Báo cáo khoa học: An antimicrobial peptide tachyplesin acts as a secondary secretagogue and amplifies lipopolysaccharide-induced hemocyte exocytosis potx

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 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 as a 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, as a 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 as a 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 and antimicrobial 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 hemocyte exocytosis in the absence of LPS. Furthermore, tachyplesin, a major antimicrobial peptide of hemocytes, was able to trig- ger exocytosis in an LPS-independent manner, which was inhibited by a phospholipase C inhibitor, U-73122, and a 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 hemocyte exocytosis also occurs via a G protein-mediating signaling pathway. We concluded that tachyplesin functions not only as an antimicrobial substance, but also as a secondary secretagogue 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. Hemocyte exocytosis 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 as a 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 as a 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 an antimicrobial 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 hemocyte exocytosis 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 hemocyte exocytosis 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 antimicrobial peptide as a secretagogue 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. An antimicrobial peptide as a 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, and a 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 exocytosis and acts as a secondary secreta- gogue released from hemocytes following stimulation by LPS. Tachyplesin induces exocytosis via a G protein- mediating signaling pathway LPS-induced hemocyte exocytosis 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, and a G protein inhibitor, pertussis toxin, strongly inhibited exocytosis at 1 lm and 1 lgÆmL )1 , respectively, indicating that the tachyple- sin-induced hemocyte exocytosis 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 as a 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 antimicrobial peptide as a secretagogue 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 tachyplesin and 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 hemocyte exocytosis 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 as a 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. An antimicrobial peptide as a 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 hemocyte exocytosis (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 an antimicrobial peptide, but also as an 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 as a secondary 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 as a 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 as a 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 as a 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 antimicrobial peptide as a secretagogue 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 as a 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. An antimicrobial peptide as a secretagogue FEBS Journal 272 (2005) 3863–3871 ª 2005 FEBS 3869 References 1 Janeway CA Jr (1989) Approaching the asymptote? Evolution and revolution in immunology. Cold Spring Harbor Symp Quant Biol 54, 1–13. 2 Hoffmann JA, Kafatos FC, Janeway CA & Ezekowitz RAB (1999) Phylogenetic perspectives in innate immu- nity. 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