
pH-dependent self-assembly to amyloid in vitro.A
natural defence strategy could involve such an amyloid
deposit, from which a temporarily cytotoxic agent could
be quickly formed triggered by an increase in pH.
Experimental procedures
Peptide synthesis and purification
All peptides were prepared using solid-phase peptide synth-
esis and were provided by Peptide Specialty Laboratories
(Heidelberg, Germany). BODIPY C1-FLÒ was from Invi-
trogen (Carlsbad, CA, USA). The peptides were purified
by RP-HPLC using a 0.1%(v ⁄ v) HCl ⁄ acetonitrile gradient
solvent system.
SUVs
A solution of 20 mgÆmL
)1
POPC (Avanti Polar Lipids, Ala-
baster, AL, USA) in water was sonicated for 10 min under

argon in a bath-type sonicator, resulting in a clear SUV
suspension.
CD spectroscopy
Peptides were dissolved in 50 mm phosphate-buffer pH 7.0 to
a final concentration of 100 lm. SDS or dodecylphosphocho-
line was added to a final concentration of 15 or 5 mm, respec-
tively. Alternatively, POPC SUVs were added to a
peptide ⁄ lipid ratio of 1 : 33 (3.25 mm POPC). CD spectra
were recorded on a Jasco (Easton, MD, USA) J-810 CD spec-
trophotometer. Spectra were measured in a 1 mm pathlength
cell at 20 °C. Five scans in the wavelength range 200–250 nm
were recorded for each spectrum at a rate of 20 nmÆmin
)1
.
Baseline was corrected using pure buffer or buffer and POPC,
respectively. Secondary structure content was estimated by
linear combination of reference spectra given in [44].
ThT assay
ThT stock solution (0.08% w ⁄ v ThT in 0.1 m HCl) was
added to aDrs gel (1 mgÆmL
)1
) and either examined directly
using fluorescence microscopy (Olympus, Tokyo, Japan,
IMT-2 microscope equipped with an FITC-filter cube) or
diluted in phosphate buffer of the desired pH for direct
fluorescence measurement at the respective pH (excitation
at 440 nm, emission 482 nm, slitwidth 5 nm). Within the
pH interval of interest detection of amyloid should not be
affected by hydroxylation of ThT [45].
Congo Red assay [46]

Lyophilized aDrs fibrils were incubated with a filtrated
Congo Red stock solution (saturated Congo Red, NaCl in
80% ethanol) [47], centrifuged and the resuspended pellet
was examined for birefringence under crossed polarisators
with a light microscope (Olympus) using a DPlanApo 20
objective.
TEM
Peptide samples were spotted on carbon-coated formvar-cov-
ered copper grids (Ager Scientific, Stansted, UK), negatively
stained with 2% (w ⁄ v) uranyl acetate (Electron Microscope
Sciences, Hatfield, PA, USA) or NanoVan (Nanoprobes,
Yaphank, NY, USA) and examined with a Jeol 2010 electron
microscope (Tokyo, Japan) operated at 100 kV.
FTIR
Hydrogen exchange of the peptides was performed by
repeated dissolution in D
2
O and lyophilization at a neutral
pD. Peptide samples were dissolved in DCl ⁄ D
2
O to a final
concentration of 2 mm and allowed to aggregate.
All spectra were collected at room temperature on a Bruker
(Billerica, MA, USA) spectrometer (model Tensor 27) as pre-
viously described [48]. The amide I’ region (1600–1700 cm
)1
)
of the sample spectrum was examined [49]. Buffers in D
2
O

were used to measure background spectra. Prior to curve fit-
ting, peaks resulting from water vapour were interactively
removed and a 13-point Savitsky–Golay smoothing filter was
applied. A straight baseline passing through the ordinates at
1600 and 1700 cm
)1
was subtracted. Second-derivative spec-
tra were calculated in order to identify peak positions. The
contributions of the individual peaks were estimated by fitting
Gauss peaks at the most likely band assignments. Secondary
structure was correlated to band frequencies according [13].
Equilibrium dialysis
Five-hundred micrlitre Micro-Equilibrium Dialyzers (Har-
vard Apparatus, Holliston, MA, USA) were equipped with
Spectra ⁄ Por RC Membranes, MWCO 12-14000 (Spectrum-
labs, Breda, Netherlands) or Spectra ⁄ Por 3, MWCO 3500.
After loading with 250 lg peptide, the chambers were
rotated at 37 °C for 24 h. The amount peptide present in
both chambers was analysed by RP-HPLC using a 0.1%
(v ⁄ v) TFA ⁄ acetonitrile gradient solvent system.
Inhibition zone assay [50]
E. coli and B. subtilis were grown at 37 °Cin10mLLB
medium pH 6.5 to a D
600
of 1.2. An agar plate was over-
lain with 150 lL of diluted (1 : 10) bacteria stock in 20 mL
of 0.7% Agarose Type I in LB medium pH 6.5. Holes were
prepared and filled with peptide dilutions (0, 4, 16 and
32 lm). The plates were incubated overnight at 37 °C and
inspected for clear inhibition zones.

Self-assembly of anionic dermaseptin R. Go
¨
ßler-Scho
¨
fberger et al.
5856 FEBS Journal 276 (2009) 5849–5859 ª 2009 The Authors Journal compilation ª 2009 FEBS
Liquid growth inhibition assay [51]
Preinocula of E. coli were prepared in LB medium (pH 6.5
or 5.5) at 37 °C and diluted to a D
600
of 0.0001 (1 : 10
6
).
Peptides were added to the bacteria suspension to final con-
centrations of 0, 4or16lm and incubated at 37 °C for
2 h. Bacteria were plated out, incubated overnight at 37 °C
and the colonies counted.
Cell culture
Sf9 cells were cultured in Insect Express SF9-S2 medium
(PAA cell culture company, Pasching, Austria) adjusted to
pH 6.0 at 27 °C. NIH-3T3 cells (mouse fibroblasts) were
cultured in Dulbecco’s modified Eagle’s medium containing
10% bovine calf serum in a 5% CO
2
humidified atmo-
sphere at 37 °C.
MTT inhibition assay [52]
Cells were seeded at a density of 4 · 10
3
cellsÆwell

)1
on
24-well plates in 500 lL of fresh medium. Fresh monomeric
aDrs or a fibril suspension were added to the cells to final
concentrations of 0, 1, 4 and 16 lm. Alternatively,
fibril suspensions preincubated at pH 5.5 for 0, 15 or 60 min
were added to the cells to final concentrations of 15 lm.
After 24 h incubation, MTT stock solution in NaCl ⁄ P
i
was
added to each well to give a final concentration of
0.5 mgÆmL
)1
and incubated for further 4 h. Five hundred
microlitres of cell lysis buffer (10% SDS, 50% N,N-dimethyl-
formamide) was added to each well. The samples were incu-
bated overnight at 27 or 37 °C, respectively, and absorbance
at 570 nm was determined with a Cary spectrophotometer.
LDH release assay [27]
Sf9 cells were seeded at a density of 4 · 10
3
cellsÆwell
)1
on
24-well plates in 500 lL of fresh medium and fibril suspen-
sion was added to the cells to a final concentration of 15 lm.
The concentration of LDH in 200 lL culture supernatants
was determined with a Cyto Tox 96Ò non-radioactive cyto-
toxicity assay (Promega, Madison, WI, USA) according to
the manufacturer’s instructions. Untreated cells were used to

determine background lysis, cells treated with 1% Triton
X-100 were used to determine total LDH (100% lysis).
Determination of reactive oxygen species
The generation of intracellular reactive oxygen species in Sf9
cells was examined by oxidation of the dye 2¢,7¢-dichloro-
fluorescin diacetate (Invitrogen). Cells were seeded in 24-well
plates and allowed to reach 50% confluence. The cells were
loaded with 5 lm 2¢,7¢-dichlorofluorescin diacetate and
incubated in the presence of 15 lm aggregated peptide for
differing time intervals. Reactive oxygen species levels were
detected by measuring the fluorescence of the oxidized dye
with a plate reader (Zenyth 3100 Multimode Spectrofluori-
meter, Anthos Mikrosysteme GmbH, Krefeld, Germany)
with excitation at 485 nm and emission at 535 nm.
Laser scanning fluorescence microscopy
Sf9 cells were seeded into an AttoFluor chamber (Invitro-
gen). Fibril suspension or soluble aDrs was added to the
cells to a final concentration of 15 lm. After 3 and 20 h of
incubation, a QLC100 Real-Time Confocal System (Visi-
Tech Int., Sunderland, UK) was used for recording fluores-
cence images. A Photometrics CoolSNAPHQ monochrome
camera (Roper Scientific, Sarasota, FL, USA) and a dual
port adapter (dichroic: 505lp; emission filter: 535 ⁄ 50;
Chroma Technology Corp., Rockingham, VT, USA) were
connected in conjunction with an argon ion multiwave-
length (514 nm) laser (Spectra Physics, Mountain View,
CA, USA). This system was attached to an Axiovert 200M
microscope (Zeiss, Oberkochen, Germany).
Acknowledgements
We thank Christa Mollay for helpful suggestions and

proofreading the manuscript, Dr Irene Frischauf for
operating the plate reader, Professor Hermann Gruber
for advice concerning the preparation of SUVs, Profes-
sor Christoph Romanin for providing access to the
confocal microscope, Professor Reinhard Vlasak for
the Sf9 cells and Professors Gu
¨
nther Kreil and Heinz
Falk for critically reading the manuscript and useful
comments. This work was supported by the FWF
grant P19393 to A. Jilek, and by grant K-151.217 ⁄
4-2007 ⁄ Lin of the ‘Land Obero
¨
sterreich’ to A. Jilek.
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Supporting information
The following supplementary material is available:
Fig. S1. CD spectra of soluble aDrs (100 lm) at var-
ious pH.
Fig. S2. aDrs* has similar self-aggregation and cyto-
toxic properties to unlabelled aDrs.
Fig. S3. Progression of the poisoning effect of aDrs
aggregates on Sf9 insect cells.
Fig. S4. No reactive oxygen species were induced by
aDrs aggregates.
Fig. S5. Amyloid-like characteristics of N4a aggregates
at neutral pH.
This supplementary material can be found in the
online article.
Please note: As a service to our authors and readers,
this journal provides supporting information supplied
by the authors. Such materials are peer-reviewed and

may be re-organized for online delivery, but are not
copy-edited or typeset. Technical support issues arising
from supporting information (other than missing files)
should be addressed to the authors.
R. Go
¨
ßler-Scho
¨
fberger et al. Self-assembly of anionic dermaseptin
FEBS Journal 276 (2009) 5849–5859 ª 2009 The Authors Journal compilation ª 2009 FEBS 5859