endothelin family of peptides

Vol. 162, No. 3, 1989 August

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

15, 1989

Pages

IMMUNOLOGICAL AND STRUCTURAL CHARACTERIZATION SARAPOTOXIN/ENDOTHELIN FAMILY OF PEPTIDES

OF

Fleminger*, Daniele Bousso-Mittler, Avner Yoel Kloog and Mordechai Sokolovskyji

Bdolah**,

Gideon

Department of Biochemistry, *Department of Biotechnology, **Department of Zoology, The George S. Wise Faculty of Life Tel Aviv University, Tel Aviv 69978, Israel Received

June

26,

1317-1323

Sciences

1989

A highly specific and sensitive radioimmunoassay (RIA) was developed for the potent vasoconstrictor peptides, sarafotoxin-b and human endothelin. The antigenic determinants of the antibodies employed in studies with these assays were found to be localized within the amino acid sequence at positions 4-7. This was confirmed by CNBr cleavage of the methionyl residue at position 6 in the sarafotoxin and at position 7 in the endothelin. The chemically characterized modified peptldes showed very low cross reactivity in the RIAs. On the other hand, the binding properties as well as the ability to induce phosphoinositide hydrolysis were very similar in the modified and native peptides, indicating that despite cleavage of the peptide bond the biologically active conformation responsible for either binding or phoshoinositide hydrolysis is retained, probably because of the disulfide bonds. Thus, structural alteration might be a valuable means of curtailing some of the various activities induced by the sarafotoxin/endothelin family of peptides. 0 1989 Academic Press, Inc.

At least toxin

(SRTX)

homology, both

six

occurring which, though

family now known

are

possessing

constrictive

two

(l-7).

sets

in the

position

4

rat

heart 7,

sents

a variable

Thus,

while

region in

methionyl

but

through the

residue

and its

cation

at

the

functions

These

also

which of the

among

location methionyl which

One such activity

relates

# To whom reprint

requests

is

two groups

(6-8). part

It

might

prove

loop

family

the

position

messenger

sequence from 11 , repre-

6 is (human)

Therefore,

therein).

occupied

by a

possesses chemical

in identifying

to the

structure

determinants

vaso-

cys3-cys

useful

immunological

only

(7 and refs.

ET-1

7 (l-3).

not

and second that

inner

only

may be attributed

should

sites

peptides

endothelins

acid-peptides,

to share

appears

peptide

is at position

to the

appear

of the

sarafotoxin

site

of 21-amino

common binding

ET/SRTX

the

of the endothelin (ET)/sarafohave a high degree of sequence

bonds,

and brain

three

residue,

peptides diverse,

of disulfide

properties

systems

logical

naturally

this modifi-

those of this

(7).

bio-

region.

In order

to

be addressed.

1317

0006-291X&39 $1.50 Copyright 8 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

BIOCHEMICAL

Vol. 162, No. 3, 1989 study

this

(RIA)

for

subjecting assayed as by the

we (i)

developed

SRTX-b, them by binding

and to

(ii)

a highly specific modified the

cyanogen studies

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

bromide

and sensitive radioimmunoassay structure of SRTX-b and ET-1

treatment.

and phosphoinositide

The modified hydrolysis

peptides turnover

by

were as well

new RIA. METHODS

Sarafotoxins (a, b, c) were prepared as described elsewhere (4, 5). was purchased from American peptide Company Inc. Human endothelin (ET-l) endothelin (ET-3) and porcine endothelin (ET-l) (Santa Monica, CA); rat 'neat' were from Peninsula Laboratories Europe Ltd. (Merseyside, antiserum England). The synthetic peptide corresponding to the C-terminal fragment His-Glu Asp-Val-Ile-Trp (referred to here as SRTX-b (16-21)) was prepared by solid phase synthesis on an Applied Biosystem model 480A peptide synthesizer in the Unit of Chemical Services at the Weizmann Institute of Science (Rehovot, The purity of all peptides was >97% as determined by HPLC analysis Israel). using gradient I as detailed below. SRTX-b (1.75 ug) was iodinated with iodogen reagent and purified as described previously (9). ET-l (4 ug) was iodinated with Enzymo Beads (Bio Rad, Richmond, CA) and purified as described (8). HPLC analysis: HPLC analysis of peptides was performed with a Gilson (Villiers le Bel, France) Model 303 HPLC system equipped with a Perkin Elmer (Nowalk, CO) Model LC-75 spectrophotometer detector. The toxin (So-100 ug in 0.1 ml of 0.1% trifluoroacetic acid (TFA) in water) was applied to a Lichrosorb RP-18 column (250x4 mm, Merck, Darmstadt, FRG). The column was developed at 1 ml/min with one of two gradients composed of 0.1% TFA (buffer A) and 80% acetonitrile in 0.1% TFA (buffer B): gradient I consisted of a 5 min wash with buffer A, followed by a linear gradient of O-100% buffer B over 35 min. The column was then washed with buffer B for 10 min and buffer A for 10 min. Gradient II consisted of a 5-min wash with buffer A, an additional followed by three steps of linear gradients of 0-35X buffer B over 10 min, 35-60% buffer B over 20 min and 60-100% buffer B over 5 min. The column was then washed with buffer B for 10 min and buffer A for an additional 10 min. Elution profiles were monitored at 230 nm and 280 nm. CNBr cleavage and purification procedures: Cyanogen bromide (CNBr) cleavages of SRTX-b and of ET-l were performed with 50 ug of each of the peptides dissolved in 50 ul of 70% formic acid. Following incubation with CNBr for 19 h at room temperature, the peptides were dried in a Speed-Vat (Savant Instruments, Farmingdale, N.Y.) resuspended in 100 1.11 and dried again. The latter procedure was repeated two more times and the peptides were then separated and purified by HPLC. The elution profile of CNBr-treated SRTX-b on reverse phase HPLC developed with gradient I indicated the presence of 3 components: the first component eluted at 31.3 min (lo-15% yield), the second and major component at 31.8 min (40-50%) and the third at 32.6 min (20-25X). Formic acid treated and untreated SRTX-b were eluted as single peaks at 30.9 min. Each of these derivatives was subjected to a second HPLC purification using gradient II. The elution profiles of the three peaks were 35.5 min, 37 and 38.5 min, respectively. Since the third peak showed low absorption at 280 nm all further studies were conducted with the second peak, which is also the major component. The elution profile of CNBr-treated ET-1 using gradient I yielded two peaks with retention times of 35.6 min (45% yield) and 36.4 min (36%). ET-l, with or without formic acid treatment for 19 h, yielded only one peak with a retention time of 36.1 min. With gradient II the first peak eluted as a single component at 43.5 min as compared to 40.9 min for elution of control samples. All further characterization was carried out with this preparation. 1318

BIOCHEMICAL

Vol. 162, No. 3, 1989

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Sequence analysis: This was performed by the Protein Analysis Group at the Hebrew University - Hadassah Medical School, Jerusalem, Israel, using a 470A Gas Phase Protein Sequence with an online 120A HPLC-AA analyzer (Applied Biosystem Inc.). Immunological procedures: Rabbits (local strain) were injected subcutaneously with 60 pg of SRTX-b emulsified with complete Freund's adjuvant (Sigma, St. Louis, MO). This was followed after 3 weeks by bimonthly subcutaneous injections of the emulsified SRTX-b. After 5-6 repeated immunizations, antisera suitable for RIA of SRTX-b were obtained. The buffer used for RIA was 62 mMNa-phosphate (pH 7.5) containing 13 mM Na EDTA, 0.02% Na azide, 0.2% BSA and 0.1% Triton X 100. This buffer was used for dilution of sera and of peptides. The incubation mixture consisted of 0.1 ml of competition peptide or 0.1 ml of assay buffer, 0.1 ml of anti SRTX-b antiserum (1:lOOO final dilution) or 0.1 ml of anti ET-1 antiserum (1:40,000 final dilution), 0.1 ml of lz51-SRTX-b for the SRTX-b RIA (6000 cpm/l5 pg) or 0.1 ml iz51-ET-1 f or the ET-1 RIA (9000 cpm/30 pg), and 0.2 ml of assay buffer. Incubation was carried out at 4°C for 16 h, followed by the addition of 0.05 ml 5% naive rabbit serum and 0.05 ml of reconstituted goat anti-rabbit IgG antiserum (Sigma, RO 881, 1000 test/vial reconstituted in 20 ml assay buffer and diluted 1:2.5). Bound and free ligands were separated after 4 h by centrifugation at 4000 x g for 30 min. The radioactivity in the precipitate was counted in a y-counter. Under the assay conditions described the ratio of bound to total iz51-ligand (B,/T) was 30-33% in the SRTX-b RIA and 28-31% in the ET-l RIA. Blanks (less than 8% of Bo) were subtracted. All assays were performed in triplicate and the data are expressed as of B/B, vs peptide concentration. Biochemical assays: Phosphoinositide hydrolysis assays in rat (Charles River derived (CD), adult male) cerebellar tissue slices and lz51-SRTX-b binding assays in rat cerebellar or atria1 homogenates were performed as described previously (8-10). RESULTSANDDISCUSSION Radioimmunoassayof SRTX-b and ET-1 Figure 1 demonstrates the RIA for SRTX-b and for ET-l. Under the comassay conditions employed here the 50% B/B, value of SRTX-b in its petition

ANTI

SRTX b

ANTI

ANTISERUM

CN&-

SRTXb

ET-l

ANTISERUM SRTXb.

SRTXb (t-21)

SRTXa

SRTXc

I 100

I 1000 PEPTIDE

(pg,hbe

I 10

10000

I 100 PEPTIDE

)

I 1000

(p&be)

Figure 1 various in the

Radioimmunoassays (RIA) peptide analogs. B, presence of competitor

for SRTX-b and ET-l, - bound trace in the (see Methods).

1319

and crossreactivities absence and B - bound

with trace

Vol.

162,

No.

3, 1989

BIOCHEMICAL

own

RIA

was

was

62

pg/tube

(76 and 24.8

its

own RIA was 40?15

ET-l

in

90%

B/B,)

value

for

et al. studies

pg/tube

was

7.5 pg/tube

the

ET-1

(11) lower

readily

RIA is

by

antisera:

peptide,

whereas

peptide.

Nonetheless,

ficiently

sensitive.

the

or

low

SRTX-c that

common

ET-1

C- terminal

sequence

of Ile-Trp

from

directed

the

to the

Trp

materials

with

the

that

region

the

by Ando et al.

residue et al.

1).

as shown

SRTX-a,

These

results

towards

the

study.

The

determinant

These

Abs thus

which

were

They do however

which

reacts

in

with

under

antigenic

(ll),

of ET-l. (12),

the native

directed

peptides

of ET-l.

to prepare

RIAs'

(Fig.

is not

RIA is

against a conjugate both RIAs were suf-

both

ET-3

here

of all

suggests

used against

no crossreactivity with

The fiveET-1

with

is

clearly

found

to be

resemble

ET(l-21)

the

but

not

ET(lS-21). The anti

SRTX-b

Ab showed

ET-l

or ET-3

SRTX-a.

These

results

SRTX-b

antibodies

used

SRTX-b

molecule,

which

fide

bonds.

Ser-Cys

3

Since

and

determinant between SRTX-b Cyanogen

indicate here

no crossreactivity

be

is

within

and ET-l ,

SRTX-b

region

and contains

with

SRTX-b likely loop

the

the that

anti

of the 2 disul-

common Cysithe

antigenic

I1 of SRTX-b

Cys'-Cys

was examined

with

of the

the N-terminal acids

share

possibility

SRTX-c,

determinant

it seemed most within the inner

localized

with

and 18% crossreactivity

antigenic

of 15 amino 11

This

the

localized

SRTX-a

Lys'-Asp-Met-Thr7.

pg/tube),

that

consists

Asps-Lys-Glu-Cys

using

CNBr-modified

and ET-l. bromide

cleavage

Cyanogen

bromide

carboxyl

group

bonds

almost

(50% B/B,>4000

both

would

residue,

located that of

the

of SRTX-b cleaves

of remain

(i)

there cleavage

and ET-1

specifically

methionine.

at position

supposedly

indicate result

21

by Suzuki

(16-21),

the

used here.

used

1-15

Abs used

the

Ab used

ET-3

N-terminal

rabbit

against

monoclonal with

the

in their

conditions

performed

(at

by Ando

condition

RIA as compared

showed

antiserum

with

within

differ

(Ab)

rabbit

crossreactivity

localized

antibody

level

recently

was raised

of

The 50% B/B,

reported

antiserum was raised purposes of this study were

value

detectable

non-equilibrium

and 16% crossreactivity

the

that

antiserum

90% B/B,)

The 50% B/B,

immunization

SRTX-b

experiments

1. The anti

indicate

different

(at

respectively).

with assay

COMMUNICATIONS

level

and the minimum

SRTX-b

anti-ET-l for the

Crossreactivity SRTX-b

the

anti

detectable

and 3 fmol/tube

competition

the

the

pg/tube comparable

of

RESEARCH

respectively).

who employed

to the

explained

Figure

fmolftube (16

sensitivity

rabbit

BIOPHYSICAL

and the minimum

(6 fmol/tube),

as compared

fold the

190+10

AND

6, and since

intact, is

the

In SRTX-b the

is

two

post-cleavage

no methionyl there

the

residue

a new N-terminal

1320

peptide there

bond is only

involved

intramolecular sequence

at position possessing

with

one methionyl disulfide

analysis

should

6 and (ii) the

as a

sequence

BIOCHEMICAL

Vol. 162, No. 3, 1989

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Thr-Asp-Lys-Glu. Sequence analysis of intact SRTX-b and CNBr-STRX-b in fact yielded the following amino acids: for intact SRTX-b (in brackets, the cycle no.) Ser(II), Lys(IV), Asp(V), Met(VI), Thr(VII), Glu(X); for the modified peptide Asp/Ser(II), Lys(III), The yields

of

the two amino acids identified

cycles were comparable. Similar analysis of CNBr-ET indicated residue

vl the

Lys(IX), Asp(V).

in the second and the fourth

the disappearance of the methio-

7, and the presence of a new N-terminal

sequence Asp-Lys-Glu-X-Val.

tide at

at position

Asp(VIII), Glu/Lys(IV),

However, the original

N-terminal

possessing heptapep-

fragment response was weaker than that expected from a single cleavage the

methionyl

partial leading

residue.

The most likely

explanation

is that additional

cleavages had occurred, probably at the cysteinyl residue, to the partial disappearance of the N-terminal heptapeptide.

thus This

possibility is currently under investigation. As shown in Figure lA, the CNBr-modified SRTX-b showed no crossreactivvity

in the anti SRTX-b RIA (50% B/B,>4000 pg/tube).

Binding properties of CNBr-SRTX-b were determined by means of competi125 tion experiments using I-SRTX-b in rat cerebellar and atria1 preparations (6, 9). As shown in Figure 2, the modified toxin competes with the intact

peptide displaying

Iso values similar

to that of the native

Thus, in the cerebellum the I50 values for the native were 15 nM and 12 nM respectively,

peptide.

and the modified toxin

while in the atria1

preparation

they were

SRTX b-0 CNBr-SRTXb-o

PEPTIDE

PiRure

(M)

2 binding by SRTX-b and Concentration-dependent inhibition of '251-SRTX-b in Methods using CNBr treated SRTX-b. Binding assays were as described membrane preparation from rat cerebellum, 2.5 nM "'1-SRTX-b and various concentrations of the two peptides.

1321

Vol. 162, No. 3, 1969

BIOCHEMICAL

7 and 20 nM respectively.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The modified ET in these two preparations

yielded

Iso values very close to those obtained for native ET-l (8). In order to determine whether modified SRTX-b and modified ET-1 can activate

phosphoinositide

and atria1

tissue

stimulated

with

hydrolysis

(6, 8-lo),

we examined rat cerebellar

slices that were prelabeled with [3H]-inositol

0.1 pM native

and then

or modified peptide in the presence of 10 mM

LiCl. The responses of the modified SRTX-b and ET were very similar to those of the respective native peptides: e.g. the level of Induced/Basal for 13HIIPl

was 4+2.3

for the modified peptide and 4.2tl.O

for the control

in

atria1 in the

preparations, n=4. Thus, cleavage of the peptide bond at residue 6 inner loop does not affect either the binding properties or the bioindicating that the rigid entity of the chemical response (PI hydrolysis), N-terminal portion in the biologically active two disulfide bonds. Reduction of the disulfide viously

shown to

result

in

conformation is held by the bonds of the toxin was pre-

reduced binding and diminished PI-hydrolysis

of the inner loop by cleavage of (61, while enzymic interruption 10 ET-1 at Lys'-Glu with lysyl endopeptidase (13), indicated that this strucpotency

ture

is

especially

alterations biologically

important for the constrictor

activity.

Thus, different

in the structure of the SRTX/ET family might result in different active or inactive forms. Preliminary Experiments have indi-

cated that the high toxicity of SRTXsand endothelins (14) is reduced when the CNBr-treated SRTX-b and endothelin derivatives are used. We are currently

investigating

the

possibility

that

this phenomenonmay be due to

increased susceptibility of the modified peptides to e.g. proteolysis. The highly specific and sensitive RIA developed in this study, for

detection

noted

that

the native antibodies

of

SRTX-b at concentrations

the polyclonal

allows

as low as 25 fmol. It should be

antibodies were obtained from immunization using

peptide, i.e., without conjugation to a carrier protein. These recognized the variable region (7). This might be related to the

fact that rabbits possess endothelins, and that the sequence homology of these peptides is strongest between residues 7 and 21 - hence if antibodies are

produced they

are

likely

to be directed

toward the variable

region.

However, it should be noted that antibodies against ET C-terminal peptide (12) were recently elicited in rabbits by immunization with BSA conjugates. ACKNOWLEDGMENT We thank Mrs. Shirley

Smith for excellent

editorial

assistance.

REFERENCES 1.

Yanagisawa, Mitsui, Y., 415.

M., Kurihara, H., Kimura, S., Tomboe, Y., Kobayashi, M., Yazaki, Y., Goto, K. & Masaki, T. (1988) Nature 332, 411-

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Vol. 162, No. 3, 1989

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Itoh, Y., Yanagisawa, M., Ohkubo, S., Kimura, C., Kosaka, T., Inoue, A Ishida, N., Mitsai, Y., Onda, H., Fujino, M. & Masaki, T. (1988) F&S Lett. 231, 440-444. Yanagisawa, M., Inoue, A., Ishikawa, T., Kasuya, Y., Kimura, S., S-I, Nakajima, K., Watanabe, T.X., Sakakibara, S., Gotto, K. K=gwe, & Masaki, T. (1988) Proc. Natl. Acad. Sci. USA 85, 6964-6967. Kochva, E., Viljonen, C.C. & Botes, D.P. (1982) Toxicon 20, 581-592. Takasaki, C., Tamiya, N., Bdolah, A., Wollberg, Z. & Kochva, E. (1988) Toxicon 26, 543-548. Kloog, Y., Ambar, I., Sokolovsky, M., Kochva, E., Wollberg, Z. & Bdolah, A. (1988) Science, 242, 268-270. Kloog, Y. & Sokolovsky, M. (1989) Trends Pharmacol. Sci. 10, 212-215. Ambar, I., Kloog, Y., Schvartz, I., Hazum, E. & Sokolovsky, M. (1989) Biochem. Biophys. Res. Commun.158, 195-201. Ambar, I., Kloog, Y., Kochva, E., Wollberg, Z., Bdolah, A., Oron, U. & Sokolovsky, M., (1988) Biochem. Biophys. Res. Commun.157, 1104-1110. Kloog, Y., Ambar, I., Kochva, E., Wollberg, Z., Bdloah, A. & Sokolovsky, M. (1988) FEBS Lett. 242, 387-390. Ando, K., Hirata, Y., Shichiri, M., Emori, T. & Marumo. F. (1989) FEBS Lett. 245, 164-166. Suzuki, N., Matsumoto, l-l., Kitada, C., Masaki, T. & Fujino, M. (1989) J. Immunol. Meth. 118, 245-250. Kimura, S., Kasuya, Y., Sawamura, T., Shinmi. D., Sugita, Y., Yanagisawa, M., Goto, K. & Masaki, T. (1988) Biochem. Biophys. Res. Commun. 156, 1182-1186. Bdolah, A., Wollberg, Z., Ambar, I., Kloog, Y., Sokolovsky, M. & Kochva, E. (1989) Biochem. Pharmacol. In press.

1323