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

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 lipopolysac

secretagogue and amplifies lipopolysaccharide-induced hemocyte exocytosis

Aya Ozaki1, Shigeru Ariki1and 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.

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

Results

Effect of hemocyte density on LPS-induced exocytosis

Different cell numbers of hemocytes from 0.5· 105 to 8.0· 105 cellsÆwell)1were treated with various concen-trations of LPS ranging from 10)13 to 10)7gÆmL)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· 106 cellsÆwell)1 were treated with 1.0· 10)12gÆmL)1LPS, and the exocyto-sed fluid was collected TL-2 in the exocytoexocyto-sed 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· 105 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· 105 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

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 fracposi-tion 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 anti-

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 replotresul-ted.

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

1 pgÆmL)1LPS 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)1LPS, as the negative control (bar 1) (B) Hemocytes (1.0 · 10 6 cells) were treated with

1 pgÆmL)1LPS 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.

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· 105 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-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 inhibinhib-itor, 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 , 1 l M ) at 23
C for 20 min, or with pertussis toxin (0.1 lgÆmL)1, 1 lgÆmL)1) at 23
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-treatrea-ted (bar 1) or nontreatrea-ted tachyplesin

(bar 2) in the absence of LPS.

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 ka¼ 2.0 · 104 m)1Æs)1and

a dissociation rate constant kd¼ 4.3 · 10)3s)1 and,

consequently, a dissociation constant Kd (kd⁄ ka)¼

2.2· 10)7m As regards tachyplesin, the following

respective values were obtained: ka¼ 7.3 · 102m)1Æs)1,

kd¼ 4.4 · 10)4s)1, and Kd (kd⁄ ka)¼ 8.8 · 10)7m;

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· 106 cellsÆmL)1, which reacts with a very small amount of LPS at about 10)13gÆmL)1 We found that LPS-induced hemocyte exocytosis is highly dependent on the cell density, namely, an increase in cell density from 0.5· 105 to 8.0· 105 cellsÆmL)1 yielded a 106 -folded change in the apparent LPS sensitivity from

10)7 to 10)13gÆmL)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.

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

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 Kd¼ 8.8 · 10)7m similar to that

of mastoparan (2.2· 10)7m), 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

secretion of a-defensin is followed by an increase in

intracellular Ca2+ and the involvement of the Ca2+

-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 Go and Gi

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 MgCl2, and 10 mm CaCl2

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 modificacomple-tion 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.)

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