Vol.

176,

May

15, 1991

No.

Hida.ka,

AND BIOLOGICAL ENTEROTOXIN

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

PROPERTIES (ST) PRODUCED Escherichia coli

Research,

+Fa.cnIt,,v of Medicine, Instit,ut,e

Osaka University,

Kyoto

of Medical

University,

Tokyo Received

March

Yamadaoka

Yoshida-konoe,

Science. The Uuiversity

958-965

OF CARBA-ANALOGS BY ENTEROTOXIGENIC

Katsuhiko Ohmori, Plkihiro Wa.da, Hiroshi Ozaki, Hicleaki Hirayama.#, ~‘oshifurni Takeda+, aud Yasutsugu Shimonishi*

Tnstitut,e for Protein

#The

AND

Pages

SYNTHESIS HEAT-STABLE

Yuji

BIOCHEMICAL

3, 1991

It,o+,

OF

Toshiya

3-2, Suita, Osaka 565, Japan Sakyo-ku,

of Tokyo,

Iiyoto

606, Japan

Shirokanedai,

Miuato-ku,

108, .Japan

18, 1991

SUMMARY: Analogs of a, heat-st,able euterotmiu (ST) that have a. CH2-S linkage instea(l of an S-S linkage in t,he molecule were synthesized by conventiona methods. The synt,hetic pcptides showed toxicity, assayed a.s inductiou of fluid secretion in suckling mice, although their tosicities were hundredth t,hat of native ST. This finding implies that ST is not. WC‘ognized 11~ its receptor protein t.hrough an exchange reaction between its disulfide lirtlta.ges alIt t,hiol-groups of its recept,or proteins(s), hut, through hydrophobic or elect,rost.at ic illt.er0 1991 AcademicPRSB, Inc. actions.

Heat.-s-t,able ent,erotosins 18 amino cret,ion

acid residues in the intestine

isolat.ed from pathogenic rio cholm7e

noii-01

produced

(named

a.re linked

strains

activity

of the toxicit,y

0006-291W91 Copyright All rights

with

bacteria

wit,11 the conserved

animals

disulfide

these 13 amino

$1.50 0 1991 by Academic Press, of reproduction in an? form

IXXK~S, which

should

Inc. reserved.

is located

acid residues.

be addressed.

958

tl1a.t iucluce

e~ct~rocoliticrr

[7]. Our previouc + studies sequence

with

19 OI

fluid

S’nni 1al: enterot.oxins

such a.s E’eminiu

w were

[.5]. I/i/i-

denmust ratrtl

from CysG to Cys18 in STh ha{1 sequences

are organized

TI 11s .: 1u d’ica t es t1ia.t the structural

of the enterotosins

correspondence

in Fig.1) [l--1].

[S], aud t,lia.t in this conserved

by three int~ramolcular

the sequeuce

* ‘To whom

a.nd domestic of euteric

E. coli are pept,ides

respectively,

[6], a.nd C’ta 70 ~ buc-t*w, f rearndii

t.oxins exa.minecl so fa.r [9,10]. generat,ion

ST!, aud ST,.

of infmts

that a 13 a.mino acid peptide full enterotoxigeuic

1:)~ enterotosigenic

the six Cys resiclues identically

element(s)

in the tert,iary

structure

in all the required f(lrllie(l

for IL))

Vol.

176,

The

No.

BIOCHEMICAL

3, 1991

initial

step in the biological

tein on the membrane complex

of intestinal

leads to stimulation

concentra.tion

of guanylate

in the cells and fluid

a thiol-disulfide

of the receptor is actually

protein.

formed

hydrophobic,

of a disulfide with

we synthesized

linkage

the receptor

protein.

Demonstration

that ST does not bind t,o its receptor non-covalent,

by increase

in cyclic GMP

cells

on t,he surface of brushborder its disulfide

unknown

whether

reaction

linkages

and thiol

the ST-receptor

or by non-cova.lently

groups complex bondetl

nneractions. of ST, which

to undergo of their

protein

of an ST-recepi.or

receptor

carba:analogs

and so were unable

pro

t,hat,

exchange

or ion-bonding

t,o it.s receptor

from the cells. de .Jonge [J5] suggested

between

it is still

by this thiol-disulfide

hydrogen-bonding

In this work,

reaction

COMMUNICATIONS

Tl re f orma.tion

cyclase and is followed

secretion

However,

RESEARCH

of ST is it.s binding

cell [II-141.

protein-like

exchange

BIOPHYSICAL

reaction

epit,helial

ST may bind to a. tissue-specific through

AND

had a. CH?-S linkage

a t,hiol-disulfide

biological

by a thiol-disulfide

activities

inst.&

exchange

reaction

provided

evitleuce

exchange

reaction

but 1,)

interaction.

MATERIALS

AND

METHODS

Synthesis of Peptides : Peptides were synthesized by conventional methods according t,o t,he scheme sllowrl in Fig. 2. Briefly, t.he C-terminal tetra.peptide was prepared step 1)~ st,ep by coupling Boc-amino acids (Ala, Pro, a.ud -4~11) to the C-terminal Cys tlerivative with t,he aid of l-ethyl-3-(3-dimethylalllinopropyl)-carbodiin~~le or p-nitrophengl ester. A cystathionine derivative (Z-H&OBut, Boc-Ala-OMe) [16] was synthesized by rraction of benzyloxycarbonyl( Z)-homocyst,eine t-hutylesi.er(But) with t-butoxq.carb[~nyl( Boc)aziridine methyl est.er [17] and converted to the corresponding hydrazide. The lrydrazitle was then coupled t.o t.he C-terminal tetra.peptide, t,he Boc-group was removed am1 Bocamino a.cids (Ala, Leu, Glu(OBzl), and Cys(l-~~lethyll~er~zyl(RIIRzl)) N-h~drosysuccillilnjtlc esters(ONSu) were coupled stepwise. iifter removal of Boc-group, the peptide was cyclizetl by coupling the peptide bond between t,he N-terminal and second amino acids, Fiually the peptide was trea.ted with a.nhydrous HF and air-oxidized aft.er removal of HF to form t,he disulficle linkage bet,ween the two Cys residues. The crude product, was purified 1)) high-performance liquid chromat,ography (HPLC), as d escribed previously [lS]. Analyses of Purified Peptides : The amino acid compositions and molecu1a.r weights of purified peptides were examined by amino acid analysis a.ud fast a.tom I,oml~a.~-cllrlel~t, ma.55 spectrometry, respectively, as described previously [19]. Biological Activity : The biological activit,y of synthetic peptides was examiued using suck ling mice of 2 days old (1.7 f 0.1 g) as described previously [‘JO]. Tl re minimum effect.ive dose (MED) was evaluat,ed as t,he minimum a.mount. tha.t generated the t0xicit.y. Receptor Binding Assay : The binding assa.y of synthet,ic peptides to t,he recept.or protein membranes isolated from Sprague-Dawlcy (70 kDa protein) was a.ssayed using brush-border rat.s (S weeks old, 200-250 g), as described [13]. 5-Azidoz-nitrobenzoyl(ANB)ST,,(5-19) a.rtd ‘“51-ANB-STr,(5-19) were synthesized as described [13]. 959

Vol.

176,

No.

BIOCHEMICAL

3, 1991

1

AND

BIOPHYSICAL

5

RESEARCH

10

We recently

synthesized

bonds and one disulfide fide linkages

between

tosic,

a. peptide

Cysll

bond

having

one disulfide

linkage

was peptide

2 in Table

all possible

R esult,s demonstrated

two disulfide

I

are essential

E. cnli.

combinations

t,hat peptides

a.nd Cys’”

1~0~1s between

of two disulfide t,hat, had t,wo disul-

for the toxicity

did not, show a.ny t,oxicity.

Cys6 a.ud Cys’l

aud Cys”

bond

of ST (Fig.

The smallest

1.

Synthetic

Analow

of ST and Their

1). Pept.ides

wit,h only

of the toxic

aualogs

Mm

I

I

0.4*

Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Gly-Cys I I 1

1

I

I2

Y2-S-S-CH

NH2-CH-CO-NH-CH-CO-Gl~-L~~-Al~-NH-CH-CO-A~~-P~~-Al~-NH-CH-COOH I CH2s S

66** I CH2

43 CH2p

s-s

CH2p

CH 2 -S

CH2

4

38

MED. minimum effective *, Cited from Ref. [8]. Cited

from

Ref.

dose.

[21],

960

CH2

that t.he Cys” and

Toxicities

PeDtide

aud

between

1.

NO

**,

iu ST,,, were

or Cys’ and Cys”

Cys7 aud Cys15 was ina.ct,ive, indicating

bond betweeu

Table

2

with

Cys7 and Cys15 and one ot,her clisulfide

and Cysl’

1

I

DISCUSSION

Cps7 aud Cys15 and Cys”

between

or Cys”

AND

ST analogs

bond [‘l].

Cpqls *c but not t,he disulfide disulfide

I

Amino acid sequences of ST isolated from enterotoxigenic a) Strain SK-1 [2,9] ; b) strain 18D [3,4,10].

1.

RESULTS

while

I

Asn-Thr-Phe-Tyr-Cys-Cysys-Asn-Pro-Ala-Cys-Ala-Gly-Cys-Tyr 1

Fig.

19

15

Asn-Ser-Ser-As"-Tyr-CyTs-Cys-Glu-Leu-Cy;~-A~~-P~~-Al~-C~~-T~~-Gl~-C~~-T~~ 1 1 STpb'

COMMUNICATIONS

of ST

Vol.

176,

No.

BIOCHEMICAL

3, 1991

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

op1 3. Boc-Glu-ONSu MBzl 4. Boc-C;s-ONSu

+ S

7"2

4, iHZ MBzl OBrl I 2 Z-NH-CH-CO-L:s-Gi"-~eu-Rla-NH-CH-CD-Asn-~~-OB~~

MBzl

;-, NH~-CH-CO-Cys-Glu-Leu-Ala-NH-CH-CO-Asn-Pro-Ala-Cys I

I

Fig. 2. Scheme for synthesis of [6-Carba,Ala”]STh(6-15).

In the initial through

step of its biological

a thiol-disulfide

of its receptor synthesized

prot,ein peptides

CH3-S a.s shown

in Table

residues,

exchange

N-terminal

side containing

was formed

gave peptide Table

1)

The toxic results

activities

are shown

in Table

and thiol

groups

one of the two disulfide

bonds in peptide

2 was repla,ced b:,

with

the receptor

of a,n CH1-S linkage protein.

that is, t.he C-terminal

a.nd finally

part consisting acid residues. the protecting

procedure

of these synthetic 1. Synthetic

portion

containing

of a cystathionine These groups

961

pre-

was st art.etl

four a,mino acid moiety,

blocks were coupled were removed

and the step 1~)

and a. disulfide

by reversed-phase

([‘i-Ca.rba.,Alal”]ST,,(6-15),

HPLC

pept.ide 4, in

to that shown in Fig. 3,.

peptides

peptides

we

The scheme for synt,hesis of one

Cys7 and Cys15. The final purification

by a similar

int.o ST should

[‘?I is shown in Fig. 2. The synthesis

3 (in Fig. 3) [Z3]. The other peptide

was synthesized

linkages

this possibilit,y,

four amino

between

its disulfide

protein

[15]. To examine

and the central

step from the C-terminus, linkage

between

with it.s receptor

complex

STh(6-15))

of three blocks;

the N-terminal

ST may interact

1, because introduction

([G-Casba,Alalo]

by const,ruction

react,ion

to form an ST-receptor in which

vent a t,hiol-disulfide of pept,ides

exchange

reaction,

were examined

3 and 4 which

in suckling

mice and

ha.ve a CHz-S linka.ge be-

Vol.

176,

No.

3, 1991

BIOCHEMICAL

”

AND

10

BIOPHYSICAL

30

“cl

RESEARCH

COMMUNICATIONS

30

Ketention time (min) Fig. 3. HPLC profile of a crude preparation of [6-Carha,Ala’“]ST~(6-15). Column, Cbsmo~il 5C18 (S x 300mm). Solvent, linear gradient from 10%) to 40% CHSCN in 0.05%) TF.4. Flow rate , 2 ml/min. tween the amino at position

xicl

residue

at position

showed

7 and Cys15, respectively,

1. However 1 these pept.ide, $ were as tcxic 1211. Tl lese - zebu _ 1t.s indicate Ct,sT and Cys I5 could I~eptide

wit,hout

affecting

linkages

eschange

of ST ( “51-ANB-ST,,

Therefore,

of ST analogs

we esarnined

a. clone encoding

from the nucleot,ide

at concent’ra,tions analog

of it,s cDN.4

of 10WG M ( while

peptides

of lo-’

proteins

that a photo-reactive t,o several

We have also found

are correla.ted

with

for ST was isolated of the receptor

h4 , as shown

in Fig.

2 and d can I)intl t,o 70 1iDa protein

962

t,hat 1,he

Keccuf I>.. lil)r;irJ

of 121 kDa. was tletlucctl is prol>a.bly

tlifferent

in size. The synthetic

pept,i(les

analog to the 70 kDa protein

nat,ive ST inhibited

ratlio-

their t,oxicit.ies.

front a cDN:1

[ 1241. Tl ie 70 kDa protein

because of it,s difference

of S’I‘

intest inal cp-

of 3 a.nd J to t.he $0 kDa. protein.

aci(l sequence

of the photo-react,ive

a.t a concent.rat,ion

the syntliet.ic

activities

of t,lle

protein.

3 ant1 4 to receptor

of 70 kDa [lU].

or between

and Cys7 a.ntl (2)~~~ in !?I‘,,

1Gnds specifically

cyclasc recepl,or

cyclase receptor,

inlribit~ecl the binding

(i;-IS))

to the 70 kDa. protein

and the amino sequence

peptides

lilllGig('S

the toxicity

wit,11 the recept.or

LVe have found

a protein

the binding

the guanylat,e

of rat sniall inlest,ine

from the guanglyl

cell inenilxanes.

especially

c’ys” and c’ys” reaction

of syntliet,ic

iotlinat,ed

cell membranes,

between

of’ l~cpli(le

has t,W0 distllfitl~

be repla.ced by a CHJ-S linkage

epit,lielia.l

activit,ies

Y& wliicli

CysG and Cys”

on rat, intest,inal

binding

as peptitle

acid resitluc

the toxicity

l~etween

We nest. t.est,ed the bindings

ithelial

one-hundredth

the anlino

linkage

in a. thiol-disulfide

analog

about

t.hat. the disulfide

and that the disulfide

are not involved

a.nd between

6 a.nd Cys”

t,he binding 4. These

almost, conlplet PI!

of t,he photc,-react results

as well as pepti(le

ivc

inclic.;rt