Báo cáo khoa học: An antimicrobial peptide tachyplesin acts as a secondary secretagogue and amplifies lipopolysaccharide-induced hemocyte exocytosis potx
Anantimicrobialpeptidetachyplesinactsasa secondary
secretagogue andamplifies lipopolysaccharide-induced
hemocyte exocytosis
Aya Ozaki
1
, Shigeru Ariki
1
and Shun-ichiro Kawabata
Department of Biology, Faculty of Sciences, Kyushu University, Fukuoka, Japan
The innate immune system is a sensitive nonself-recog-
nizing cascade triggered by microbial cell wall consti-
tuents referred to as pathogen-associated molecular
patterns (PAMPs), which include the lipopolysaccha-
ride (LPS) of Gram-negative bacteria, b-1,3-glucan of
fungi, and peptidoglycan of Gram-positive bacteria
[1,2]. PAMPs are recognized via a set of pattern-recog-
nition receptors and proteins that are germline-enco-
ded receptors of the innate immune system. Recent
studies have revealed that insects and mammals have
a conserved signaling pathway of the innate immune
system that functions through cell-surface receptors
referred to as Toll and Toll-like receptors [3,4]. Ini-
tially, Toll was identified asa transmembrane protein
that controls dorsoventral patterning in the Drosophila
embryo [5]. In the embryo, a proteolytic cascade con-
taining four proteases produces a cytokine-like protein,
Spaetzle, asa ligand for Toll. During infection, the
cleaved form of Spaetzle is produced via another pro-
teolytic cascade that includes Persephone, a newly
identified serine protease [6]. In the case of Drosophila,
Toll controls the host defense against fungal and
Gram-positive bacterial infections, but it does not
function asa pattern-recognition receptor for PAMPs
[7]. The Drosophila immune system also detects bac-
teria via peptidoglycan-recognition proteins. Gram-
negative diaminopimelic-acid-type peptidoglycan is
recognized as the most potent inducer of the Imd
Keywords
exocytosis; horseshoe crab; innate
immunity; secretagogue; tachyplesin
Correspondence
S. Kawabata, Department of Biology,
Faculty of Sciences, Kyushu University,
Fukuoka 812-8581, Japan
Tel ⁄ Fax: +81 92 642 2632
E-mail: skawascb@mbox.nc.kyushu-u.ac.jp
1
Note
A. Ozaki and S. Ariki contributed equally to
this work
(Received 30 March 2005, revised 25 May
2005, accepted 31 May 2005)
doi:10.1111/j.1742-4658.2005.04800.x
In the horseshoe crab, bacterial lipopolysaccharide (LPS) induces exocyto-
sis by granular hemocytes, resulting in the secretion of various defense
molecules, such as lectins andantimicrobial peptides, via a G protein-
mediating signaling pathway. This response is a key component of the
horseshoe crab innate immune response against infectious microorganisms.
Here, we report an endogenous amplification mechanism for LPS-induced
hemocytes exocytosis. The concentration of LPS required for maximal
secretion decreased in proportion to the density of hemocytes, suggesting
the presence of a positive feedback mechanism for secretion via a mediator
secreted from hemocytes. The exocytosed fluid of hemocytes was found
able to induce hemocyteexocytosis in the absence of LPS. Furthermore,
tachyplesin, a major antimicrobialpeptide of hemocytes, was able to trig-
ger exocytosis in an LPS-independent manner, which was inhibited by a
phospholipase C inhibitor, U-73122, anda G protein inhibitor, pertussis
toxin. Surface plasmon resonance analysis showed that tachyplesin directly
interacts with bovine G protein. These findings suggest that the tachyple-
sin-induced hemocyteexocytosis also occurs via a G protein-mediating
signaling pathway. We concluded that tachyplesin functions not only as an
antimicrobial substance, but also asasecondarysecretagogue of LPS-
induced hemocyte exocytosis, leading to the amplification of the innate
immune reaction at sites of injury.
Abbreviations
LPS, lipopolysaccharide; PAMP, pathogen-associated molecular pattern; TL-2, tachylectin-2.
FEBS Journal 272 (2005) 3863–3871 ª 2005 FEBS 3863
pathway [8,9]. In mammals, Toll-like receptors on
specialized antigen-presenting cells function as signal
transducers by way of nuclear factor (NF)- jB, thus
leading to the production of pro-inflammatory cyto-
kines and the expression of costimulatory molecules on
the cell surface. These cytokines and costimulatory
molecules are necessary to activate naive T cells; it has
been suggested that Toll-like receptors are assembled
in the cell membrane as signaling receptor complexes
[10].
Circulating hemocytes are essential to invertebrate
innate immunity, which includes the performance of
functions such as nonself recognition, phagocytosis,
encapsulation, and melanization [11]. In the horseshoe
crab Tachypleus tridentatus, granular hemocytes
account for 99% of all hemocytes and are involved in
the storage and release of defense molecules, including
serine protease zymogens, a clottable protein coagulo-
gen, protease inhibitors, antimicrobial peptides, and
lectins [12–14]. Horseshoe crab hemocytes are highly
sensitive to LPS [15]. In response to stimulation by
LPS, the defense molecules stored in granules are
immediately secreted by exocytosis [12,16]. This exocy-
tosis reaction is important for the host defense ability
to engulf and kill invading microbes; hemolymph
coagulation prevents the leakage of hemolymph and
the spread of infectious pathogens, while lectins and
antimicrobial peptides aggregate and lyse the patho-
gens. Hemocyteexocytosis is specifically induced by
LPS, but not by other PAMPs such as b-1,3-glucan
and peptidoglycan [17]. A cDNA coding for a Toll-like
receptor has been identified from horseshoe crab
hemocytes and is most closely related to the Drosophila
Toll in terms of both its domain architecture and over-
all length [18]. Human Toll-like receptors have been
suggested to contain numerous PAMP-binding inser-
tions located in the leucine-rich repeats of their ecto-
domains [19]. However, we found that the leucine-rich
repeats of the horseshoe crab Toll and those of the
Drosophila Toll contained no obvious PAMP-binding
insertions, suggesting that the horseshoe crab Toll does
not function asa PAMP receptor on granular hemo-
cytes [18].
We recently established a quantitative assay for the
LPS-induced exocytosis of granular hemocytes and
reported that a granular protein factor C, an LPS-
recognizing serine protease zymogen that initiates the
hemolymph coagulation cascade, also exists on the
hemocyte surface asa biosensor for LPS [17]. The pro-
teolytic activity of factor C is both necessary and suffi-
cient to trigger exocytosis via a heterotrimeric G
protein-mediating signaling pathway. Using this assay,
we found that the reactivity of hemocytes to LPS
increases in proportion to the density of the hemo-
cytes. This finding prompted us to speculate the pres-
ence of an amplification mechanism of exocytosis via
mediators secreted from hemocytes. Here, we report
that anantimicrobial peptide, tachyplesin, which is a
component of the small granule of hemocytes that is
secreted in response to stimulation by LPS, induces
hemocyte exocytosis via a G protein-mediating signa-
ling pathway and thereby amplifies LPS-induced exo-
cytosis.
Results
Effect of hemocyte density on LPS-induced
exocytosis
Different cell numbers of hemocytes from 0.5 · 10
5
to
8.0 · 10
5
cellsÆwell
)1
were treated with various concen-
trations of LPS ranging from 10
)13
to 10
)7
gÆmL
)1
,
and hemocyteexocytosis was quantitatively assayed by
ELISA using an antibody against tachylectin-2 (TL-2)
(Fig. 1A). An optimal concentration of LPS was
observed for each cell density to obtain the maximal
secretion. This bell-shaped, dose-dependent curve was
quite similar to that of LPS for the activation of factor
C in vitro [20]; therefore, the curve appears to indicate
that factor C molecules are clusterized on the hemo-
cyte surface by interaction with LPS, thus triggering
exocytosis. The re-plot of the optimal concentration
vs. cell density showed that the optimal LPS concen-
tration decreases with increasing cell density (Fig. 1B).
These data suggest the presence of a positive feedback
mechanism for secretion via an unknown secretagogue
secreted from hemocytes in response to stimulation by
LPS.
Tachyplesin induces the exocytosis of granular
hemocytes
To examine whether or not exocytosed fluid induces
exocytosis, hemocytes at 1.0 · 10
6
cellsÆwell
)1
were
treated with 1.0 · 10
)12
gÆmL
)1
LPS, and the exocyto-
sed fluid was collected. TL-2 in the exocytosed fluid
was removed by immunoprecipitation using anti-(TL-
2) polyclonal antibody to avoid contamination in the
exocytosis assay. The resulting TL-2-free exocytosed
fluid induced hemocyteexocytosis at 0.5 · 10
5
cellsÆ
well
)1
(Fig. 2A, bar 2). In contrast, LPS at 1.0 · 10
)12
gÆmL
)1
was unable to trigger exocytosis under the
same conditions, since the cell density at 0.5 · 10
5
cellsÆwell
)1
was too low for the induction of exocytosis
(Figs 1A and 2A, bar 1). These results suggest that the
exocytosed fluid contained an unknown factor capable
An antimicrobialpeptideasasecretagogue A. Ozaki et al.
3864 FEBS Journal 272 (2005) 3863–3871 ª 2005 FEBS
of inducing hemocyte exocytosis. To identify this
unknown factor, the exocytosed fluid was fractionated
by gel filtration, and each fraction obtained was exam-
ined in terms of its ability to induce exocytosis
(Fig. 2B). The results revealed that fraction 24 was
able to efficiently induce exocytosis. The elution posi-
tion of this fraction exactly corresponded to that of
tachyplesin (Fig. 2C). Tachyplesin is a major compo-
nent of the small granules of hemocytes, and is an
antimicrobial peptide with a broad spectrum of activity
against fungi, Gram-positive bacteria, and Gram-neg-
ative bacteria [21]. When tachyplesin in the exocytosed
fluid was removed by immunoprecipitation using anti-
tachyplesin polyclonal antibody, the tachyplesin-deple-
ted exocytosed fluid led to a 60% decrease in TL-2
secretion (Fig. 2D).
Fig. 1. Effects of hemocyte density on exocytosis. (A) Five different
numbers of hemocytes adsorbed on sterilized plastic wells were
treated with various concentrations of LPS at 23 °C for 1 h. The
amount of TL-2 secreted in the supernatant was determined by
ELISA. h,8.0· 10
5
; d,4.0· 10
5
; s,2.0· 10
5
; m,1.0· 10
5
; n,
0.5 · 10
5
cellsÆwell
)1
. (B) The optimal LPS concentration that resul-
ted in the maximal secretion at each cell density was replotted.
Each symbol corresponds to that defined in (A).
Fig. 2. Exocytosed fluid induces exocytosis. (A) Hemocytes
(1.0 · 10
6
cells) adsorbed on plastic wells were treated with
1pgÆmL
)1
LPS at 23 °C for 1 h. TL-2 in the exocytosed fluid was
removed by immunoprecipitation using anti-(TL-2) Ig. Hemocytes
(0.5 · 10
5
cells) were treated with the resulting supernatant at
23 °C for 1 h (bar 2) or with 1 pgÆmL
)1
LPS, as the negative control
(bar 1). (B) Hemocytes (1.0 · 10
6
cells) were treated with
1pgÆmL
)1
LPS at 23 °C for 1 h. The exocytosed fluid was fraction-
ated by gel filtration. Hemocytes (0.5 · 10
5
cells) were treated with
each fraction obtained at 23 °C for 1 h. After treatment, the
amount of exocytosed TL-2 was determined. (C) Purified tachyple-
sin (50 lg) was subjected to gel filtration under the same condi-
tions. (D) The exocytosed fluid was treated with anti-TL-2 (bar 1) or
both anti-tachyplesin and anti-(TL-2) polyclonal antibody (bar 2).
Each supernatant was used for the exocytosis assay at 0.3 · 10
5
cellsÆwell
)1
.
A. Ozaki et al. Anantimicrobialpeptideasa secretagogue
FEBS Journal 272 (2005) 3863–3871 ª 2005 FEBS 3865
Next, the purified tachyplesin was examined in terms
of whether or not it could induce hemocyte exocytosis.
As expected, tachyplesin was indeed able to induce
exocytosis in a concentration-dependent manner in the
absence of LPS (Fig. 3A). To eliminate the possibility
of LPS contamination in the assay sample, 10 lm
tachyplesin was adsorbed onto CM sepharose, and the
supernatant was collected by centrifugation. The exo-
cytosis activity of the resulting tachyplesin-depleted
supernatant decreased to about 15% of that of the
nontreated sample (Fig. 3B). To confirm further whe-
ther or not the tachyplesin sample was contaminated
with LPS, hemolymph was collected into sterilized
glass tubes and incubated at 23 °C for 45 min with
10 lm tachyplesin. To serve as positive controls,
hemolymph was mixed with various concentrations
of LPS, anda minimum concentration of LPS at
1.0 · 10
)10
gÆmL
)1
triggered hemolymph coagulation
via the autocatalytic activation of factor C by LPS. In
contrast, 10 lm tachyplesin also induced exocytosis,
but hemolymph coagulation did not occur, indicating
that LPS contamination of the tachyplesin sample
amounted to less than 1.0 · 10
)10
gÆmL
)1
. In the exo-
cytosis assay carried out using a low density of hemo-
cytes (0.5 · 10
5
cellsÆwell
)1
), a concentration of LPS at
1.0 · 10
)10
gÆmL
)1
was found unable to induce exo-
cytosis (Fig. 1A). Therefore, the influence of LPS con-
tamination in the test samples for tachyplesin-induced
exocytosis was negligible in the present study. These
findings clearly demonstrated that tachyplesin induces
hemocyte exocytosisandactsasasecondary secreta-
gogue released from hemocytes following stimulation
by LPS.
Tachyplesin induces exocytosis via a G protein-
mediating signaling pathway
LPS-induced hemocyteexocytosis has been shown to
take place via a signaling pathway mediated by per-
tussis toxin-sensitive-G protein that activates phos-
pholipase C [17]. To examine whether or not
tachyplesin induces exocytosis via the same pathway as
that associated with the activity of LPS, two types of
inhibitors were tested for their effects on the exocytosis
induced by tachyplesin. Both a phospholipase C inhib-
itor, U-73122, anda G protein inhibitor, pertussis
toxin, strongly inhibited exocytosis at 1 lm and
1 lgÆmL
)1
, respectively, indicating that the tachyple-
sin-induced hemocyteexocytosis occurs via a G pro-
tein-mediating signaling pathway similar to that which
mediates exocytosis in response to stimulation by LPS
(Fig. 4).
Binding parameters of mastoparan and
tachyplesin to G protein
Mastoparan, a basic tetradecapeptide from wasp
venom, directly interacts with G protein and receptor-
independently induces the exocytosis of mast cells [22].
Previously, we found that mastoparan is able to induce
the exocytosis of the granular hemocytes of horseshoe
crabs [17]. Tachyplesin has structural properties in
common with mastoparan, such asa high content of
basic amino acids, an amphiphilic structure, and an
Fig. 4. Effects of inhibitors on exocytosis induced by tachyplesin.
Hemocytes (0.5 · 10
5
cellsÆwell
)1
) were preincubated with
U-73122 (0.1 l
M,1lM)at23°C for 20 min, or with pertussis toxin
(0.1 lgÆmL
)1
,1lgÆmL
)1
)at23°C for 1 h. Hemocyte exocytosis
was induced by 10 l
M tachyplesin. A control experiment was per-
formed without the inhibitor treatment.
Fig. 3. Tachyplesin induces exocytosis. (A) Hemocytes (0.5 · 10
5
cellsÆwell
)1
) were treated with various concentrations of tachyplesin
in the absence of LPS at 23 °C for 1 h, and the amount of exocyto-
sed TL-2 was determined. (B) Tachyplesin (10 l
M) was incubated
with CM sepharose at 4 °C for 2 h, and the supernatant was collec-
ted by centrifugation. Hemocytes (0.5 · 10
5
cellsÆwell
)1
) were trea-
ted with CM sepharose-treated (bar 1) or nontreated tachyplesin
(bar 2) in the absence of LPS.
An antimicrobialpeptideasasecretagogue A. Ozaki et al.
3866 FEBS Journal 272 (2005) 3863–3871 ª 2005 FEBS
amidated carboxyl terminus, suggesting that tachyple-
sin might interact with G protein in a manner similar
to that of mastoparan, although we have not yet iden-
tified horseshoe crab G protein(s) involved in hemo-
cyte exocytosis. The binding parameters of mastoparan
and tachyplesin to bovine G protein were determined
by surface plasmon resonance analysis (Fig. 5A,B).
The passage of mastoparan at various concentrations
over G protein immobilized on a sensor chip yielded
an association rate constant k
a
¼ 2.0 · 10
4
m
)1
Æs
)1
and
a dissociation rate constant k
d
¼ 4.3 · 10
)3
s
)1
and,
consequently, a dissociation constant K
d
(k
d
⁄ k
a
) ¼
2.2 · 10
)7
m. As regards tachyplesin, the following
respective values were obtained: k
a
¼ 7.3 · 10
2
m
)1
Æs
)1
,
k
d
¼ 4.4 · 10
)4
s
)1
, and K
d
(k
d
⁄ k
a
) ¼ 8.8 · 10
)7
m;
these results thus indicated that tachyplesin is able to
bind directly to G protein.
Higashijima et al. reported that a cationic property
of the amphiphilic helical structure of mastoparan is
required to activate G protein [23]. Tachyplesin, which
consists of 17 amino acid residues, forms a rigid hair-
pin loop constrained by two disulfide bridges and
adopts the conformation of an antiparallel b-sheet con-
nected to a b-turn [24]. In the planar conformation
of tachyplesin, the six hydrophobic side chains are
thought to be localized at one face, and the six cat-
ionic side chains, one Lys and five Arg residues, are
thought to be distributed at another face. As shown in
Fig. 5C, the chemical modification of these Arg resi-
dues with 1,2-cyclohexanedione led to the complete
loss of the original affinity to G protein, suggesting
that the Arg residues play an important role in the
interaction between tachyplesinand G protein. The
modification at Arg residues did not appear to have
any effect on the overall conformation of tachyplesin,
since the same chemical modification of tachyplesin
has not been found to have an effect on the kinetic
parameters of the interaction between tachyplesin and
hemocyanin according to the surface plasmon reson-
ance analysis [25].
Discussion
In horseshoe crabs, the exocytosis of granular hemo-
cytes is one of the most important reactions of the
innate immune system against infectious microorgan-
isms. The hemolymph of horseshoe crabs contains
granular hemocytes at approximately 5.0 · 10
6
cellsÆmL
)1
, which reacts with a very small amount of
LPS at about 10
)13
gÆmL
)1
. We found that LPS-
induced hemocyteexocytosis is highly dependent on
the cell density, namely, an increase in cell density
from 0.5 · 10
5
to 8.0 · 10
5
cellsÆmL
)1
yielded a 10
6
-
folded change in the apparent LPS sensitivity from
10
)7
to 10
)13
gÆmL
)1
(Fig. 1). Here, we demonstrated
that a major granular component, tachyplesin, induced
exocytosis in an LPS-independent manner. For each
density of hemocytes, there was an optimal LPS con-
centration for the induction of exocytosis, and the
LPS-dependence of TL-2 secretion approximately con-
formed to a bell-shaped curve (Fig. 1A). The amount
of secreted TL-2 appears to be exclusively dependent
upon the initial amount of tachyplesin released under
the assay conditions asa 1-h incubation of hemocytes
with assay buffer is not long enough for tachyplesin to
induce maximal secretion. The initial trigger of hemo-
cyte exocytosis has been shown to be regulated by the
LPS-dependent autocatalytic activation of hemocyte-
bound factor C, and this autocatalytic activation of
factor C requires an optimal LPS concentration with a
bell-shaped curve [20]. Thus, the amount of initially
secreted tachyplesin decreases at levels above the
Fig. 5. Association and dissociation of tachyplesin or mastoparan with immobilized G-protein. Sensorgrams for the binding of mastoparan (A)
and tachyplesin (B) to G-protein immobilized on a sensor chip were superposed at various concentrations. (C) Sensorgrams of tachyplesin
(native) and Arg-modified tachyplesin at 500 n
M were superposed.
A. Ozaki et al. Anantimicrobialpeptideasa secretagogue
FEBS Journal 272 (2005) 3863–3871 ª 2005 FEBS 3867
optimal LPS concentration. Thus, the bell-shaped
curves in Fig. 1A reflect how much factor C is initially
activated. These findings suggest that the activation of
hemocyte-bound factor C, which leads to a chain reac-
tion of tachyplesin-potentiated exocytosis, requires an
optimal LPS concentration. In contrast, in the absence
of activated factor C, tachyplesin induces exocytosis in
a concentration-dependent manner without the require-
ment of an optimal concentration (Fig. 3A). Moreover,
tachyplesin most likely interacts directly with a G pro-
tein, leading to the enhancement of exocytosis, much
as in the case of mastoparan (Fig. 5). Other horseshoe
crab antimicrobial peptides such as tachycitin [26] and
big defensin [27] exhibited similar types of activity with
respect to hemocyteexocytosis (data not shown). How-
ever, depletion of tachyplesin from the exocytosed fluid
results in a considerable reduction of the original activ-
ity (Fig. 2D). Therefore, tachyplesin functions not only
as anantimicrobial peptide, but also asan effective
endogenous secretagogue of hemocytes, thereby enhan-
cing the sensitivity of the hemocytes to LPS.
Tachyplesin effectively induces the exocytosis of
granular hemocytes at concentrations ranging from 5
to 10 lm, indicating that a high concentration of tachy-
plesin is required to function asasecondary secreta-
gogue (Fig. 3A). Tachyplesin has been shown to bind
with hemocyanin and functionally converts hemocya-
nin to phenoloxidase [25]. It is possible that tachyple-
sin is trapped by hemocyanin in the hemolymph and
thus the spread of tachyplesin is limited to the site of
infection. Hemocyanin might prevent over-amplified
exocytosis by tachyplesin. Once defense molecules are
released from immune cells, their activities should be
tightly regulated at appropriate places. Crayfish per-
oxinectin, a 76-kDa protein identified asa multifunc-
tional protein (i.e. a cell adhesion factor, opsonin,
encapsulation factor, and peroxidase), is stored in
granular and semigranular hemocytes and is released
concomitant with activation of prophenoloxidase [28].
The activities of peroxinectin seem to be controlled
partly by proteolysis, since peroxinectin is rapidly
degraded to a less active 30-kDa fragment [29]. The
acid extract of horseshoe crab cuticles contains degra-
dation products of tachyplesin (unpublished data),
suggesting the possibility of proteolytic regulation of
tachyplesin activity.
In mammals, cytokines and chemokines, as modula-
tors of the inflammatory response, transmit complex
signals among immune cells [30,31]. Also in Dro-
sophila, the importance of communication between
hemocytes in the course of the immune response has
been reported, although the actual substance remains
unidentified [32]. Tachyplesin is a pluripotent peptide
and possibly functions asa modulator of the innate
immune reaction of horseshoe crabs at the site of
infection.
Mastoparan, a cationic tetradecapeptide from wasp
venom, induces the exocytosis of granular hemocytes
of the horseshoe crab [17]. Mastoparan was first identi-
fied asa stimulator that led to the release of histamine
from rat mast cells [33]. Mastoparan forms an amphi-
philic a-helix, and its carboxyl terminus is amidated.
Mastoparan is a potent stimulator of exocytosis from
diverse mammalian cells such as b-cells, platelets, neu-
trophils, and pneumocytes [34–40]. For the release of
histamine from mast cells, mastoparan interacts
directly with G protein and receptor-independently
induces exocytosis, whereas no direct evidence has yet
been reported that mastoparan enters the intracellular
space through the lipid bilayer membrane [41,42].
Tachyplesin induces exocytosis of granular hemocytes
via a G protein-mediating signaling pathway that is
likely to be the same pathway as that involved in LPS-
induced exocytosis (Fig. 4). The ability of tachyplesin
to bind to G protein was evaluated here by surface
plasmon resonance analysis (Fig. 5A,B). Although
tachyplesin shows a K
d
¼ 8.8 · 10
)7
m similar to that
of mastoparan (2.2 · 10
)7
m), both the association and
dissociation rate constants of tachyplesin were found
to be much lower than those of mastoparan. The phy-
siological meaning of these differences remains unclear
at present.
In mammals, endogenous basic secretagogues, inclu-
ding antimicrobial peptides defensins and cathelicidins,
activate G protein, resulting in the secretion of hista-
mine from mast cells; this scenario indicates that the
basic secretagogues directly interact with G proteins,
thus implicating the entry of secretagogues into mast
cells [43–45]. This account appears to be applicable to
the exocytosis-inducing activity of tachyplesin, since
the chemical modification of Arg residues in tachyple-
sin dramatically reduces its affinity to G protein
(Fig. 5C). Mast cells and horseshoe crab hemocytes
play central roles in the primary step of the immune
response, and these cells resemble each other function-
ally. The finding that an endogenous basic peptide,
tachyplesin, induces the exocytosis of granular hemo-
cytes is quite interesting in the context of a comparat-
ive examination of the innate immune mechanisms of
mammals and horseshoe crabs.
The PAMP-induced exocytosis of immune cells is
not a phenomenon specific to horseshoe crab hemo-
cytes. For example, mouse Paneth cells in the small
intestinal crypts secrete antimicrobial a-defensins in
response to stimulation by PAMPs such as LPS,
lipoteicholic acid, and muramyl dipeptide [46]. The
An antimicrobialpeptideasasecretagogue A. Ozaki et al.
3868 FEBS Journal 272 (2005) 3863–3871 ª 2005 FEBS
secretion of a-defensin is followed by an increase in
intracellular Ca
2+
and the involvement of the Ca
2+
-
activated K
+
channel mIKCa1 has been suggested
[47]. Paneth cells not only play a role in the innate
host defense as effector cells that produce antimicro-
bial factors and release them into the intestinal lumen,
but these cells may also communicate and coordinate
host defense signals with other cell types [48]. Further-
more, a-defensin induces interleukin 8 secretion in the
human intestinal cell line T84 via signaling cascades
that involve both NF-jB and p38 mitogen-activated
protein kinase. The mechanism of secretion may
require the reversible formation of ion-conductive
channels by peptides in the apical membrane. Such
findings suggest that a-defensin may amplify the roles
played by Paneth cells in innate immunity by acting
as paracrine agonists in order to coordinate an inflam-
matory response [49,50]. Such amplification of the
inflammatory response, mediated by multifunctional
molecules, may be conserved both in vertebrates and
arthropod.
Experimental procedures
Materials
Lipopolysaccharide (Salmonella minnesota R595) was from
List Biological Laboratories (Campbell, CA, USA). Per-
tussis toxin was from Wako Pure Chemical (Osaka, Japan).
U-73122 and bovine G protein (a mixture of G
o
and G
i
proteins from bovine brain) were from Calbiochem.
Mastoparan was from Bachem (Bubendorf, Switzerland).
Tachyplesin was purified as previously described [21,27],
and was further purified by reverse-phase HPLC on a
TSK-GEL Phenyl-5PW RP column (TOSOH, Tokyo,
Japan) [51].
Assay of exocytosis
Hemolymph (1 mL) was collected into 50 mL of pyrogen-
free 10 mm Hepes ⁄ NaOH pH 7.0, containing 0.5 m NaCl.
The diluted hemolymph (200 lL) was applied to pyrogen-
free 24-well plates filled with 800 lL of the same buffer,
and then was incubated at 23 °C for 10 min to allow for
the attachment of hemocytes. After removing the superna-
tant from each well, tachyplesin or LPS in the same buffer
containing 0.5 m NaCl, 50 mm MgCl
2
, and 10 mm CaCl
2
was added to the wells. For the inhibition studies, hemo-
cytes were pretreated with U-73122 for 20 min or with per-
tussis toxin for 1 h. The hemocytes were then stimulated
with 10 lm tachyplesin. After incubation at 23 °C for 1 h,
each exocytosed fluid was collected by centrifugation at
2000 g for 5 min. The amount of TL-2 in the exocytosed
fluid was quantitated by sandwich ELISA as previously
described [52].
Gel filtration of exocytosed fluid
Fluid that was exocytosed from the hemocytes was subjec-
ted to FPLC on a Superdex 75 HR 10 ⁄ 30 column (Amer-
sham Biosciences, Piscataway, NJ, USA) equilibrated with
10 mm Hepes-NaOH, pH 7.0, containing 0.5 m NaCl. The
eluate was collected and each fraction was subjected to the
exocytosis assay.
Chemical modification
The Arg residues of tachyplesin were modified with 100 mm
1,2-cyclohexanedione in 0.2 m boric acid, pH 9.0, at 37 ° C
for 2 h [53]. The modified tachyplesin was desalted by gel fil-
tration on a Sephadex G-15 column equilibrated with 30%
acetic acid and then the sample was lyophilized. The comple-
tion of chemical modification was confirmed by amino acid
and sequence analyses. This method of chemical modification
produced a 90% yield. Amino acid analysis was performed
on an AccQ-Tag system (Waters, Milford, MA). Amino acid
sequence was determined by using an Applied Biosystems
Procise 491-HT gas-phase protein sequencer.
Surface plasmon resonance analysis
G protein (20 lgÆmL
)1
in 10 mm sodium acetate, pH 5.5)
was immobilized on a CM5 sensor chip of the BIAcore
1000 system (BIAcore, Uppsala, Sweden), according to the
manufacturer’s specifications. After washing the sensor chip
with 10 mm Hepes ⁄ NaOH, pH 7.0, containing 0.15 m
NaCl, tachyplesin or mastoparan was injected at a flow rate
of 20 lLÆmin
)1
. The change in the mass concentration on
the sensor chip was monitored asa resonance signal by
using the program supplied by the manufacturer. Sensor-
grams of the interactions obtained using the various con-
centrations of peptides were analyzed by the BIAevaluation
program, version 3.0.
Acknowledgements
We thank Dr Takumi Koshiba (Kyushu University)
and Dr John Kulman (University of Washington Seat-
tle, WA, USA) for helpful discussions and suggestions,
and Dr Tsukasa Osaki (Kyushn University) for assis-
tance in amino acid analysis. This work was supported
by a Grant-in-Aid for Scientific Research on Priority
Area 839 from the Ministry of Education, Culture,
Sports, Science and Technology of Japan and by the
Naito Foundation and Japan Foundation for Applied
Enzymology (to S. K.).
A. Ozaki et al. Anantimicrobialpeptideasa secretagogue
FEBS Journal 272 (2005) 3863–3871 ª 2005 FEBS 3869
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A. Ozaki et al. Anantimicrobialpeptideasa secretagogue
FEBS Journal 272 (2005) 3863–3871 ª 2005 FEBS 3871
. An antimicrobial peptide tachyplesin acts as a secondary secretagogue and amplifies lipopolysaccharide-induced hemocyte exocytosis Aya Ozaki 1 , Shigeru Ariki 1 and Shun-ichiro Kawabata Department. horse- shoe crab antimicrobial peptides. J Biol Chem 276, 27166–27170. 26 Kawabata S, Nagayama R, Hirata M, Shigenaga T, Agarwala KL, Saito T, Cho J, Nakajima H, Takagi T & Iwanaga S (1996) Tachycitin,. lipid A analogues and acidic phospholipids. Eur J Biochem 176, 89–94. 21 Nakamura T, Furunaka H, Miyata T, Tokunaga F, Muta T & Iwanaga S (1988) Tachyplesin, a class of antimicrobial peptide