Vol. 78, No. 2, 1977

Synthesis

BIOCHEMICAL

of the Mating

truncated

Factor

peptides

of Saccharomyces

Sakakibara"

Naoyoshi

Takaharu

Tanaka**,

Taeko

Protein

Minoh,

August

Shumpei

and Hiroshi

Research

Osaka 562,

Research

Shimamoto-cho,

Chino*, Murakami**

Institute,

**Central

and its

Relationship

Masui*,

Peptide

cerevisiae

: The Structure-Activity

Yoshihiro

*The

Received

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Kita**

Foundation

Japan

Institute,

Suntory

Mishima-gun,

Ltd,

Osaka 618,

Japan

2,1977

SUMMARY : The mating factor of Saccharomyces cerevisiae, Trp-His-Trp-LeuGln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr, has been synthesized to confirm the structure of the natural mating factor, The tridecapeptide has the same biological activity as the natural mating factor, From the studies on the biological activity of its truncated peptide synthesized the minimum sequence of the peptide require for the mating factor was deduced to be as His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gin,

In 1956,

INTRODUCTION : chemical

factor

Levi

in a culture

recognized

fluid

which

mating

of Saccharomyces cerevisiae,

(2)

indicated

biological

that

four

function,

involved

different

and they

among them as below

first

of thea-mating

myces cerevisiae,

type cells

is

(1)

in

type

the mating

peptides proposed

the presence

process

were

responsible

the structure

et al. --

type

(3)

cells

isolated

which

not recognize

the presence

fluid,

Instead,

isolated

and a longer

of the mating

Copyright All rights

they

must have arisen

Lys-7,

0 1977

et al ---

to the same components

(II)

by cleavage peptide

which

a mating

factor

of the

peptide

two inactive of the peptide was considered

from

the culture

as the

II in their

fragments

culture

of the

bond between

I,

peptide

I

peptide Leu-6

to be a precursor

and peptide

factor,

by Academic in any

of reproduction

Stt)tzler

(I)

had the same sequence

They could

which

and a-

:

Tanaka

of&-mating

between&-

of two major

His-Trp-Leu-Gin-Leu-Lys-Pro-Gly-Gin-Pro-Met-Tyr

fluid

of Saccharo-

Recently,

Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr

Independently,

cells

of a

Press, Inc. resen,ed.

form

534 ISSN

0006ZYIX

Vol. 78, No. 2, 1977

BIOCHEMICAL

Boc-Gly-Gln-Pro

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

(IV)

Z(C1)

Bzl(C12)

Boc-Trp-Hi~-Trp-Leu-Gln-Leu-L~~-Pro-Gly-G,"-P~~-~~t-T~~-~G~, HF

(VI)

Anisole/Me2S/HS-(CH2)*-SH

1 Trp-His-Trp-Leu-Gln-Leu-Lys-Pm-Gly-Gly-Gln-Pra-Met-Tyr

Fig,

1.

Synthesis

of a Mating

A : Trifluoroacetic

Factor acid

of Saccharomyces

treatment

--cerevisiae

in a presence

of Me2S

B : I-Ethyl-3-(3-Dimethylaminoprophyl)-Carbodiimide in a presence of 1-Hydroxybenzotriazole C : Trifluoroacetic

acid

treatment

D : Trifluoroacetic

acid

treatment

treatment

in a presence

of Me2S and

HS-(CH2)2-SH

The synthesis the

structure

activities

of the

of the and the

several

truncated

peptide

required

tridecapeptide

natural

mating

factor

chromatographic peptides

for

I was undertaken

were

the mating

by comparing

behavior

of the

synthesized factor

in order the

two.

and the minimum

activity

to confirm

biological Furthermore, sequence

of the

was elucidated,

MATERIALS and METHODS : Biological activity of the mating factor synthesized chemically and its truncated peptides was determined by incubating Saccharomyces cerevisiae X-2180 lA, a-mating type cell, at 27°C for 5 hr in the presence of different amounts of each peptide. The activity was expressed as the minimum amount of the peptide necessary for the induction of cellular deformation on the test organisms, RESULTS and DISCUSSION Synthesis out

of Peptides

by the conventional

:

Synthesis solution

of the tridecapeptide

procedure

as shown

in Fig,

has been carried 1.

Abbreviations Boc, t-butyloxycarbonyl ; OBzl, benzyl ester ; Bzl(C12), 2,6-dichlorobenzyl ; Z(Cl), 2-chlorobenzyloxycarbonyl ; ONp, p-nitrophenyl ester ; ONSu, succinimido ester ; TOS, tosyl All amino acids are L-configuration,

535

Boc-amino

;

Vol. 78, No. 2, 1977

acids

which

are

BIOCHEMICAL

commonly

the

synthesis,

in which

the

stabilized

protective

used for

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

solid

phase

the side-chain groups

functional

for

IV and V were

synthesized

by the stepwise

starting

proline

sor

of compound

precursor

In each removal acid,

( 10 eq. with

) for

dimethyl

protected [b.]i5-27.3"

( c 0,5,

in a HF-reaction dimethly the excess with

material

was removed

column

LH-20

purified

further

using

the main

by lyophilization of the

peptide

from final

had the

Homogeneity graphy,

lyophilizate acid

Rf value

of the natural

shown

activity

synthesized

as the

was confirmed

factor

as shown 536

well

and insoluble through

a

by gel

filtration

The main

fraction

G-25

using

Fractions reduced

covering followed

trideca-

product,

by thin

in Table

was

and biological

natural

was found

on

a mixture

pressure,

The synthetic

1.

of

HF, and the effluent

).

under

of anisole,

was washed

was passed bound

(7)

removal

acid,

on Sephadex

of the tridecapeptide

fluoride

residue

The analytical

in Table

) and

After

as a solvent,

acid,

) together

in the presence

was purified

and concentrated

mating

in the

The final

( decomp,

O"C, the

( 4 : 1 : 5 v/v/v

factor

precur-

by tri-

( 10 eq.

in 2 N acetic

chromatography

are

same mating

of the peptides

and the that

product

in the

: Dimethylsulfide

hydrogen

The filtrate

1 N acetic

acid

peptide.

one hour

below

by filtration,

combined

(4),

and that

as the scavengers.

acid-water

peak were

group

added

by anhydrous

at 0°C for

2 N acetic

by

Fragments

intermediates

mp 175°C

was redissolved

by partition

of n-butanol-acetic

with

showed

The crude

Sephadex

(6),

ester

of Dowex 1 x 2 ( AcO- Form ) to remove the

was lyophilized,

data

which

and ethanedithiol

The residue

were

Trp-containing

HF by vaccum distillation,

ether,

Tyr

of each amino

of the

) for

(8)

His

for

protected for

and ethanedithiol

DMF ), was treated

apparatus

sulfide,

Boc-group

peptide

(VI),

were

hydrogenolysis

scavengers

( 20 eq.

tridecapeptide

utilized

by saponification.

of the

Met-containing sulfide

addition

by catalytic

the following

for

The benzyl

V was removed

reaction

groups

and tosyl

ester.

IV was removed

of compound

fluoroacetic

Lys (5),

benzyl

were

such as 2,6-dichlorobenzyl

2-chlorobenzyloxycarbonyl

from

synthesis

layer

chromato-

to be identical 1.

Data and Biological

[d$

-2005"

**)

*I

-48.5"

-29.8"

-38.6"

-30.8"

0,57 0.54

0.53 0.50

0.54

0.69

0.47

0.54

0.58

0.42

0.54

0,41

0.49

0.49

x

lo-6

ughl

500

500

500

500

6

6

4

100 x 1o-3

708 x lO-3

500

500

6 x 1O-6

6

Activity

Biological

Deyeloped on Cellulose plate ( Merck Art 5552 ) Rf : 1-BuOH : AcOH : H 0 ( 4 : 1 : 5 v/v/v ) Upper Phase : H20 ( 15 : 3 : 10 : 12 v/v/v/v Rf2 : 1-BuOH : AcOH : Pjridine

)

Elemental Anal. ; H,6.94 ; N,14.91%. Calcd for C82H114017N20S'2AcOH'6H20 ; C, Found : C,54.18 54.02 ; H,7.06 ; N,14.65% The molar ratio of amino acid was determined after hydrolysis of this material at 108°C for 24 hr, using 6 N hydrochloric acid in the presence of 2% thioglycolic acid ; Lys,l.07 ; His,0,95 ; Trp,0,95x2 ; Glu,l.OOx2 ; Pro,0.95x2 ; Gly,l.OO ; Met,0.93 ; Leu,l.O3x2 ; Tyr,0.98.

His-Trp-Leu-Gln-Leu-Lys-Pro-Gly

Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly

Leu-Gin-Leu-Lys-Pro-Gly-Gln

Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln

-39.0"

-29.8"

0.61

0.63

-33.5"

0.54

0.51

-40,O"

0.69

0.55

0,77

0.72

0.74

-42.3"

0.57

0.59 0.68

Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln

His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln

Laver

Chromatogr.** Rfl Rf2

Thin

-43.30

Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro

His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met

Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met

Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr

Trp-Leu-Gin-Leu-Lys-Pro-Gly-Gin-Pro-Met-Tyr

Peptides

(c 0.5,AcOH)

Rotation

Synthetic

Optical

of the

-38.4"

Activities

His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gin-Pro-Met-Tyr

Peptides

Analytical

-35.6"

1.

Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr*

Table

Vol. 78, No. 2, 1977

Mating were

Factor

BIOCHEMICAL

Activity

synthesized

order

of Truncated

similary,

activity.

on thin biological

the dodecapeptide

Trp-His

devoid

residues

of the

When the C-terminal the dodecapeptide acid

residues

natural

the mating

1,

factor

results

factor

activity

active

as

inactive,

the

biological

Further

removal

activity

gave an inactive sequence

of

of the amino

no decrease

can be deduced

1,

the N-terminal

was virtually

the minimum

from Table

is equally

produced

Rf values

and their

lacking

was removed,

in

the mating

their

values

undecapeptide

of Gln-10

expresses

residue

peptides

assayed

As is obvious

up to Pro-11

loss

which

rotation

dramatically,

the

were

synthesized,

Trp

mating

residue

From these

for

the

the C-terminus

factor,

required

in Table

though

Tyr

However,

optical

truncated

activities

peptides

of the N-terminal

decreased from

activity. mating

shown

tridecapeptide

Several

of the peptide

their

were

:

biological

of those

chromatography,

activities

the natural

sequence

Structure

layer

Peptides

and their

to know the minimum

factor

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in

peptide

as a

of the

peptide

as His-Trp-Leu-Gln-

Leu-Lys-Pro-Gly-Gln. REFERENCES 1,

Levi,

2.

Stlltzler,

J.D.

3,

Tanaka,

4.

Erickson,

5,

Erickson,

6.

3757-3601 Fujii, T,,

(1956) D.,

Nature

Kiltz,

177, 753-754. and Duntze, W. (1976)

H.,

Eur,

J. Biochem.

69,

397-400. T,,

Kita,

H.,

and Narita,

K. (1977)

Proc.

Japan

Acad.

53, 60-70.

B-W.,

and Merrified,

R.B.

(1973)

J. Am. Chem, Sot,

95,

B.W.,

and Merrified,

R.B.

(1973)

J. Am. Chem, Sot.

95,

3750-3756

49, 7,

Kimura,

T.,

and Sakakibara,

S. (1976)

Bull.

Chem, Sot,

Japan

1595-1601.

Sakakibara,

S,,

and Shimonishi,

S.,

Shimonishi,

Y. (1965)

Bull,

Chem, Sot.

Japan

38,

1412-1413, 8.

Sakakibara, (1967)

Bull.

Chem. Sot,

Y., Japan

Kishida,

Y.,

40, 2164-2167.

538

Okada,

M,,

and Sugihara,

H.

Synthesis of the mating factor of Saccharomyces cerevisiae and its truncated peptides : the structure-activity relationship.

Vol. 78, No. 2, 1977 Synthesis BIOCHEMICAL of the Mating truncated Factor peptides of Saccharomyces Sakakibara" Naoyoshi Takaharu Tanaka**,...
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