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.