A possible physiological function and the tertiary structureof a 4-kDa peptide in legumes Toshimasa Yamazaki1, Motoko Takaoka2,3, Etsuko Katoh1, Kazuki Hanada2, Masashi Sakita2, Kyoko Sa
Trang 1A possible physiological function and the tertiary structure
of a 4-kDa peptide in legumes
Toshimasa Yamazaki1, Motoko Takaoka2,3, Etsuko Katoh1, Kazuki Hanada2, Masashi Sakita2,
Kyoko Sakata2, Yuji Nishiuchi4and Hisashi Hirano2
1
National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan;2Yokohama City University, Kihara Institute
for Biological Research/Graduate School of Integrated Science, Totsuka, Yokohama, Japan;3Kamakura Woman’s University, Iwase, Kamakura, Japan;4Peptide 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-4Bcolumn 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: hirano@yokohama-cu.ac.jp
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)
Trang 2experiments 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 nM) 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 25C 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)1of
2,4-dichlorophenoxy acetic acid (2,4-D) After 2 days, the
explants were transferred to MS medium containing
30 gÆL)1of sucrose, 3 gÆL)1of 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-faciensstrain LBA4404 containing pBI 121was grown for
2 days on LBmedium (10 gÆL)1of 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 25C and subcultured at 25 C at 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)1of 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 25C in MS liquid medium containing
30 gÆL)1of sucrose and 2 mgÆL)1of 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 pM, 1, 100 nM, 1, 10 and 100 lM) of the 4-kDa peptide at a density of 0.5· 105 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¢) and legR1 (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 0.1M AcONH4 buffer (pH 7.4) in a 50% (v/v) aqueous isopropyl alcohol solution containing 0.5Mguanidine hydrochloride at a peptide concentration
of 10)5M, 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
Trang 3mass spectrometry As both natural and chemically
synthesized 4-kDa peptides provided the same NMR
spectra at concentration of 200 lM, the synthesized
peptide was used for further detailed NMR analysis
The solution used for NMR structure determination
contained about 4 mM synthesized 4-kDa peptide in
70% H2O and 30% CD3COOD 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 50C 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 withinX-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 /, 14 w, 19 v1and 2 v2) 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
101C 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 and 5, 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 usingMOLMOL[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¢, Variant V29A 5¢-GGACT ATTTGCTGGTTTCTGC-3¢, Variant F31A 5¢-CTATTT GTTGGTGCCTGCATACATC-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/Pi (Tris/ NaCl/Pi) for 5 min, and in Tris/NaCl/Pi containing 1% (w/v) skimmed milk for 1 h, then in Tris/NaCl/Pi 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/Pi The membrane was incubated overnight at 4C and washed twice with Tris/NaCl/Pifor 5 min Then, rabbit anti-(4-kDa peptide) Ig in 5 mL of Tris/NaCl/Pi was added The membrane was incubated for 1 h at 4C, and washed twice with Tris/NaCl/Pi for 5 min Goat anti-(rabbit IgG) Ig conjugated with alkaline phosphatase in 5 mL of Tris/ NaCl/Piwas then added The membrane was incubated for
1 h at 4C, and washed twice with Tris/NaCl/Pifor 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
Trang 4peptide, 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 transfecactiva-ted
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)1of 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 lM 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 by1H-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) andhistochemical 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.
Trang 5bC: Gly30–His34) Two adjacent b-strands, bBand 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-IVBfrom 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 using125I-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, 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 Results represent means ± SE, n ¼ 6.
Fig 3 Amino-acidsequences 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.
Trang 6chain 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.
Trang 7signalling pathway This signalling mechanism seems to be
different from that of the 4-kDa peptide Phytosulfokines A
and Bwhich 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.
References
1 Yamauchi, F., Sato, K & Yamagishi, T (1984) Isolation and partial characterization of a salt-extractable globulin from soy-bean seeds Agric Biol Chem 48, 645–650.
2 Watanabe, Y & Hirano, H (1994) Nucleotide sequence of the basic 7S globulin gene from soybean Plant Physiol 105, 1019– 1020.
3 Komatsu, S., Koshio, O & Hirano, H (1994) Protein kinase activity and insulin-binding activity in plant basic 7S globulin Biosci Biotechn Biochem 58, 1705–1706.
4 Nishizawa, N.K., Mori, S., Watanabe, Y & Hirano, H (1994) Ultrastructural localization of the basic 7S globulin in soybean (Glycine max) cotyledons Plant Cell Physiol 35, 1079–1085.
5 Kagawa, H., Yamauchi, F & Hirano, H (1987) Soybean basic 7S globulin represents a protein widely distributed in legume species FEBS Lett 226, 145–149.
6 Kolivas, S & Gayler, K.R (1993) Structure of the cDNA coding for conglutin gamma, a sulphur-rich protein from Lupinus angu-stifolius Plant Mol Biol 21, 397–401.
7 Satoh, S., Sturm, A., Fujii, T & Chrispeels, M.J (1992) cDNA cloning of an extracellular dermal glycoprotein of carrot and its expression in response to wounding Planta 188, 432–438.
8 Watanabe, Y., Barbashov, S.F., Komatsu, S., Hemmings, A.M., Miyagi, M., Tsunasawa, S & Hirano, H (1994) A peptide that stimulates phosphorylation of the plant insulin-binding protein Isolation, primary structure and cDNA cloning Eur J Biochem.
224, 167–172.
9 Nishizawa, N.K., Mori, S., Kajiwara, H., Komatsu, S & Hirano,
H (1995) Subcellular localization of leginsulin in the immature seeds of soybean Plant Cell Physiol (Supplement)36, 42.
Fig 5 Ribbon representation of insulin andthe 4-kDa peptide (A) Solution structure of human insulin corresponds to the locked, inactive state (B) Solution structure of [GlyB24]human insulin represents a model of the unlocked, active state In a and b, side chains of the insulin pharmacophore are shown as ball-and stick models (C) Solution structure of the 4-kDa peptide Hydrophobic residues, potentially involving in binding to the 43-kDa protein, are shown as ball-and-stick models.
Fig 6 Binding activity of the normal and mutant 4-kDa peptides to the
43-kDa protein The 43-kDa protein was separated by SDS/PAGE
and electroblotted onto a poly(vinylidene difluoride) membrane The
membrane was incubated with the mutant 4-kDa peptides, and the
binding activity of the mutant 4-kDa peptides to the 43-kDa protein
was detected with anti-(4-kDa peptide) Ig Ligand: (A) normal 4-kDa
peptide; (B) Arg16fiAla mutant; (C) Val29fiAla mutant; (D)
Phe31fiAla mutant.
Trang 810 Hoekema, A., Hooykaas, P.J & Schilperoort, R.A (1984) Transfer
of the octopine T-DNA segment to plant cells mediated by
dif-ferent types of Agrobacterium tumor- or root-inducing plasmids:
generality of virulence systems J Bacteriol 158, 383–385.
11 Jefferson, R.A., Kavanagh, T.A & Bevan, M.W (1987) GUS
fusions: beta-glucuronidase as a sensitive and versatile gene fusion
marker in higher plants EMBO J 6, 3901–3907.
12 McCabe, D.E., Swain, W.F., Martinell, B.J & Christou, P (1988)
Stable transformation of soybean (Glycine max) by particle
acceleration Bio/Technol 6, 923–926.
13 Satoh, S., Kamada, H., Harada, H & Fujii, T (1986)
Auxin-controlled glycoprotein release into the medium of embryogenic
carrot cells Plant Physiol 81, 931–933.
14 Brunger, A.T (1992) A System for X-Ray Crystallography and
NMR Yale University Press, New Haven, CT.
15 Koradi, R., B illeter, M & Wuthrich, K (1996) MOLMOL: a
program for display and analysis of macromolecular structures.
J Mol Graph 14, 51–55.
16 Hashimoto-Gotoh, T., Mizuno, T., Ogasahara, Y & Nakagawa,
M (1995) An oligodeoxyribonucleotide-directed dual amber
method for site-directed mutagenesis Gene 152, 271–275.
17 Tan, J., Lou, C & Hirano, H (1999) Analysis of leginsulin gene in
soybean cultivar (Glycine max) and wild species (Glycine Soja).
Chin J Appl Environ Biol 5, 259–263.
18 Higgins, T.J.V., Chandler, P.M., Randall, P.J., Spencer, D.,
Beach, L.R., Blagrove, R.J., Kortt, A.A & Inglis, A.S (1986)
Gene structure, protein structure, and regulation of the synthesis
of sulfur-rich protein in pea seeds J Biol Chem 261, 11124–
11130.
19 Ilgoutz, S.C., Knittel, N., Lin, J.M., Sterle, S & Gayler, K.R.
(1997) Transcription of genes for conglutin c and a
leginsulin-like protein in narrow-leafed lupin Plant Mol Biol 34, 613–
627.
20 Hua, Q & Weiss, M.A (1990) Toward the solution structure of
human insulin: Sequential 2D 1H NMR assignment of a
des-pentapeptide analogue and comparison with crystal structure.
Biochemistry 29, 10545–10555.
21 Nagata, K., Hatanaka, H., Kohda, D., Kataoka, H., Nagasawa,
H., Isogai, A., Ishizaki, H., Suzuki, A & Inagaki, F (1995)
Three-dimensional solution structure of bombyxin-II an insulin-like
peptide of the silkmoth Bombyx mori: structural comparison with
insulin and relaxin J Mol Biol 253, 749–758.
22 Rees, D.C & Lipscomb, W.N (1982) Refined crystal structure of
the potato inhibitor complex of carboxypeptidase A at 2.5 A˚
resolution J Mol Biol 160, 475–498.
23 Narasimhan, L., Singh, J., Humblet, C., Guruptasad, K &
Blundell, T (1994) Snail and spider toxins share a similar tertiary
structure and cystine motif Nat Struct Biol 1, 850–852.
24 Nakagawa, S.H & Tager, H.S (1992) Importance of aliphatic side-chain structure at positions 2 and 3 of the insulin A chain in insulin–receptor interactions Biochemistry 31, 3204–3214.
25 Kitagawa, K., Ogawa, H., Burke, G.T., Chanley, J.D & Katsoyannis, P.G (1984) Critical role of the A2 amino acid residue in the biological activity of insulin: [2-glycine-A]- and [2-alanine-A]insulins Biochemistry 23, 1405–1413.
26 Carpenter, F.H (1966) Relationship of structure to biological activity of insulin as revealed by degradative studies Am J Med.
40, 750–758.
27 Hu, S.Q., Burke, G.T & Katsoyannis, P.G (1993) Contribution
of the B16 and B26 tyrosine residues to the biological activity of insulin J Protein Chem 12, 741–747.
28 Mirmira, R.G., Nakagawa, S.H & Tager, H.S (1991) Importance
of the character and configuration of residues B24, B25, and B26
in insulin–receptor interactions J Biol Chem 266, 1428–1436.
29 Mirmira, R.G & Tager, H.S (1989) Role of the phenylalanine B24 side chain in directing insulin interaction with its receptor Importance of main chain conformation J Biol Chem 264, 6349–6354.
30 Nakagawa, S.H & Tager, H.S (1986) Role of the phenylalanine B25 side chain in directing insulin interaction with its receptor Steric and conformational effects J Biol Chem 261, 7332–7341.
31 Hua, Q.X., Gozani, S.N., Chance, R.E., Hoffmann, J.A., Frank, B.H & Weiss, M.A (1995) Structure of a protein in a kinetic trap Nat Struct Biol 2, 129–138.
32 Hua, Q.X., Shoelson, S.E., Kochoyan, M & Weiss, M.A (1991) Receptor binding redefined by a structural switch in a mutant human insulin Nature 354, 238–241.
33 Fischer, W.H., Saunders, D., Brandenburg, D., Wollmer, A & Zahn, H (1985) A shortened insulin with full in vitro potency Biol Chem Hoppe Seyler 366, 521–525.
34 Pearce, G., Strydom, D., Johnson, S & Ryan, C.A (1991) A polypeptide from tomato leaves induces wound-inducible protei-nase inhibitor proteins Science 253, 895–898.
35 Matsubayashi, Y & Sakagami, Y (1996) Phytosulfokine, sulfated peptides that induce the proliferation of single mesophyll cells of Asparagus officinalis L Proc Natl Acad Sci USA 93, 7623–7627.
36 Matsubayashi, Y., Ogawa, M., Morita, A & Sakagami, Y (2002)
An LRR receptor kinase involved in perception of a peptide plant hormone, phytosulfokine Science 296, 1470–1472.
37 Pearce, G., Moura, D.S., Stratmann, J & Ryan, C.A (2001) RALF, a 5-kDa ubiquitous polypeptide in plants, arrests root growth and development Proc Natl Acad Sci USA 98, 12843– 12847.
38 Fletcher, J.C., Brand, U., Running, M.P., Simon, R & Meyero-witz, E.M (1999) Signaling of cell fate decisions by CLAVATA3
in Arabidopsis shoot meristems Science 283, 1911–1914.