Fulicin, a novel neuropeptide containing a D-amino acid residue isolated from the ganglia of Achatina fulica

Vol. 178, No. 2, 1991 July 31, 1991

FULICIN,

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 486-493

A NOVEL NEUROPEPTIDE CONTAINING A D-AMINO ACID RESIDUE ISOLATED FROM THE GANGLIA OF ACHATINA FULICA

Noriyuki Ohta’, Ichiro Kubota*, Toshifumi Takao3, Yasutsugu Shimonishi3, Yoshimi Yasuda-Kamatani4, Hiroyuki Minakata4, Kyosuke Nomoto4, Yojiro Muneoka’ and Makoto Kobayashi’* ‘Physiological Sciences, *Suntory 3Division

Laboratory, Hiroshima

Bio-Pharma

of

Faculty University,

Tech Center, Gunma 370-05.

of Integrated Hiroshima 730, Chiyoda-machi, Japan

Arts Japan

and

Ohra-gun,

Institute for Protein Organic Chemistry, Osaka University, Suita, Osaka 565, Japan

Research,

4Suntory Institute for Bioorganic Research, Shimamoto-cho, Mishima-gun, Osaka 618, Japan Received June 10, 1991 A novel pentapeptide containing a D-amino acid residue was purified from the central ganglia of the African giant snail Achatina fulica Ferussac, and it was named fulicin. The primary structure of the peptide was determined to be Phe-o-Asn-Glu-Phe-Val-NHz. Fulicin potentiated tetanic contraction of the at very low concentrations, and also penis retractor muscle of this snail showed modulatory actions on the activity of the buccal and ventricular 0 1991Academic Press,Inc. muscles and the central ganglionic neurons.

It is well known that contraction of regulated by multiple bioactive substances peptides

[1,2].

In

several

neuropeptides

isolated

from

the

the African giant such as achatin-I

ganglia

or the

has been investigated [3-61. characteristic in possessing

atria,

molluscan including

muscle is neuro-

snail Achatina fulica, and ACEP-1 have been and their

mode of action

Among these peptides, achatin-I a D-phenylalanine residue [31.

is In

*To whomcorrespondence should be addressed. Abbreviations: ABRM. anterior byssus retractor muscle; ACEP-1, Achatina cardio-excitatory peptide-l (H-Ser-Gly-Gln-Ser-Trp-Arg-Pro-Gln-Gly-Arg-PheNH,); FAB-MS, fast atom bombardment mass spectrometry; Hepes. N-2-hydroxyethyl-piperazine-N’-2-ethanesulfonic acid; HPLC, high performance liquid chromatography; MIPS, Mytilus inhibitory peptides; PTH. phenylthiohydantoin; TFA, trifluoroacetic acid; Tris, tris-(hydroxymethyl) aminomethane. ODO6-291X/91 $1.50 Copyright All rights

0 1991 by Academic Press, Inc. of reproduction in any form reserved.

486

Vol.

178,

No.

the

present

from

the

2, 1991

BIOCHEMICAL

study,

a biologically

cerebral

and

using

the

system. mined

The substance to be a novel

This

peptide

penis

was

AND

active

suboesophageal

retractor

RESEARCH

substance ganglia

of

muscle

was purified pentapeptide

named

BIOPHYSICAL

this

and having

snail

COMMUNICATIONS

was of as

its structure a D-asparagine

&

isolated fulica

the

by

bioassay was

deterresidue.

fulicin. Materials

and Methods

Purification The African giant snail, Achatina fulica Ferussac, was collected in Okinawa and transported by air to Hiroshima. The cerebral and suboesophageal ganglia excised from 1,600 snails were immediately frozen with dry ice, and the ganglia were steeped in 100% acetone and homogenized. The homogenates were centrifuged at 15,000 g for 30 min at 2’C. The pellet was re-extracted with 80% acetone twice. The extracts were pooled and evaporated to dryness. The dried material was taken up in O.lN hydrochloric acid and again centrifuged. The supernatant was forced through two disposable C-18 cartridges (Waters Sep-Pak). The retained material was eluted with methanol and gel-filtrated with a column of Sephadex G-15 (26 x 400 mm). Each of the gel-filtrated fractions was applied to the preparation of the penis retractor muscle of the snail to examine the bioactivity. The active fractions were divided into two groups, fractions 24-30 and fractions 31-52. Fulicin was obtained from the latter group. The fractions of the latter group were pooled, concentrated and subjected to HPLC (Shimazu LCGAD-SCLGB) separation with a C-18 reversed-phase column (Tosoh ODS-80TM. 4.6 x 150 mm). The column was eluted with a 60-min linear gradient of O-60 % acetonitrile in 0.1 % TFA at pH 2.2. The active fractions were applied to an anion-exchange column (Tosoh, DEAE-5PW, 7.5 x 75 mm) and eluted with a 70-min linear gradient of O-O.7 M NaCl in 10 mM Trisbuffer at pH 9.6. The active material was again applied to the C-18 reversed-phase column and eluted with a 60-min linear gradient of lo-30 % acetonitrile in 0.1 % TFA at pH 2.2. Final purification was performed by applying the active material to the C-18 reversed-phase column and eluting iso-cratically with 17 % acetonitrile in 0.1 % TFA. Bioassay After each purification step, the bioactivity of each fraction was examined on the tetanic contraction of the penis retractor muscle isolated from the snail. Following excising the penis retractor muscle from the body, both ends of the muscle were tied with threads and mounted in an experimental chamber, one end being connected to a silver hook in the chamber and the other to a strain gauge for tension recording. Methods of the muscle stimulation and the tension recording were essentially the same as those employed previously for ABHM by Muneoka and Twarog (71. The chamber was filled with physiological saline 1.5 ml in volume, the composition of which was as 5.0; follows (mm): NaCl, 61.0; KCl, 3.3; CaCl,, 10.7; MgCl,, 13.0; glucose, For examining and Hepes, 10.0 (pH adjusted to 7.5 by titration with NaOH). the test solution was substituted for the bioactivity of each fraction, physiological saline in the chamber. The purified active substance was subjected to Structure determination amino acid sequence analysis by the automated Edman degradation with a gas477A) coupled with a PTH-amino acid phase sequencer (Applied Biosystems (Hitachi L-8500) and analyzer (Applied Biosystems 120A), amino acid analysis Determination of amino acid enantiomers FAB-MS anal-ysis (JEOL JMS HX-100). was made by a highly sensitive resolution method using a chiral fluorogen. The chiral fluorogen was derivatized by mixing a small amount of peptide hydrolysate solution with o-phthaldialdehyde-N-acetyl-L-cysteine (OPA-NAC) Synthesis of reagent followed by HPLC analysis as Aswad’s method [8,91. peptides was performed by the solid phase method. After treatment with liquid HF and ether extraction, peptides were purified by a reverse-phase HPLC. The 487

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AND

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synthetic and native peptides were compared in HPLC profiles and biological activities on the penis retractor muscle of this snail. Biological action Biological action of fulicin was examined not only on the penis retractor muscle but also on the radula retractor muscle and ventricle of L fulica, and a muscle (ABRM) of Mytilus edulis (bivalvia). except the ventricle, were isolated and mounted in the same These muscles, Methods of stimulating the muscle chamber as used in the bioassay experiment. and recording the tension were also the same as those employed in the bioassay experiment. Fulicin was applied to the muscle by replacing the saline in the chamber with a solution containing the peptide. The ventricle preparation was suspended in a vertical bath of 2 ml in volume, which was aerated continuously Fulicin, dissolved in the saline, was applied to the muscle by injecting [41. the solution into the bath. Electrical activity of ganglionic neurons was recorded by the intracellular microelectrode method [lo]. Results shows

Figure purification the

penis

of

fulicin,

retractor

of each fraction coincident with

Asp 1.0, analysis

HPLC profile together

with

analysis

amino acid

Glu 0.9, Val was performed

1.0, twice

of

composition

Phe 1. . by using

The two

step of results

the on

the excitatory effect peak was found to be

peak monitored

acid

A E

the final the bioassay

By examining the active muscle,

amino

following

at

muscle.

on the the absorbance

Quantitative showed the

the

and D scussion

at the

220 nm. purified

peptide

normalizing amino separate

on Val=l:

acid

sequence

samples.

The

E

r

s 0.03 M

Fulicin

--.

1

0

I

I

lo

I 30

I

40

Tiilnhl,

t pg..;

I wash

z 10-a M Flkhl

lmin

IP

Fig. 1. A. The final HPLC purification of fulicin from the central ganglia of Achatina fulica. The active fraction obtained by the preceding chromatography was applied to the reversed-phase HPLC and eluted isocratically with 17% acetonitrile in 0.1% TFA (PH 2.2). UV absorption was monitored at 220 nm. The peak eluting at 28.2 min was active on the penis retractor muscle as shown in B. B: Effects of native fulicin on the penis retractor muscle. lo-lo M fulicin enhanced tetanic contraction of the muscle evoked by repetitive electrical stimulations (20 v, 1 msec, 40 Hz, for 1 see) at 10 min intervals. The peptide was applied to the muscle 8 fin prior to the stimulation (upward arrow) and washed out soon after it (downward arrow). In this preparation, lO* #l fulicin elicited repetitive contractions. The concentration of native fulicin in the stock solution was calculated from the result of amino acid analysis. 488

Vol.

178,

No.

sequence

2, 1991

and

samples

was

BIOCHEMICAL

detected

amount

determined

up to

AND

BIOPHYSICAL

(pmol) the

of

fourth

RESEARCH

each

COMMUNICATIONS

amino

residue

acid

as

in

both

Phe31.5-Asn16.L-

and Phe30.3-Asn 15.7-Glu26.3-Phe6.4, respectively, but the Glu4.4-Phel.l, fifth residue was not detected in both cases. However, since one valine exists in the molecule from the amino acid composition the or

sequence its

of

fulicin

C-terminal

was

amide

suggested

to

derivative.

be

Phe-Asn-Glu-Phe-Val

A molecular

FAB-MS spectrum of fulicin was the peptide was of an amidated

at

654.2

form,

m/z that

ion

peak

in

the

(M+H)’ indicating Phe-Asn-Glu-Pheis,

Val-NH,. A pentapeptide The synthesized native fulicin (Fig.

2A).

anion

exchange

the

sequence

of

fulicin

was

peptide behaved chromatographically on the C-18 reversed-phase column However,

synthetic

peptide

hundredth

that

with

with

it

column of

behaved (Fig.

2B).

on the

penis

native

fulicin.

Thus, we considered Lor D-amino

different Moreover, retractor

32 possible acid residues

Phe-Asn-Glu-Phe-Val-NH*.

However,

Tll

from

(mh)

equal to mentioned above native

the muscle

synthesized.

one

activity was

on the of

about

combinations of the peptides which have the sequence five kinds of peptides with

The

(fail)

Fig. 2. The chroaatograas of native fulicin (nP), synthetic peptide Phe-Asn-Glu-Phe-Val-NH2 (sP) and a mixture (nP + sP). A. The reversed-phase colunn was eluted isocratically with 18% acetonitrile in 0.1% TFA (ptl 2.2). B. The anion-exchange column was eluted isocratically with 0.03 M NaCl in 10 mM Tris-buffer (pH 9.6). 489

the

a one-

Vol.

178,

each

No.

2, 1991

one

D-amino

Phe-Val-NH*

only

identical Val-NH, three (Fig.

residue peptides

positions. also eluted

at

the

D-amino

acids,

I and second applied

eluted as NH*behaved

[131

the

they

to

a single differently

were

fulicin

absorbance (Fig.

peak 3B).

naturally

dermorphin

[ll],

and achatin-I have

[3], just

one

fulicin

eluted

at

peptide D-Phe-Asn-Glu-Phenative fulicin. The other Phe-Asn-Glu-o-Phe-Valat

to

Phe-n-Asn-Glu-Phe-Val-NH* native

the

in their sequence. anion-exchange HPLC

native

eluted applied

Phe-Asn-Glu-

animals

position to the and

COMMUNICATIONS

of of

Phe-Asn-o-Glu-Phe-Val-NH*,

peptide

manner

II

from

Another synthetic very close to

When

synthetic

similar

sequence all

isolated the were

RESEARCH

because

Phe-Asn-Glu-Phe-o-Val-NHz, 3A).

the

been

BIOPHYSICAL

Phe-a-Asn-Glu-Phe-Val-NH*

peptides,

NH, and

in

deltorphins

hitherto

D-amino acid The synthetic

residue

containing

[12],

have

AND

synthesized

peptides

dermenkephalin

and

acid

were

occurring which

BIOCHEMICAL

different

the

reversed-phase again

and whereas

positions

a mixture

HPLC

behaved of

in

the

D-Phe-Asn-Glu-Phe-Val-

FdEFH

I 0

I 10

I 20 Time (ti)

Fig3. The chromatograas of native fulicin (np) and the five synthetic peptides. The structure of the synthetic peptides is shown with one-letter symbol of amino acid sequences. d and a nean D-form amino acid and C-terminal amide, respectively. A. The anion-exchange column was eluted lsocratlcally with 0.03 M NaCl in 10 ml Tris-buffer (pH 9.6). B. The reversed-phase column was eluted isocratically with 18% acetonitrile in 0.1% TFA (pH 2.2).

490

two

a

Vol.

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A

BIOCHEMICAL

0-o t- *

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

native peptiinP) synthetic psptide(sP)

4or

T

r

SP 1o-g M

SPJX16gM

Fig. 4. Effects of native fulicin (nP) and synthetic peptide Phe-o-Asn-Glu-Phe-Val-NH2 (sP) on the penis retractor muscle. A. Dose-response relationship for native (solid circle) and synthetic (solid square) peptides. Enhancement of tetanic contraction was expressed as a percentage of increased amount of peak tension of the contraction to the control peak tension. B. Enhancement of tetanic contraction and elicitation of repetitive contractions by native (upper) and synthetic (lower) peptides. The concentration of native fulicin in the stock solution was calculated from the result of amino acid analysis. Procedures for applying the peptides and stimulating the muscle were the same as in Fig. 1.

Both

fulicin

native

Glu-Phe-Val-NH2 retractor

enhanced in

muscle

lo-l1

M

(Fig.

peptides

4). (Fig.

more

than

one

to

ten

a similar

that

of

manner

with

The

activity

the

other

higher

concluded

that

the

10eg

M

of

these

synthetic

the

structure

native

for

which were

analysis the

of peptide

Thus,

D-Asp. is

far

fulicin

HPLC

fulicin

the

was

of

a

both

after

than

of

about

more,

peptides

The

presence of

or

peptides,

responses.

penis

at

immediately

derivatives

demonstrated

the

threshold

concentrations

comparable

Phe-o-Asnof

the of

o-phthaldialdehyde-N-acetyl-L-cysteine hydrolysate

peptide

contraction

contractions

times

obtain

synthetic

concentrations

4B).

thousand to

the

tetanic

repetitive

application potent

the

At

elicited

required

and

we

Phe-D-Asn-Glu-

Phe-Val-NH2. Fulicin neurons

of

showed L

fulica.

concentrations

of

contraction enhance the

other

in the

lOma

response

contraction in

hand,

modulatory In the to the

M

action on radula retractor or

more

electrical

depressed

the

stimulations

and

after washing the ventricle fulicin 491

several muscles muscle fulicin

peptide at a

and at

tetanic tended

(Fig. 5A). concentration

to On of

Vol.

178,

No.

BIOCHEMICAL

2, 1991

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

C

newon

Fig. 5. Divers effects of A. Effects of & fulica. radula retractor muscle stimulations (20 V, 0.6 intervals. B. Effects of of the ventricle. C. spontaneous activity of buccal ganglion.

lo-”

M clearly

enhanced

significant

change

ganglia,

fulicin

neurons tions

Bl

Phe-Val-NH, the

Fulicin but it

the

B4 (Fig. buccal

with

ganglia

the

produced

and

of the Structurally,

in

fulicin on the muscles and neurons fulicin on tetanic contraction of elicited by repetitive electrical msec, 40 Hz, for 1 set) at 10 10-s H fulicin on the spontaneous Effects of 10-s M fulicin on identified neurons Bl and B4 in

amplitude

frequency rhythmic

5C),

which

muscle

of (Fig. of

depressed was about

ABRM of

In

firing

known

to

with

the in

induce

ruin beat the the

no

central

identified contrac-

[lo].

fulicin shares the Mytilus inhibitory peptides

and

beat

5B).

bursts are

the

of the

a bivalve

C-terminal (MIPS)

Mytilus

portion isolated from

edulis

114,

the phasic contraction of ABRM like the 10,000 times less potent than the MIPS.

151. MIPS, This

could be due to the fact that fulicin lacks a Pro-residue in the sequence which is very important for the inhibitory activity of the MIPS [15]. Therefore, fulicin may not be a member of MIPrelated peptides. The present study has shown that a pentapeptide fulicin containing ganglia

of

a D-asparagine A- fulica and

it

muscles

and

neurons

snail.

of

the

residue shows

is present in diverse effects Fulicin

exhibits

the central on several the

activity

Vol.

178,

No.

very

at

2, 1991

low

muscle,

and

BIOCHEMICAL

concentrations it

is

roles in function

phenylalanine

residue

BIOPHYSICAL

particularly

highly

physiological physiological

AND

in

possible

this of which

RESEARCH

that

snail. achatin-I

In

was also

a D-amino

investigated. progress peptides

Studies in

our

acid

isolated

laboratories,

containing

D-amino

whose it

is

residue(s)

plays

paper [51 the having a D-

from

A _ fulica was ganglionic neurons

second

neuropeptide

function

neuropeptides

and acid

retractor

peptide

our previous a tetrapeptide

residue

on molluscan

penis

this

demonstrated, and the effects on the central were also studied [5,16]. Fulicin is the containing

the

COMMUNICATIONS

has are

expected will

been

still that

in more

be found.

Acknowledgments We would like critical

to express our sincere thanks to Dr. P.T.M. Kenny for his

reading of the manuscript.

Yoshida, Y. Fujisawa, animals.

We also thank M.

K. Fujimoto and Y. Kuroki for

This work was supported

Research from the Ministry

in part

of Education,

T. Ikeda,

M.

help with dissection

of

Sakata,

by Grants-in-Aid

for

Scientific

Science and Culture in Japan.

References 1. 2. 3.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

14. 15.

16.

Walker, R.J. (1986) In The Mollusca (A.O.D. Willows, Ed.), Vol. 9, pp. 279-485. Academic Press, New York. Kobayashi, M. and Muneoka, Y. (1990) Zool. Sci. 7, 801-814. Kamatani, Y., Minakata, H., Kenny, P.T.M., Iwashita, T., Watanabe, K., Funase, K., Sun, X.P., Yongsiri, A., Kim, K.H., Novales-Li, P., Novales, E.T., Kanapi, C.G., Takeuchi, H. and Nomoto, K. (1989) Biochem. Biophysic. Res. Commun.160, 1015-1020. Fujimoto, K., Ohta, N., Yoshida, M., Kubota, I., Muneoka, Y. and Kobayashi, M. (1990) Biochem. Biophysic. Res. Commun.167, 777-783. Kobayashi, M., Fujimoto, K. and Kubota, I. (1991) In Molluscan Neurobiology (K.S. Kits, H.H. Boer and J.Joosse, Eds.) North Holland Publishing Co., Amsterdam. in press. Fujimoto, K., Kubota, I., Yasuda-Kamatani, Y., Minakata, H., Nomoto, K., Yoshida, M., Harada, A., Muneoka, Y. and Kobayashi, M. (1991) Biochem. Biophysic. Res. Commun. 177, 847-853. Muneoka, Y. and Twarog, B.M. (1977) J. Pharmacol. Exp. Ther. 202, 601609. Buck, R.H. and Krummen, K. (1984) J. Chromatography 315, 279-285. Aswad, D.W. (1984) Analytical Biochem. 137, 405-409. Yoshida, M. and Kobayashi, M. (1991) J. exp. Biol. 155, 415-433. Montecucchi, P.C., de Castiglione, R., Piani, S, Gozzini, L. and Erspamer, V. (1981) Int. J. Pept. Protein Res. 17, 275-283. Mor, A., Delfour, A., Sagan, S., Amiche, M., Pradelles, P., Rossier, J. and Nicolas, P. (1989) Febs Lett. 255, 269-274. Erspamer, V., Melchiorri, P., Falconieri-Erspamer, G., Negri, L., Corsi, R Severini, C., Barra, D., Siwmaco, M. and Kreil, G. (1989) Proc. Nail. Acad. Sci. USA 86, 5188-5192. Hirata, T., Kubota, I., Iwasawa, N., Takabatake, I., Ikeda, T. and Muneoka, Y. (1988) Biochem. Biophysic. Res. Commun.152, 1376-1382. Fujisawa, Y., Kubota, I., Kanda, T., Kuroki, Y. and Muneoka, Y. (1991) and G.B. In Comparative Aspects of Neuropeptide Function (E. Florey Stefano, Eds.) pp. 97-114. Manchester Univ. Press, Manchester. Kim, K.H., Takeuchi, H., Kamatani, Y., Minakata, H. and Nomoto, K. (1991) Eur. J. Pharmac. 194, 99-106. 493