Báo cáo khoa học: A possible physiological function and the tertiary structure of a 4-kDa peptide in legumes potx

A possible physiological function and the tertiary structure
of a 4-kDa peptide in legumes
Toshimasa Yamazaki
1
, Motoko Takaoka
2,3
, Etsuko Katoh
1
, Kazuki Hanada
2
, Masashi Sakita
2
,
Kyoko Sakata
2
, Yuji Nishiuchi
4
and Hisashi Hirano
2
1
National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan;
2
Yokohama City University, Kihara Institute
for Biological Research/Graduate School of Integrated Science, Totsuka, Yokohama, Japan;
3
Kamakura Woman’s University,
Iwase, Kamakura, Japan;
4
Peptide Institute Inc, Protein Research Foundation, Minoh, Osaka, Japan
Previously, we isolated a 4-kDa peptide capable of binding
to a 43-kDa receptor-like protein and stimulating protein

kinase activity of the 43-kDa protein in soybean. Both of
them were found to localize in the plasma membranes and
cell walls. Here, we report the physiological effects of
4-kDa peptide expressed transiently in the cultured carrot
and bird’s-foot trefoil cells transfected with pBI 121 plas-
mid containing the 4-kDa peptide gene. At early devel-
opmental stage, the transgenic callus grew rapidly
compared to the wild callus in both species. Cell prolifer-
ation of in vitro cultured nonembryogenic carrot callus was
apparently affected with the 4-kDa peptide in the medium.
Complementary DNAs encoding the 4-kDa peptide from
mung bean and azuki bean were cloned by PCR and
sequenced. The amino-acid sequences deduced from the
nucleotide sequences are homologous among legume spe-
cies, particularly, the sites of cysteine residues are highly
conserved. This conserved sequence reflects the importance
of intradisulfide bonds required for the 4-kDa peptide to
perform its function. Three dimensional structure of the
4-kDa peptide determined by NMR spectroscopy suggests
that this peptide is a T-knot scaffold containing three
b-strands, and the specific binding activity to the 43-kDa
protein and stimulatory effect on the protein phosphory-
lation could be attributed to the spatial arrangements
of hydrophobic residues at the solvent-exposed surface of
two-stranded b-sheet of 4-kDa peptide. The importance of
these residues for the 4-kDa peptide to bind to the 43-kDa
protein was indicated by site-directed mutagenesis. These
results suggest that the 4-kDa peptide is a hormone-like
peptide and the 43-kDa protein is involved in cellular
signal transduction of the peptide.

Keywords: hormone-like peptide; plants; NMR; three-
dimensional structure; site-directed mutagenesis; physio-
logical function.
A 43-kDa protein found in the soybean seeds is a
glycoprotein with sedimentation coefficient of 7S and
isoelectric point ranging from 9.05 to 9.26 [1]. This protein
has been classified into the category of globulin, which is
soluble only in high ionic strength of salt solutions [1]. It
consists of a and b subunits linked by disulfide bridge(s).
There are a cysteine-rich domain in the N-terminal side of
a subunit, a putative transmembrane domain in the b sub-
unit [2], and a consensus sequence of ATP-binding site
indispensable for protein phosphorylation activity [2]. The
43-kDa protein has autophosphorylation activity and
protein kinase activity about two thirds of tyrosine kinase
activity of the rat insulin receptor [3]. Immunocytochemistry
has indicated that the 43-kDa protein is localized in the
plasma membranes and the middle lamellae of cell walls [4],
suggesting that it is a receptor-like protein. Western blotting
and DNA cloning experiments revealed that these proteins
are structurally similar to the 43-kDa protein and distribute
in a number of legume species such as azuki bean, cowpea,
French bean, lupin, mung bean and winged bean [5,6], and
nonlegume species such as carrot [7].
The presence of this receptor-like protein has allowed us
to predict that the physiologically active peptides, which are
capable of binding to the 43-kDa protein, may also be
present in plants. To isolate such peptides, affinity chroma-
tography using Sepharose CL-4B column immobilized the
43-kDa protein as a ligand was conducted [8]. By this

chromatography, we purified a 4-kDa peptide from the
fractionated extract of soybean radicles. Ligand blotting
Correspondence to H. Hirano, Yokohama City University,
Kihara Institute for Biological Research/Graduate School
of Integrated Science, Totsuka, Yokohama, 244–0813 Japan.
Fax: + 81 45 8201901, Tel.: + 81 45 8201904,
E-mail:
Abbreviations: CPA, carboxypeptidase A; CPI, carboxypeptidase A
inhibitor; 2,4-D, 2,4-dichlorophenoxy acetic acid; GUS,
b-glucronidase; MS medium, Murashige & Skoog’s medium.
Note: The nucleotide sequences reported in this paper has been
submitted to the GenBankTM/EMBL Data Bank with accession
numbers AB052880 and AB052881. The structure reported in this
paper has been submitted to the Protein Data Bank with accession
number 1JU8, BMRB 5098.
Note: As the capability of binding of insulin and the 4-kDa peptide to
the 43-kDa protein is similar, in our previous paper we named the
4-kDa peptide as leginsulin. But the 4-kDa peptide is not insulin, and
one must discriminate between them. To avoid confusion, we use
4-kDa peptide as the name of the peptide instead of leginsulin in this
paper.
(Received 6 November 2002, revised 27 December 2002,
accepted 28 January 2003)
Eur. J. Biochem. 270, 1269–1276 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03489.x
experiments using the radioiodinated 4-kDa peptide con-
firmed that this peptide is capable of binding to the 43-kDa
protein [8]. Maximum stimulatory effect was observed at
relatively low concentration (1 n
M
) of the 4-kDa peptide,

indicating possible involvement of the 4-kDa peptide and
43-kDa protein in some cellular signal transduction [8].
Immunocytochemical studies revealed that a small
amount of 4-kDa peptide is localized around the plasma
membranes and cell walls [9]. The subcellular localization of
4-kDa peptide is similar to that of the 43-kDa protein,
suggesting that the 4-kDa peptide is localized at the site
suitable for interaction with the 43-kDa protein.
The present study is performed to understand the
physiological function of the 4-kDa peptide, and suggests
that this peptide is involved in the regulation of callus
growth and cell proliferation. Tertiary structure of the
4-kDa peptide has revealed that this peptide is a T-knot
scaffold containing three b-strands, and the specific binding
activity to the 43-kDa protein and stimulatory effect on
the protein phosphorylation, which could be attributed to
the spatial arrangements of the hydrophobic residues at the
solvent-exposed surface of the two-stranded b-sheet of
4-kDa peptide. The site-directed mutagenesis suggests the
importance of these residues in binding it to the 43-kDa
protein.
Experimental procedures
Transformation of the 4-kDa peptide gene
Seeds of carrot (Doucus carota L., cvs. Benibijin and
Harumakisanzun) were surface-sterilized in 2.5% (v/v)
hypochlorite solution containing 0.02% (v/v) Tween 20
with shaking for 20 min. After washing with deionized
water, the seeds were planted on Murashige & Skoog (MS)
medium containing 30 gÆL
)1

of sucrose and 3 gÆL
)1
of
Gelrite, and incubated at 25 °C under continuous illumi-
nation. After 1–2 weeks, hypocotyls of the developed
seedlings were sliced into 3 mm segments and placed on
MS medium containing 30 gÆL
)1
of sucrose, 2 mgÆL
)1
of
2,4-dichlorophenoxy acetic acid (2,4-D). After 2 days, the
explants were transferred to MS medium containing
30 gÆL
)1
of sucrose, 3 gÆL
)1
of Gelrite without 2,4-D, and
cultured for 10 days. Agrobacterium tumefaciens strains
LBA4404 [10] and the binary vector pBI 121 [11] obtained
from Clontech, CA were used for the transformation. pBI
121 contains a udi coding region of the Escherichia coli
b-glucronidase reporter gene (udiA) under the control of the
cauliflower mosaic virus (CaMV) 35S promoter and a
polyadenylation signal of nopaline synthetase gene (nos)
region. The 4-kDa peptide gene was inserted, in either sense
(545 bp DNA) or antisense directions, between the 35S
promoter and udiA gene of pBI 121 plasmid. The pBI 121
was introduced into A. tumefaciens LBA 4404 by triparental
mating with E. coli pRK 2013 as a helper strain. A. tume-

faciens strain LBA4404 containing pBI 121was grown for
2 days on LB medium (10 gÆL
)1
of Bacto tryptone, 5 gÆL
)1
of Bacto yeast extract, 10 gÆL
)1
of NaCl) containing
100 mgÆL
)1
of kanamycin and 50 mgÆL
)1
of rifampicin.
The hypocotyl segments of carrot described above were
immersed in the bacterial suspension for 2 h and then
transferred to MS medium containing 100 mgÆL
)1
of
kanamycin, 100 mgÆL
)1
of cefotaxime and 2 mgÆL
)1
of
2,4-D, incubated at 25 °C and subcultured at 25 °Cat
2-week intervals. After eight weeks, the callus regenerated
from hypocotyl on the first selection medium was explanted
for induction adventitious embryo on MS medium con-
taining 100 mgÆL
)1
of kanamycin, 250 mgÆL

)1
of cefota-
xime. The induced embryogenic callus was subcultured on
the same medium to develop the plantlets.
Transformation was also performed in bird’s-foot trefoil
(Lotus cornialatus) by the methods as described above
except that cotyledons were used as materials and 1 mgÆL
)1
of benzyladenine instead of 2,4-D was added into the
medium. Histochemical and fluorometric assays for GUS
activity were performed as described [12].
Carrot cell culture
The carrot nonembryogenic cells gifted by S. Satoh [13]
were grown at 25 °C in MS liquid medium containing
30 gÆL
)1
of sucrose and 2 mgÆL
)1
of 2,4-D. The suspension
was subcultured at two-week intervals. Three days after the
final transplanting, the cells were precipitated by centrifu-
gation (100 g, 5 min) and resuspended in the same medium
containing different concentrations (0.1 p
M
,1,100 n
M
,1,10
and 100 l
M
) of the 4-kDa peptide at a density of 0.5 · 10

5
cellsÆmL
)1
. After 3, 7, 10 and 14 days, the cells were
harvested to determine the density. Experiments were
repeated six times.
Nucleotide sequence of DNAs encoding
the 4-kDa peptide superfamilies
The genomic sequences coding for the 4-kDa peptide
precursor polypeptides were amplified by PCR strategy
using the azuki bean and mung bean genomic DNAs as
templates and the synthetic primers legF1 (5¢-AGC
AGCAGATTGTAATGGTG-3¢)andlegR1(5¢-CAGC
ACTTCAGAATCAGAGTC-3¢). PCR products were
cloned on pT7Blue T-vector (Novagen, Darmstadt) and
their nucleotide sequences were determined. The amino-acid
sequences of 4-kDa peptides were deduced from the
nucleotide sequences.
Tertiary structure of the 4-kDa peptide
Natural 4-kDa peptide purified from soybean radicles as
described in [8] and chemically synthesized one were used
for NMR studies. The reduced peptide obtained by solid-
phase synthesis using Boc strategy was subjected to
oxidative folding in 0.1
M
AcONH
4
buffer (pH 7.4) in a
50% (v/v) aqueous isopropyl alcohol solution containing
0.5

M
guanidine hydrochloride at a peptide concentration
of 10
)5
M
, in the presence of reduced and oxidized
glutathione (GSH/GSSG) as redox reagents at room tem-
perature for 60 h. The molar ratio of peptide/GSH/GSSG
was set to 1 : 100 : 10. The crude cyclic peptide was
purified on preparative high performance liquid chroma-
tography (HPLC) with a C18 column. The homogeneity
of the synthesized product purified by reversed phase-
HPLC was further confirmed by amino-acid analysis,
ion-exchange-HPLC, capillary zone electrophoresis and
matrix assisted laser desorption ionization time-of-flight
1270 T. Yamazaki et al. (Eur. J. Biochem. 270) Ó FEBS 2003
mass spectrometry. As both natural and chemically
synthesized 4-kDa peptides provided the same NMR
spectra at concentration of 200 l
M
, the synthesized
peptide was used for further detailed NMR analysis.
The solution used for NMR structure determination
contained about 4 m
M
synthesized 4-kDa peptide in
70% H
2
Oand30%CD
3

COOD at pH 1.8. We obtained
no evidence for any conformational changes and aggre-
gation of the 4-kDa peptide even at the higher peptide
concentration. All NMR spectra were recorded at 25, 40
and 50 °C on a Bruker DMX750 spectrometer equipped
with a x,y,z-shielded gradient probe. Complete sequence-
specific assignments for all backbone and side-chain
protons were obtained using two-dimensional DQF-
COSY, HOHAHA and NOESY experiments.
Structures of the 4-kDa peptide were calculated using the
hybrid distance geometry-dynamical simulated annealing
protocol within
X
-
PLOR
[14]. For structure calculations, we
used 541 interproton distance restraints [comprising 229
intraresidue, 161 sequential (|i – j| ¼ 1), 56 medium-range
(1 < |i – j| < 5) and 95 long-range (|i – j| > 5)] obtained
from NOESY spectra with a mixing time of 150 ms. In
addition to the NOE-derived distance restraints, 16 distance
restraints for eight hydrogen bonds and 55 dihedral angle
restraints (20 /,14w,19v
1
and 2 v
2
) were included in the
structure calculation. A peptide bond between Val12 and
Pro13 was set to a cis configuration, i.e. x  0°, based upon
observation of an extremely strong sequential NOE between

the Val12 Ha and Pro13 Ha. Structure calculations were
first carried out without restraints regarding disulfide
bridges. Analysis of Ca–Ca and Sc–Sc distances between
cysteines observed for the resultant structures led to
identification of disulfide bond pairings of the 4-kDa
peptide as Cys3–Cys20, Cys7–Cys22 and Cys15–Cys32.
The disulfide bond between Cys15 and Cys32 was experi-
mentally confirmed by amino-acid sequence analysis of
several peptide fragments generated from hydrolysis of the
natural 4-kDa peptide with 10% (v/v) phosphoric acid at
101 °C for 15 h. Hence, the final structure calculations
included disulfide bond restraints in addition to the NMR-
derived distance and dihedral angle restraints. A final set of
15 lowest-energy structures was selected from 100 calcula-
tions. None of them had NOE and dihedral angle violations
>0.05 nm and5°, respectively. The average coordinates of
ensembles of the final 15 structures were subjected to 500
cycles of Powell restrained energy minimization to improve
stereochemistry and nonbonded contacts. Figures were
generated using
MOLMOL
[15].
Site-directed mutagenesis
The wild-type DNA sequence of 4-kDa peptide was
amplified from the soybean 4-kDa peptide cDNA by
polymerase chain reaction (PCR) using the following
oligonucleotide primers: the 4-kDa peptide N-terminal
primer: 5¢-AACCATGGCTAAAGCAGATTGTAATGG
TGCATGT-3¢; the 4-kDa peptide C-terminal primer:
5¢-AAGAATTCTTATTATCCAGTTGGATGTATGCA

GAA-3¢. The amplified sequence was cloned into the
plasmid pKF18 via the EcoRI and SalI restriction sites in
the multicloning site. This plasmid was designated as
pKF18/LEG.
Site-directed mutagenesis was performed using pKF18/
LEG as a template using the commercial kit of oligonu-
cleotide-directed dual amber method (Mutan-Super Express
Km, Takara Biochemicals, Osaka) [16]. Arg16, Val29 and
Phe31 in the 4-kDa peptide were singly replaced by Ala with
pKF18/LEG, selection primer included in the Mutan-Super
Express Km and the following oligonucleotide primers
(mismatches are underlined): Variant R16A 5¢-CCACCGT
GCGCCTCACGTGATTG-3¢,VariantV29A5¢-GGACT
ATTTG
CTGGTTTCTGC-3¢, Variant F31A 5¢-CTATTT
GTTGGT
GCCTGCATACATC-3¢. All variants were veri-
fied to be correctly constructed by dideoxy sequencing. Each
DNA fragment of the 4-kDa peptide variants was removed
by the EcoRI and SalI restriction enzymes and recloned into
pET-32a(+). The 4-kDa peptide and its variants were
prepared by the Escherichia coli protein expression system.
The assay of binding activity of the mutant 4-kDa
peptides to the 43-kDa protein was carried out by ligand
blotting. The 43-kDa protein was separated by SDS/gel
electrophoresis and electroblotted onto a poly(vinylidene
difluoride) membrane. The poly(vinylidene difluoride)
membrane was soaked in Tris buffered NaCl/P
i
(Tris/

NaCl/P
i
) for 5 min, and in Tris/NaCl/P
i
containing 1%
(w/v) skimmed milk for 1 h, then in Tris/NaCl/P
i
for
10 min. The poly(vinylidene difluoride) membrane was
packed in the plastic bag with 5 lg of the 4-kDa peptide or
mutant 4-kDa peptides in 2 mL of Tris/NaCl/P
i
.The
membrane was incubated overnight at 4 °Candwashed
twice with Tris/NaCl/P
i
for 5 min. Then, rabbit anti-(4-kDa
peptide) Ig in 5 mL of Tris/NaCl/P
i
was added. The
membrane was incubated for 1 h at 4 °C, and washed twice
with Tris/NaCl/P
i
for 5 min. Goat anti-(rabbit IgG) Ig
conjugated with alkaline phosphatase in 5 mL of Tris/
NaCl/P
i
was then added. The membrane was incubated for
1hat4°C,andwashedtwicewithTris/NaCl/P
i

for 5 min.
Finally, the cross-reacted bands were detected with alkaline
phosphatase substrate (Moss, Maryland).
Results and discussion
Possible physiological function of the 4-kDa peptide
To investigate the physiological functions of the 4-kDa
peptide, we transiently expressed the peptide in the cultured
carrot cells transfected with pBI 121 plasmid containing the
4-kDa peptide gene and GUS gene as a reporter gene using
Agrobacterium transformation system. The presence of
4-kDa peptide gene in the transgenic plants was confirmed
by Southern blotting (Fig. 1). The GUS activity was
constitutively detected in the roots and leaves of the
transgenic plants (Fig. 1). The transgenic plant has two
integration sites for the 4-kDa peptide gene, as two bands
were detected when the DNA was digested with HindIII. As
shown in Fig. 1, at early developmental stage, the transgenic
callus rapidly grew compared with the wild callus. Three
weeks after transplanting, the growth ratio of the callus was
162.8 ± 84.6 for the transformant to 15.3 ± 5.3 for the
nontransformant (control). However, the phenotype of
intact transgenic plants regenerated from the calli was not
noticeably different from that of the wild plant. The 43-kDa
protein has been detected in the wild carrot cells [13], but not
the 4-kDa peptide. This result suggests that the 4-kDa
Ó FEBS 2003 Possible function and structure of a 4-kDa legume peptide (Eur. J. Biochem. 270) 1271
peptide, which was synthesized by the transfected gene,
stimulated protein kinase activity of the carrot 43-kDa
protein, and the signal transduction pathway was activa-
ted for the regulation of growth of callus. We transfected

the carrot cells with pBI 121 plasmid containing the anti-
sense 4-kDa peptide gene, but could not detect any
significant effect of the antisense gene on the development
of callus, probably because there was no 4-kDa peptide-
like peptide gene which could interact with the anisense gene
in carrot.
We tried to culture the soybean callus in vitro to use it for
the 4-kDa peptide gene transformation. However, the
shoots and roots were not easily differentiated from the
callus, and consequently we could not obtain the transgenic
plants. However, we could construct the transgenic ones in
bird’s-foot trefoil instead of soybean. In this case, the 4-kDa
peptide showed a similar effect on growth of the callus in the
transgenic bird’s-foot trefoil to that of the transgenic carrot
(data not shown).
On the other hand, we investigated the effects of 4-kDa
peptide on proliferation of the carrot auxin-autotropic
nonembryogenic cells, which lost embryogenic competence.
When the 4-kDa peptide was added into the liquid culture
medium containing 2 mgÆL
)1
of 2,4-D, the cell proliferation
was stimulated depending on the 4-kDa peptide concentra-
tion. The optimum concentration for maximum cell proli-
feration was 1 l
M
in the culture medium (Fig. 2). This
indicates that the 4-kDa peptide may also be involved in the
regulation of carrot cell proliferation.
Tertiary structure of the 4-kDa peptide in solution

The 4-kDa peptide consisting of 37 residues contains 6 half-
cystines in three disulfide bridges (Fig. 3). Disruption of the
disulfide bridges leads to a complete loss of the stimulatory
effect of 4-kDa peptide on the phosphorylation activity of
43-kDa protein [8], indicating that the disulfide bridges
might play an important role in maintaining the correct
three-dimensional structure of 4-kDa peptide required for
its function. Complementary DNAs encoding the 4-kDa
peptide from mung bean and azuki bean were cloned by
PCR and sequenced. The amino-acid sequences deduced
from the nucleotide sequences are homologous among
legume species, particularly, the sites of cysteine residues are
highly conserved (Fig. 3). This conserved sequence reflects
the importance of intradisulfide bonds required for the
4-kDa peptide to perform its function.
To investigate the structural basis for the 4-kDa peptide
function, we have determined its three-dimensional struc-
ture by
1
H-NMR spectroscopy. In the present study, the
NMR structure was determined at pH 1.8, as the purified
and lyophilized 4-kDa peptide is soluble only at this pH.
Similar to the 4-kDa peptide, the higher solubility of the
purified animal insulin and invertebrate insulin-like peptides
have been reported elsewhere [20,21]. No information of
these peptides at higher pH, which might cause chemical sift
related to structural rearrangements, is available. As shown
in Fig. 4, the 4-kDa peptide was found as a T-knot scaffold
containing 3 b-strands (bA: Ala6–Ser8; bB: Cys20–Pro24;
Fig. 1. Growth of transgenic callus (A), Southern blot analysis of transgeneic plant (B) and histochemical detection of GUS expression in the carrot

tissue. (A) A, wild callus; B, transgenic callus containing the fused protein gene but lacking the 4-kDa peptide gene; C, transgenic callus containing
the GUS/the 4-kDa peptide fused protein gene. The transgenic and nontransgenic callus generated was explanted on MS medium. Three weeks
after transplanting, the growth of the callus was compared. (B) total DNA (20 lg per lane) isolated from 1, transformant; 2, nontransformant; 3,
soybean. (C) Top, adventitious embryo of the transformant, GUS activity was detected basal (dark) part of embryo; bottom, leaf of the
transformant, GUS activity was clearly detected in the vein.
1272 T. Yamazaki et al. (Eur. J. Biochem. 270) Ó FEBS 2003
bC: Gly30–His34). Two adjacent b-strands, bB and bC,
connected by a distorted type-I b-turn around Gly26-Val29,
make up a two-stranded antiparallel b-sheet which is
stacked by a long N-terminal loop containing bA, 2 type-I
b-turns around Ser8–Glu11 and Ser17–Cys20, and a cis
proline at position 13. The stacked structure is stabilized by
3 disulfide bridges formed between the b-sheet and the
N-terminal loop. It has been reported that the T-knot
scaffold [15] is shared by several small, disulfide-rich
proteins with diverse functions, such as potato CPI [22]
and calcium channel blockers x-conotoxin GVIA from the
venom of cone snail and x-agatoxin-IVB from the venom of
funnel web spider [23] (Fig. 6C). The X-ray crystal structure
of CPA–CPI complex has revealed that CPI recognizes the
enzyme using the C-terminal tetrapeptide and residues at
the solvent-exposed surface of strand bB [22].
By ligand blotting experiments using
125
I-labelled 4-kDa
peptide, Watanabe et al. [8] demonstrated that the
4-kDa peptide competes with insulin for binding to the
43-kDa protein. This suggests that the 4-kDa peptide and
insulin bind to the same sites of the 43-kDa protein in a
similar manner, although both peptides were considered to

have totally different folds. Hence, we have assumed that
the 4-kDa peptide and insulin may possess similar spatial
arrangements of functional residues, and searched topo-
chemical similarity in local structures between them.
In the case of insulin, residues at the A-chain N-terminus
(GlyA1–IleA2–Val13A) [24,25], the A-chain C-terminus
(TyrA19 and AsnA21) [26], the B-chain central helix
(ValB12 and TyrB16) [27], and the B-chain C-terminal
b strand (PheB24–PheB25–TyrB26) [28–30] have been
shown to be important for receptor recognition. In the
solution structure of human insulin (Fig. 5) [31], which is
considered as the locked and inactive state, most of these
residues form an extensive hydrophobic core at the interface
between the B-chain b-strand and the B-chain central helix
as well as the A-chain N- and C-terminal regions. It is
generally believed that receptor binding is accompanied by
some degree of conformational change of insulin from the
locked, inactive state to the active state. Although the
receptor bound conformation of insulin has not yet been
experimentally determined, a model has been proposed
based on the solution structure of the biologically active
insulin analog, [GlyB24]human insulin, where the orienta-
tion of the disordered B-chain C-terminal region relative to
the rest of molecule is not well defined [32]. The proposed
model is described as the unlocked state where the B-chain
C-terminal b-strand is detached from the rest of molecule,
resulting in exposure of the insulin pharmacophore to the
receptor. This model is further supported by the high
potency of des-(B26-B30)-insulin amide in which the B
Fig. 2. Effect of the 4-kDa peptide on the cell proliferation of carrot

nonembryogenic cells. The carrot nonembryogenic cells were grown in
MS liquid medium. The cells were suspended in the MS medium
containing different concentrations (0.1 p
M
,1,100n
M
,1,10and
100 l
M
) of the 4-kDa peptide at a density of 0.5 · 10
5
cellsÆmL
)1
.
After 3, 7, 10 and 14 days, the cells were harvested to determine the
density. Results represent means ± SE, n ¼ 6.
Fig. 3. Amino-acid sequences of the 4-kDa peptide superfamilies. The genomic sequences coding for the 4-kDa peptide precursor polypeptides were
amplified by PCR strategy using the azuki bean and mung bean genomic DNAs as templates the synthetic primers. PCR products were cloned on
pT7Blue T-vector and their nucleotide sequences were determined. The sequences for soybean [8]; Glycine soja (wild Glycine sp.) [17]; pea [18];
lupin [19] 4-kDa peptides are cited. Underlined Ser, possibly sequencing error; X, not determined.
Ó FEBS 2003 Possible function and structure of a 4-kDa legume peptide (Eur. J. Biochem. 270) 1273
chain from TyrB26 to the C-terminus is truncated but the
resultant new C-terminal PheB25 is amidated [33]. It is
worthwhile mentioning that among the insulin pharmaco-
phore, IleA2-ValA3 at the A-chain N-terminus, TyrA19 at
the A-chain C-terminus, and ValB12 and TyrB16 at the
B-chain central helix assume essentially the same spatial
arrangements both in the locked, inactive state and in the
unlocked state (Fig. 5). Therefore, we first searched a
tetragonal arrangement made of hydrophobic residues,

TyrA19, ValB12, TyrB16 and either one of IleA2 or ValA3
of insulin, for the 4-kDa peptide structure.
We found that only the 4 hydrophobic residues, Val23,
Val29, Phe31 and Ile33, at the solvent-exposed surface of
the 4-kDa peptide b-sheet could fulfill essentially the same
tetragonal arrangement as TyrA19, TyrB16, ValB12 and
ValA3 of insulin (Fig. 5). Furthermore, it was turned out
that when the 4-kDa peptide was superimposed against the
unlocked, active state model of insulin using these tetrago-
nal arrangements, Leu27 and Phe28 of the 4-kDa peptide
could occupy similar space as PheB25 and TyrB26 of the
insulin pharmacophore. These results suggest that the
specific binding activity of 4-kDa peptide to the 43-kDa
protein and its stimulatory effect on the protein phosphory-
lation are attributed to the spatial arrangements of the
hydrophobic residues at the solvent-exposed surface of the
two-stranded b sheet.
The preliminary experiments using site-directed muta-
genesis suggested that the substitution of the hydrophobic
residues at the solvent-exposed surface of the two-stranded
b sheet caused a significant change in its binding activity to
the 43-kDa protein (Fig. 6). Although there was no great
difference in the activity between the normal and the
Arg16fiAla mutant 4-kDa peptides, the Val29fiAla and
Phe31fiAla mutant 4-kDa peptides bound to the 43-kDa
protein less strongly than the normal 4-kDa peptide. These
results show the importance of these residues for the 4-kDa
peptide to function.
There are several reports on hormone-like peptides in
plants. Systemin is an 18 residue-peptide which can induce

the transcription of the tomato protease inhibitor gene in
response to insect damage [34]. The systemin signal has been
considered to be transduced through the octadecanoid
Fig. 4. Structure of the 4-kDa peptide. A, the best-fit superposition of the backbone (N, Ca and ¢C) atoms of the 15 NMR-derived structures of the
4-kDa peptide. The structures are superimposed against the energy-minimized average structure using the backbone coordinates of residues 3–35
(r.m.s.d of 0.62 ± 0.14 A
˚
for backbone atoms and 1.16 ± 0.14 A
˚
for all heavy atoms). B, Ribbon drawing of the energy-minimized average
structure of the 4-kDa peptide. The disulfide bridges are shown as ball-and-stick models. C, Structure-based sequence alignment of the 4-kDa
peptide with carboxypeptidase A inhibitor from potato (CPI), and P-type calcium channel blocker from venom of the funnel web spider (x-Aga-
IVB), all of which possess T-knot scaffold. Secondary structural elements are indicated as arrows.
1274 T. Yamazaki et al. (Eur. J. Biochem. 270) Ó FEBS 2003
signalling pathway. This signalling mechanism seems to be
different from that of the 4-kDa peptide. Phytosulfokines A
and B which are eight- and five-residue peptides, respect-
ively, can stimulate the proliferation of asparagus and rice
cultured cells [35]. Recently, the putative receptors for
phytosulfokine were identified in the rice plasma membrane
[36], and RALF, a 5-kDa peptide from tobacco leaves was
also reported to reduce the root growth and development of
tomato and Arabidopsis [37]. A potential receptor-like
serine/threonine protein kinase CLAVATA 1 and its ligand
CLAVATA 3, which regulates cell proliferation and differ-
entiation at the shoot meristem [38], were identified in
Arabidopsis. The finding of these hormone-like peptides
including the 4-kDa peptide strongly suggest the presence of
hormone peptides in plants and their function as signal
transduction systems, which are similar to the animal

systems.
Acknowledgements
This work was supported in part by Grants-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science and
Technology, and National Project on Protein Structural and Func-
tional Analyses to H. H., and a grant from the Bio-oriented
Technology Research Advancement Institution, Japan to T. Y. We
thank Nazrul Islam for his help in preparing the manuscript.
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