Eur. J. Biochem. 239, 67-73 (1996)
0 FEBS 1996
Primary structure of co-hordothionin, a member of a novel family
of thionins from barley endosperm, and its inhibition of protein synthesis
in eukaryotic and prokaryotic cell-free systems
Enrique MENDEZ’
’, Asuncidn ROCHER
I,
Miguel CALERO I , Tomas GIRBES ’, Lucia CITORES’ and Fernando SORIANO’
’ Unidad de Aniilisis Estructural de Proteinas, Centro Nacional de Biotecnologia, Campus Universidad Authoma, Madrid, Spain
’ Servicio de Endocrinologia, Hospital Ramcin y Cajal, Madrid, Spain
Departamento de Bioquimica y Biologia Molecular, Facultad de Ciencias, Universidad de Valladolid, Spain
(Received 8 January/l6 February 1996) - EJB 96 0025/3
A new sulfur-rich basic polypeptide, so called ru-hordothionin, has been isolated from barley endosperm by extractions with NaCl and ammonium bicarbonate followed by reverse-phase high performance
liquid chromatography. Purified o-hordothionin was found to be homogeneous by SDS/polyacrylamide
gel electrophoresis, N-terminal amino-acid sequencing and electrospray-ionization mass spectrometric
analysis. The complete primary structure of cu-hordothionin was determined by automatic degradation of
the intact molecule and peptides obtained by proteolytic cleavage. cu-hordothionin consists of a single
polypeptide chain of 48 amino acids with a molecular mass of 5508 Da deduced from its amino acid
sequence, which fully coincides with the 5508.2 Da determined by electrospray-ionization mass spectrometry. The isolated polypeptide showed a characteristic composition with a high content of basic amino
acids (five arginine residues, two lysine residues and six histidine residues) and eight cysteine residues,
and has strong sequence identity (66 %) with the sorghum SIcrl a-amylase inhibitor. cu-hordothionin,
like y-hordothionin, exhibited translation inhibitory activity on both eukaryotic cell-free systems from
mammalian (rat liver and rabbit reticulocyte lysates) and prokaryotic cell-free systems (Escherichia coli).
However, in contrast to y-hordothionin, w-hordothionin did not inhibit plant systems such as Triticurn
aestivunz, Cucumis sativus, Vicia sativa and Hordeum vulgare. ;i-hordothionin also inhibited the a-amylase
activity from human saliva, while w-hordothionin and the other different genetic variants of thionins, ahordothionin and [j-hordothionin, failed to show any inhibitory effect.
Keywords: w-hordothionin; y-thionin; barley; a-amylase inhibitor.
Thionins are basic and cysteine-rich low-molecular-mass
polypeptides of about 5 kDa, prevalently found in the endosperm
of several Gramineae such as wheat and barley (aand /? purothionins and hordothionins), as well as in a variety of plant species, including leaves and stems (e.g. viscotoxin, pyrularia). Although orally innocuous, the thionins are toxic to injected animals [ l , 21, some yeast strains [3, 41, bacterial [3, 51, animal
and plant cells [6, 71, fungi [8, 91 and insect larvae [lo]. They
modify membrane permeability in cultured mammalian cells
[ I l l and inhibit in vitro protein synthesis in cell-free systems
derived from wheat germ, rabbit reticulocytes and Artemiu [ 121.
Despite the fact that thionins are all believed to play a role in the
defense system of plants against pathogens, the role and precise
functional relationship between these polypeptides is still unclear.
Recently, we described a new family of thionins, named 1’thionins of about 5 kDa, found in the endosperm of wheat [13]
and barley 1121. The three-dimensional structure of two members of the y-thionin family, y,-hordothionin (y,-H) and y,-puroCorrespondence to E. MCndez, Unidad de Aniilisis Estructurdl de
Proteinas, Centro Nacional de Biotecnologia, Campua Universidad Autitnoma, Cantoblanco, E-28049 Madrid, Spain
Abbrevinrions. RS-AFP, plant antifungal protein from radish; FST.
flower-specific thionin ; IC,,,, protein concentration required for 50% cell
growth inhibition; STa, sorghum a-amylase inhibitor; ATA, aurintricarboxylic acid.
Enzymes. Chymotrypsin (EC 32.4.21.1); pepsin (EC 3.4.23.1).
thionin (y,-P) from barley and wheat has been established [14]
using two-dimensional NMR spectroscopy. These structures,
which differ considerably from those obtained for the related
cysteine-rich polypeptides a,-purothionin [I51 and crambin [16],
show a great analogy with scorpion toxins and insect defensins
[17]. Although these y-thionins possess inhibitory activity in vitro against protein synthesis in different cell-free systems 1121,
their mode of action is unknown. Several structurally related
polypeptides exhibiting sequence identity, including the three
sorghum STal, 2 and 3 a-amylase inhibitors [18], two antifungal
plant antifungal proteins from radish (RS-AFPI) and two polypeptides from radish [19], a new family of antifungal RS-AFPlike polypeptides from Brassicaceae [20] and the flower-specific
thionin (FST) from tobacco cDNA [21], have been also described.
In this paper, we report the complete amino acid sequence of
a new basic and cysteine-rich polypeptide isolated from barley
endosperm, which we have called co-hordothionin, exhibiting a
high degree of sequence identity (66%) with the sorghum S2nl
cr-amylase inhibitor.
The in vitro inhibitory activities of a-,/?-,y- and oj-hordothionins againts a-amylase and protein synthesis are compared.
MATERIALS AND METHODS
Barley seeds (Hordeum vulgare) were used in this study to
purify cu-thionin. Wheat germ (Trificum aestivunz), cucumber
68
Mtndez et al. ( E m J. Biochem. 239)
(Cucumis sativus) and Wciu sativa seeds purchased from a local
store were bleach sterilized, imbibed in sterile tap water, and
incubated on moist filter paper for 3-5 days in the dark.
Protein extraction and o-thionin purification. Barley endosperm was obtained by hand dissection. The flour was extracted with NaCl and the NaC1-soluble proteins were removed
from the salt and lyophilized as described [12, 131. The dried
NaC1-soluble protein extract was fractionated with SO mM ammonium bicarbonate by stirring overnight at room temperature
[13]. After centrifugation at 17000Xg for 10 min, the pellet was
lyophilized. Aliqucits of the bicarbonate-insoluble extract were
dissolved i n 0.1 % trifluoroacetic acid and loaded directly onto
a reverse-phase HF'LC column. Separation was performed with
a semi-preparative Nucleosil C, silica column (particle size,
10 pm; pore size 30 nm; 16 mmX2S0 mm) fitted with a guard
column (16 mm X 30 mm) packed with the same support. The
column was eluted with an acetonitrile gradient of 10-20%
containing 0.1 % trifluoroacetic acid and operated at room temperature at a flow rate of 5.0 ml/min. The purity of w-thionin
was analyzed by SDS/PAGE, amino acid analysis and amino
acid sequencing. The individual genetic variants a-,p- and yhordothionins were. obtained from H. vulgare seeds by reversephase HPLC as described [12, 131.
Reduction and alkylation. Native w-hordothionin was reduced and S-carboxymethylated as described [12, 131.
Proteolytic cleavage. The S-carboxymethylated w-hordothionin was digesl-ed with chymotrypsin in 0.2 M N-methylmorpholine, pH 8.5, at a protein/enzyme ratio of 100: 1 for 4 h
at 37 "C. Pepsin digestion of S-carboxymethylated w-hordothionin was performed in 5 % formic acid for 18 h at 37°C at the
same protein/enzyrne ratio as described above. In every case, at
the end of the digestion the peptide mixture was adjusted to
0.1 c/o trifluoroacetic acid and loaded directly onto a reversephase HPLC column.
Peptide fractionation by reverse-phase HPLC. Peptides
were fractionated Iby reverse-phase HPLC on a Novapack C,,
column (particle size 4 pm, 3.9 mmX1SO mm) protected by a
guard column pack.ed with pBondapak C,,/Corasil. The column
was eluted at room temperature at a flow rate of 0.5 ml/min with
a gradient of acetonitrile containing 0.1 % trifluoroacetic acid.
The effluent absorbance was monitored at 220 nm.
Amino acid analysis. The hydrolysis of purified w-hordothionin and peptides derived from the enzymic digestions were
carried out as described [13] in a Beckman 3600 amino acid
analyzer. Performic acid oxidation was carried out as described
1131.
Sequencing procedure. Automatic amino acid sequence
analysis was carried out on a Knauer modular liquid-phase sequencer model 819 equipped on line with a Knauer phenylthiohydantoin amino acid analyzer as described [12].
a-amylase assay. a-amylase from human saliva, porcine
pancreas, Aspergill'us oryzae, Bacillus licheniforinis, barley malt
and a-amylase inhibitor from human saliva and porcine pancreas, were purchased from Sigma Chem. Co. The activity of
every a-amylase was measured as described [22] with the
following modifications: 0.1, 0.2, 0.6 and 1 U each enzyme in
a final volume of 20 p1 were added to 230 p1 assay buffer
(45 pM NaCl, 0.801 pM CaC12, 0.4 mM sodium phosphate,
pH 7.0) containing, 1 % starch. After S min incubation at room
temperature, the reaction was arrested by the addition of 750 pl
dinitrosalicylate reagent, kept in a boiling water bath for 10 min,
then cooled to room temperature. The amylolytic activity was
calculated as maltose equivalents liberated.
To measure the amylase inhibitory activity, a suitable
amount of a-, /I-, and o-hordothionins, purified as described
above, or a-amylase inhibitor as a control, were incubated for
;J-
30 min at room temperature with 0.5 U enzyme in a final volume of SO pl assay buffer. The enzyme reaction was initiated by
the addition of starch solution and the assay was processed as
described above. We used the decrease in enzyme activity as a
measure of the inhibitory activity.
Electrospray-ionization mass spectrometry. The samples
were analyzed on a Finnigan SSQ 710 C, an instrument that
essentially consists of an atmospheric pressure electrospray positive-ion source, attached to a triple-quadrupole mass analyzer.
The lyophilized w-hordothionin was dissolved in 50: SO acetonitrile/water containing 0.1 % trifluoroacetic acid and introduced
via loop injection to the electrospray interface through a PLRP-S
Milchrom Bioresources (0.5 mmXSO mm, 40000 nm, 8 pm)
column equilibrated in the same solvent. Masdcharge values
were acquired by scanning m/z 1000-2000. The spectra (positive-ion mode) were transformed in order to obtain the molecular size of the w-hordothionin on a mass scale. Mass-scale calibration was performed with horse myoglobulin.
One-dimensional electrophoresis. Slab-gel SDSPAGE was
carried out as described [13, 231.
Preparation of cell-free translation systems. All cell-free
translation systems were obtained under RNase-free conditions
at 0-2°C as follows. Rabbit reticulocyte lysates were prepared
as described elsewhere 1241. In the case of rat liver, a standard
procedure was used 1251. Briefly, the liver was perfused for 30 s
with homogenization buffer (1.50 mM KC1, 3 mM magnesium
diacetate, S mM dithiothreitol and 20 mM Tris/HCl, pH 7.8). After homogenization with Dounce for 5 min at a ratio of 1.25 ml
homogenization buffedg tissue, the paste was centrifuged first
at 7S0Xg for S min to remove cellular debris and unbroken tissue, and thereafter at 3OOOOXg for 15 min. The supernatant
(S-30) was used for translation assays. In the case of plants,
3-5 g 3-5-day embryonic axes were dissected by hand and
processed exactly as described elsewhere [26, 281; the S-30 supernatants were also used. E. coli MRE600 endogenous polysomes were isolated and purified as described previously 1281.
The E. coli MRE600 lOOOOOXg supernatant (S-100) was obtained and precipitated with streptomycin sulfate and ammonium
sulfate as reported 1281. All S-30 supernatants were filtered
through Sephadex G-25 to remove low molecular-mass compounds, stored at -90°C until use and thawed once.
Assays of cell-free translation. In vitro translation was carried out in cell-free translation systems obtained from mammalians, plants and bacteria by current procedures, as follows.
Translation by rat liver was carried out at 37°C for 60 min in a
total volume of 2.5 p1 containing 8 mM MgCl,, SO mM KCl,
20 mM Tris/HCl, pH 7.8, 100 mM NH,Cl, S mM dithiothreitol,
2 mM ATP, 1 mM GTP, 0.2 mM CTP, 2 mM phosphoenolpyruvate, 40 pg/ml pyruvate kinase, 0.1 mM standard amino-acids,
except for methionine; 6.5 nM ~-["S]methionine (specific activity 1112 Ci/mmol); 111 pg S-30 supernatant. Translations by
plant systems (7:aestivum, H. vulgare, K sativa, C. sativus were
carried out at 30°C for 30 min in a total volume of SO p1 containing 9.8 mM Mg(CH,),; 30 mM KCI, 28 mM Tris/HCI
(pH 7.8), 28 mM NH,Cl, S mM dithiothreitol, 4 mM ATP, 1 mM
GTP, 8 mM creatine phosphate, 72 pg/ml creatine kinase,
400 pg/ml wheat germ tRNA mixture (200 pg/ml in the case of
C. sativcis), 0.1 mM of all standard amino acids except for Lvaline, 68 nM ~-['H]valine (specific activity 65.1 Ci/mmol) ;
58 pg S-30 supernatant. Translation by the Escherichia coli system was carried out at 37°C for IS min in a total volume of
SO p1 containing 10 mM Mg(CH,)?, SO mM Tris/HCl, pH 7.8,
80 mM NH,Cl, 1 mM dithiothreitol, 1 mM ATP, 0.02 mM GTP,
0.02 mM CTP, 5 mM phosphoenolpyruvate, 30 pg/ml pyruvate
kinase, 100 pg/ml E. coli tRNA mixture, 0.05 mM of all standard amino acids except for L-valine, 68 nM ~-['H]valine (spe-
MCndez et al. (EuI: J . Biochem. 239)
230 nrn (--)
% Acetonitrile ( - - -)
1 .o
W
V
z
Q
30
0.5
0
v)
m
4:
10
0
0
50
TIME
100
150
(min)
Fig. 1. Fractionation by reverse-phase HPLC of the bicarbonate extract from the salt-soluble protein fraction from barley endospenn.
A solution of 8.0 mg total protein in 5.0 ml 10% acetonitrile, 0.1 % trifluoroacetic acid was injected onto a Nucleosil C, column and eluted
with a gradient of acetonitrile as indicated.
cific activity 65.1 Ci/mmol), 58 pg S-100 supernatant, 43.8 pg
E. coli polysomes. Translation by rabbit reticulocyte lysates was
carried out at 30°C for 20 min in a total volume of 50 pl containing 1.5 mM MgCl,, 50 mM KC1, 20 mM Tris/HCI, pH 7.8,
5 mM dithiothreitol, 1 mM ATP, 0.2 mM GTP, 20 mM creatine
phosphate, 40 pg/ml creatine kinase, 0.04 mM of all standard
amino acids except for L-valine, 68 nM L-[’H]valine (specific
activity 65.1 Ci/ininol), 20 pM hemine, 850 mg S-30 supernatant.
The reactions were started by the addition of the supernatants to the other mixed components and, after incubation, the
reactions were stopped by addition of 0.5 mlO.1 M KOH to each
sample. After 10 min, 0.5 ml 20% (madvol.) trichloroacetic
acid was added, and the precipitates were collected by filtration
on glass-fiber filters (Whatmann GF/A). Radioactivity was measured by scintillation counting using Ready Safe as scintillation
cocktail.
RESULTS
Purification of w-hordothionin. The total NaCI-soluble extract
protein mixture from barley kernel flour was fractionated by extraction with a diluted ammonium bicarbonate solution as indicated in the Materials and Methods. Aliquots of 8.0-40.0 mg
protein from the bicarbonate-insoluble extract were fractionated
by reverse-phase HPLC (Fig. 1). w-hordothionin was found in a
peak with a retention time around 130 min. The purity was examined by SDYPAGE (data not shown), N-terminal sequencing
and mass spectrometry. Amino-acid analysis of the purified whordothionin showed a characteristic composition with a high
content in basic amino acids (five arginine residues, two lysine
residues and six histidine residues), eight residues of cysteines
and the absence of methionine.
Complete amino acid sequence of w-hordothionin. The strategy applied in the sequence determination of w-hordothionin by
automatic degradation of the intact molecule and peptides obtained by enzymic cleavage with chymotrypsin and pepsin is
shown (Fig. 2). The N-terminal sequence analysis of native whordothionin was determined as far as 34 residues with gaps at
positions 3, 13, 20 and 24. The S-carboxymethylated o-hordothionin was digested with chymotrypsin and pepsin, and the resulting peptides were separated by reverse-phase HPLC. The sequences of four chymotryptic peptides Ch-I -4 and two pepsin
(P) peptides P-1 and P-2 (Fig. 3) allowed the identification of
69
all amino acid residues and were used as a second probe in order
to identify residues and provide some overlapping (Fig. 2). The
sequential degradation of carboxymethylated P-2 allowed the
confirmation of the N-terminal half of the molecule up to residue
34, and confirmed gaps at positions 3, 13, 20 and 24 as cysteine
residues, while P-I confirmed the C-terminal region at residues
37-48. Supporting data and overlapping were achieved by degradation of the chymotryptic peptides Ch-I -4. w-hordothionin
consists of 48 amino acids with a calculated molecular mass of
5508 Da.
No free sulphydryl groups could be found by titration with
4-vinylpyridine in the absence of dithiothreitol (data not shown),
thus suggesting the presence of four disulfide bridges in oJ-hordothionin.
Mass spectrometry analysis. Electrospray-ionization mass
spectrometric analysis of purified w-hordothionin yielded high
quality spectra (Fig. 4). m/z values were acquired by scanning
m/z 1000-2000. Ions at m/z 1102.7, 1378.1 and 1837.1 (Fig. 4,
top) correspond to the molecule attached to five, four and three
protons, respectively. The spectra (positive-ion mode) from the
three ions ranging from 5 ’ (m/z 1102.7) to 3’ (mlz 1837.1) were
transformed to give the molecular size of the w-hordothionin on
a mass scale (Fig. 4, bottom). The molecular size determined by
mass spectrometry, 5508.2 Da, (Fig. 4) fully coincides with that
of 5508 Da calculated from the amino-acid sequence of the purified w-hordothionin (Fig. 2).
Effects of w-hordothionin and y-hordothionin on translation.
Despite the fact that the biological role of thionins in the plant
producer is unknown, they are believed to inhibit protein synthesis in a variety of translation systems. In order to better understand the action mechanism of w-hordothionin, we assayed its
effect on protein synthesis in several cell-free systems derived
from mammalian (rat liver), bacteria (E. coli), monocotyledonous (7: aestivum, H. vulgare) and dicotyledonous plants ( K sativa and c. sativa). For comparison, y-hordothionin was also
tested in the same systems as a control, since it is known to
inhibit translation in cell-free systems derived from mammalians
and non-mammalians at several levels.
y-hordothionin strongly inhibited protein synthesis in the
eukaryotic systems from a mammalian [rat liver; protein concentration required for 50% cell growth inhibition (IC,,,) 127 pg/
ml] and from four plants (7: aestivum, IC,,, 162 pg/ml; C. sativus, IC,,, 140 pg/ml; V sativa, IC,,, 126 pg/ml; H. vulgare, IC,,,
> 400 pg/ml). In the bacterial system, the inhibition (IC,,,
400 pg/ml) was lower than in the other systems except for the
system for H. vulgare.
Since the cell-free translation systems used in this work except for the rabbit reticulocytes lysates, lack initiation of new
polypeptide chains, the inhibitory effect seems to be exerted on
the elongation step of polypeptide synthesis. Our eukaryotic
translation system, rabbit reticulocyte lysates and wheat germ
were not treated with S1 nuclease. Therefore, they were not
completely dependent on initiation since some ribosomes retained attached pieces of broken mRNA of variable length. In
all cases, an important fraction of the protein synthesis activity,
accounting for 60% in H. vulgare and 18-27% in the other
eukaryotic systems, remained refractory to inhibition. However,
surprisingly, in the bacterial system, almost the same extent of
inhibition was achieved in the 50-400-pg/ml range for both
hordothionins.
In contrast to y-hordothionin, co-hordothionin did not exert
any inhibition in plant systems (Fig. 5), while in rar liver, rabbit
reticulocytes and bacteria, it inhibited translation with nearly the
same efficiency (rat liver, IC,, 179 pg/ml; rabbit reticulocyte ly-
Mtndez et al. ( E m J. Biochem. 239)
70
Fig. 2. Alignment of peptides used for determining the amino acid sequence of o-hordothionin. Results from automatic sequencing of native
(Na) w-hordothionin '(w-H), pepsin (P) and chymotryptic (Ch) peptides from S-carboxymethylatedw-hordothionin. Open boxes indicate unidentified
residues.
Ch-4
lool
A
......
w
,
% Acetonitrile ( - . - -
220 nm (-)
0.2
40
_.'
0.1
80
20
V
Q:
137L
40
0.05
20
I
0
50
TIME
(rnin)
150
Fig. 3. Fractionation by reverse-phase HPLC of chymotryptic (Ch)
and pepsin (P) peptides from the S-carboxymethylated co-hordothionin. Chymotryptic (8.0nmol) and pepsin (5.0 nmol) peptide digests were
chromatographed on a reverse-phase C,, column and eluted with acetonitrile as indicated. The flow rilte was 0.5 mllmin.
-1
W
cc
sates, lC,,, 300 mg,'ml; E. cnli, IC,,, 375 pg/ml). This behaviour
of thionin with respect to the bacterial system is described for
the first time and clearly points out important differences between plant systems and other systems.
To ascertain whether w-thionin affected the step of initiation
of new polypeptide chains, a time course of protein synthesis
was carried out using rabbit reticulocyte lysates able to carry on
initiation of new polypeptide chains either in the absence or the
presence of aurintricarboxylic acid (ATA), an inhibitor of the
initiation step [30]; the thionin inhibited protein synthesis despite the presence of ATA (Fig. 6). The kinetics indicate that the
thionin in this cell-free system inhibited both the initiation and
the elongation steps.
Effects of cu-hordothionin and y-hordothionin in the inhibition of n-amylase activity. In order to reveal whether the purified m-hordothionin as well as the previously described a-, [jand y-hordothionins, presented any a-amylase inhibitory activity,
several u-amylases from mammalians (pancreatic and salivary),
bacterial and fungi were tested. While y-hordothionin exhibited
an inhibitory effecl. of40% on human saliva a-amylase (Fig. 6),
which is appi-oxiinxtcly 50% of the effect exerted by the specific
inhibitor used as ii control, w-hordothionin, along with a-hordothionin and p-hordothionin, failed to show any inhibition. All
purified hordothioriins were negative as inhibitors of a-amylase
from porcine pancreas, fungi and bacteria (data not shown).
*O
60
40
20
Wz)
Fig. 4. Electrospray-ionization mass spectrum of w-hordothionin.
High resolution of inultiply charged molecular ions (top). The ions at nil
z 1102.7, 1378.1 and 1837.1 correspond to the molecule with 5, 4 and
3 protons attached, respectively. Mass determination from the three ions
ranging from 5 ' (ndz 1102.7) to 3' (mlz 1837.1) yields a molecular mass
of 5508.2 Da (bottom).
DISCUSSION
The complete covalent structure of a new barley thionin,
called w-hordothionin (Fig. 2), is based essentially on the characterization and alignment of fragments obtained by enzymic
cleavage with chymotrypsin and pepsin, as well as from the Nterminal amino-acid sequence of the native protein (Fig. 2). The
polypeptide chain consists of 48 amino acids with a calculated
71
Mende7 et al. (ELII:J . Biochrm. 23Y)
I
I
I
I
I
I
I
I
I
2
I
TOXIN (rng/ml)
Fig. 5. Effect of w-hordothionin and y-hordothionin on polypeptide
synthesis in several cell-free translation systems coded by endogenous messengers. The experiments were carried out as indicated in the
Materials and Methods. ( 0 )Rat liver system (control polymerization
59000 dpm . mg-' protein); (*) rabbit reticulocyte lysates (control polymerization 16000 dpm . mg ' protein); A, E. coli system (control polymerization 36000 dpm mg-' protein); A, H. vulgure system (control
polymerization 35 000 dpm mg-' protein); 0 , 1/: sutiva (control polymerization 94000 dpm . mg-' protein); 7: aestivum (control polymerization 67000 dpm ' mg-' protein); A, C. suriuus (control polymerization
170000 dpm mg-' protein).
+,
w-H
5
10
INHIBITOR / ENZYME
Y
+ATA
I
TIME (rnin.)
Fig. 6. Effect of co-hordothionin (w-H) on polypeptide synthesis in
rabbit reticulocyte lysates either in the absence (left) or in the presence (right) of ATA. The experiments were carried out as indicated in
Materials and Methods. Reaction mixtures of 50 ml containing 0.94 mg
lysate protein were incubated at 37°C and, at the indicated times, aliquots of 10 in1 were taken and proccessed for the radioactivity incorporated into protein. ATA was used at 10 p o l a r . (O),(W) Control without
m-hordothionin; (O),(0)in the presence of 290 pg/ml o-hordothionin
molecular mass of 5508 Da, which is in good agreement with
that determined by mass spectrometric analysis (Fig. 4). w-hordothionin contains eight cysteine residues and has a high content
(27%) of basic amino acids (two lysine residues, five arginine
residues and six histidine residues). Comparison with sequences
in a data bank revealed that w-hordothionin exhibits a strong
sequence identity (66%) with the sorghum S l a l a-amylase inhibitor (Fig. 7). The amino acid sequence of w-hordothionin also
shows a close resemblance to the earlier described barley and
wheat ?I-H and yl-P thionins and the sorghum SIa2 and SIa3 aamylase inhibitors, although exhibits several peculiar structural
characteristics distinguishing it from these thionins such as : whordothionin presents only 34-36% identity with the pthionins
and the two cx-amylase inhibitors ; o-hordothionin displays a
high content of histidine residues located at positions 9, 10, 11,
28, 31 and 38, while y-thionins and the a-amylase inhibitors
have no histidine residues and, indeed, o-hordothionin contains
no methionine residues ; in w-hordothionin, the distribution of
seven of the eight cysteine residues is identical to that in 7'-
Fig.7. Comparison of the inhibitory effect of hordothionins to human saliva a-amylase. a-H (*), P H (+), y-H (A),w-H ( 0 )and namylase inhibitor (W).
thionine, except for the cysteine residue at position 37 (position
34 in y-thionin). In contrast, w-hordothionin also displays a certain degree of similarity with RS-AFPI, polypeptides from radish, RS-AFP-like polypeptide and FST from tobacco cDNA
(Fig. 7). These structurally related polypeptides probably display
the same distribution of disulfide bridges, but are notably different from a-type and j3-type thionins.
On the basis of these differences, the presently described
barley thionin represents a new family of thionins which we
have tentatively called w-hordothionin, differing from the y type.
Thus, we have organized the thionins into several groups.
Group I would comprise the classical a-puro and Bpuro and
hordothionins. These polypeptides contain 45 -46 amino-acid
residues and four disulfide bridges ( m y s 3 9 , m y s 3 1 ,
m c y s 2 9 and w y s 2 5 ) , symmetrically distributed.
This group could also include the related toxic crambrin and
pyrularia (not included in Fig. 7) and the hydrophobic viscotoxin
(52 % identity with a-hordothionin and /l-hordothionin and purothionins; Fig. 7). Group I1 would contain the newly described
w-hordothionin and the S1a1 a-amylase inhibitor which has 48
amino-acid residues (65 9% identity); they present a high content
of histidine residues (six residues in w-hordothionin and three
residues in S l a l ) and share three disulfide bridges with group
111, while in the fourth disulfide bridge the C y s l 4 linked to
Cys37 in the w-liordothionin instead to Cys34 as for 7-thionins.
Group 111 would comprise y-purothionins and hordothionins)
and the sorghum S1a3/2 a-amylase inhibitors, which display
85% identity and share four disulfide bridges ( m y s 4 7 ,
m y s 3 4 , -ys41
and -ys43)
[14]. Finally,
group IV is represented by FST [21], the 10-kDa peptide [31],
the potato p322 polypeptide [32] and RS-AFPI [19].
Groups 111 and IV present 16 conserved amino acids, including eight cysteine residues, 12 of which are also present at
equivalent positions in group 11 (Fig. 7). The organization of disulfide bridges for most of the polypeptides in groups ll-IV has
not been established yet, although it is likely to be very similar
to that in y-thionins 1141 or w-thionins [33] (Fig. 7) according
to the conservative backbone of the eight cysteine residues along
their linear polypeptide chains. In contrast, polypeptides from
group I present a different, more symmetrical disulfide-bridge
organization (Fig. 7).
The existence of common structural characteristics between
these groups of thionins or related thionins could explain certain
similarities at the functional level. Both oJ-hordothionin and yhordothionin exhibited translational inhibitory activity on an
eukaryotic and on a prokaryotic cell-free system. The most sensitive system to both thionins was that derived from rat liver and
the rabbit reticulocytes lysates. In contrast, both thionins inhib-
72
Mtndez et al. ( E m J. Biochem. 23Y)
AFPl
10 kDa
P 322
FST
Y-Y-H
511x3
I
CYS-31
cys-3
cys-39
Fig. 8. Amino acid sequence similarity between w-hordothionin and related thionins. Only the thionins a1 -P, the sorghum SIal and 3, the 7 H; FST. AFP1, P322, the 10-kDa peptide (10-kDa germination-relatedprotein from cowpea) and V-A3 have been included in the figure. Common
sequences in groups are indicated in dashed boxes and conserved amino acids are indicated by shaded boxes. Gaps are included to achieve maximal
sequence identity. Sequence and cysteine residues numbering are as in 7-H, w-H and ul-P.
ited the bacterial system to the same extent. Nonetheless, they
must possess some kind of structural difference that makes yhordothionin very active on plant systems and o-hordothionin
inactive on such systems. The reason for this difference could
be the amino-acid sequence or, more likely, the spatial folding.
Since the concentration of y-hordothionin required to inhibit
the cell-free translation systems, including the homologous systems, is relatively high, the nature of the effect might be stoichiometric. Additionally, ous results suggest that, at least in the
rabbit retictilocyte lysates, the w-hordothionin, like y-hordothionin [IZj, acted on both the initiation and the elongation steps of
protein synthesis.
The strong sequence similarity between the a-amylase iiihibitor SInl and o-hordothionin (Fig. 7) agrees with their low inhibitory effect on human saliva a-amylase. Thus, cu-hordothionin
displays no inhibitory effect on human saliva a-amylase (Fig. 6),
while SInl has a very faint inhibitory effect [18]. Furthermore,
no inhibitory effect on porcine pancreas a-amylase of both ohordothionin (data not shown) and SIal thionin [18] was detected. In contrast, y-hordothionin clearly inhibited the human
saliva a-amylase, while a-hordothionin and P-hordothionin, as
well as w-hordothionin, also failed to show any inhibitory effect.
In accordance with these data, ;I-hordothionin is, to our knowledge, the first mernber of the y-thionin family described from
barley endosperm displaying a-amylase inhibitory activity.
The different functional effects observed between w-hordothionin and y-hosdothionin can be explained not only on the
basis of their primary structures (Fig. 7), but also by their threedimensional structure motifs. In fact, w-hordothionin, unlike yhordothionin, has been shown to exist in aqueous solution as a
mixture of two dil'fesent proline ci.s-tvuns, as revealed by the
analysis of its two-dimensional NMR spectra 1331. Both cu-hor-
dothionin and 11-hordothionin share common structural motifs
including the cysteine-stabilized a-helix [14]. The three-dimensional structural similarity of this new w-hordothionin [33] with
ythionins [14], as well as with some scorpion toxins and insect
defensins [17], suggests a common mode of functional activity.
Nevertheless, more experimental data are needed in order to determine the precise action mode of thionins.
This work was supported by grants from Comisio'n Inferministerial
de Ciencia y Tecnologiu BI094-0025 and BI095-0610. We are indebted
to Mr Emilio Camafeita and Ms Shirley Macgraph for the revision of
the manuscript.
REFERENCES
1. Coulson, E. J., Harris, T. H. & Axelrod. B. (1942). Effect on small
laboratory animals of the injection of the crystalline hydrochloride
of a sulfur protein from wheat flour, Cereal Chem. 19, 301 -307.
2. Samuelsson, G. (1974) Mistletoe toxins, Sjist. Zool. 22, 566-569.
3. Stuart, L. S. & Harris, T. H. (1942) Bactericidal and fungicidal prop
erties of a crystalline protein isolated from unbleached wheat
flour, Cereal Clzem. 19, 288-300.
4. Hernandez-Lucas, C., Carbonero, P. & Garcia-Olmedo, F. (1974)
Inhibition of brewer's yeast by wheat purothionins, Appl. Microhiol. 28, 165-168.
5. Ferndndez de Caleya, R.. Gonzalez-Pascual, B.. Garcia-Olmedo,
F. & Carbanero, P. (1972) Susceptibility of phytopathogenic bacteria to wheat purothionins in vitro, Appl. Microhiol. 23, 9981000.
6. Evans, J., Wang, Y., Shaw, K. P. & Vernon, L. P. (1989) Cellular
responses to Pyrularia thionin are mediated by Ca'+ influx and
phospholipase A2 activation and are inhibited by thionin tyrosine
iodination, Proc. Nut/ Acad. Sci. USA 86, 5849-5853.
MCndez et al. (Eur: J. Biochem. 239)
7. Reimann-Philipp, U., Schrader, G., Martinoia, E., Barkholt, V. &
Apel, K. (1989) Intracellular thionins of barley. A second group
of leaf thionins closely related to but distinct from cell wall-bound
thionins, J. Bid. Chem. 264, 8978-8984.
8. Bohlmann, H., Clausen, S., Behnke, S., Giese, H., Hiller, C., Reimann-Philipp, U., Schrader, G., Barkholt, V. & Apel, K. (1988)
Leaf specifics thionins of barley - a novel class of cell wall
proteins toxic to plant-pathogenic fungi and possibly involved in
the defense mechanism of plants, EMBO J . 7, 1559-1565.
9. Ebraim-Nesbat, F., Behnke, S., Kleinhofs, A. & Apel, K. (1989)
Cultivar-related differences in the distribution of cell-wall-bound
thionins in compatible and incompatible interactions between barley and powdery mildew, Planta (Berl.) 179, 203-210.
10. Kramer, K. J., Klassen, L. W., Jones, B. L., Speirs, R. D. & Kammer,
A. E. (1979) Toxicity of purothionin and its homologues to the
tobacco hornworm, Manduca sextu (L.j (Lepidoptera: Sphingidae), Toxicol. Appl. Pharmucol. 48, 179-183.
11. Carrasco, L., Vizquez, D., Hemindez-Lucas, C., Carbonero, P. &
Garcia-Olmedo, F. (1981) Thionins : plant peptides that modify
membrane permeability in cultured mammalian cells, Eur: J.
Biochem. 116, 185-189.
12. Mtndez, E., Moreno, A., Colilla, F. J., Peliez, F., Limas, G. G.,
Mendez, R., Soriano, F., Salinas, M. & de Haro, C. (1990) Primary structure and inhibition of protein synthesis in eukaryotic
cell-free system of a novel thionin, y-hordothionin, from barley
endosperm, ELK J . Biochem. 194, 533-539.
23. Colilla, F. J., Rocher, A. & Mtndez, E. (1990) y-purothionins: amino
acid sequence of two polypeptides of a new family of thionins
from wheat endosperm, FEBS Lett. 270, 191-194.
14. Bruix, M., Jimtnez, M. A,, Santoro, J., Gonzrilez, C., Colilla, F. J.,
Mtndez, E. & Rico, M. (1993) Solution structure of yl-H and ylP thionins from barley and wheat endosperm determined by 'HNMR: a structural motif common to toxic arthropod proteins,
Biochemistry 32, 715-724.
15. Clore, G. M., Sukumaran, D. K., Gronenborn, A. M., Teeter, M. M.,
Whitlow, M. & Jones, B. L. (1987) Nuclear magnetic resonance
study of the solution structure of a-1 -purothinonin. Sequential resonance assignment, secondary structure and low resolution tertiary structure, J. Mol. Bid. 193, 571 -578.
16. Hendrickson, W. A. & Teeter, M. M. (1981) Structure of the hydrophobic protein crambin determined directly from the anomalous scattering of sulphur, Nufitre 290, 107- 113.
17. Martins, J. C., Zhang, W., Tartar, A,, Lazdunski, M. & Borremans,
E A. M. (1990) Solution conformation of Leiurotoxin I (Scyllatoxin) by 'H nuclear magnetic resonance, FEBS Lett. 260, 249253.
18. Bloch, C. & Richardson, M. (1991) A new family of small ( 5 kDa)
protein inhibitors of insect a-amylases from seeds or sorghum
(Sorghum bicolor (L) Moench) have sequence homologies with
wheat ypurothionins, FEBS Lett. 279, 101-- 104.
19. Terras, F. R. G., Schoofs, H. M. E., De Bolle, M. F. C., Van Leuven,
F., Rees, S. B., Vanderleyden, J., Cammue, B. P. A. & Broekaert,
W. F. (1992) Analysis of two novel classes of plant antifungal
proteins from radish (Raphunms sativus L.) seeds, J. Bid. Chem.
67, 15301 15 309.
-
73
20. Terras, F. R. G., Torrekens, S . , Van Leuven, F., Osborn, R. W., Vanderleyden, J., Cammue, B. P. A. & Broekaert, W. F. (1993) A
new family of basic cysteine-rich plant antifungal proteins from
Brassicaceae species, FEBS Lett. 316. 233 -240.
21. Gu, Q.. Kawata, E. E., Morse, M.-J., Wu, H.-M. & Cheung, A. Y.
(1992) A flower-specific cDNA encoding a novel thionin in tobacco, Mol. Gen. Genet. 234, 89-96.
22. Rocher, A., Colilla, F., Ortiz, M. L. & MCndez, E. (1992) Identification of the three major coeliac immunoreactive proteins and one
a-amylase inhibitor from oat endosperm, FEBS Lett. 310, 37-40.
23. Laemmli, U. K. (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4, Nature 227,680-685.
24. GirbCs, T., Citores, L., Iglesias, R., Ferreras, J. M., Mufioz, R., Rojo,
M. A., Arias, F. J., Garcia, J. R., MCndez, E. & Calonge, M .
(1993) Ebulin 1, a nontoxic novel type 2 ribosome-inactivating
protein from Sambucus ebulus L. leaves, J , B i d . Chenz. 268,
I 8 195-18 199.
25. Martin, A,, Perez, J., Ayuso, M. S. & Parrilla, R. (1979) On the
mechanism of the glucagon-induced inhibition of hepatic protein
synthesis, Arch. Biochem. Biophys. 195, 223 -234.
26. Arias, F. J., Rojo, M. A,, Ferreras, J. M., Iglesias, R., Mutioz, R.,
Rocher, A., MCndez, E., Barbieri, L. & Girbts, T. (1992) Isolation
and partial characterization of a new ribosome-inactivating protein from Petrocoptis glaucifolia (lag.) Boiss, Planfa (Bed.) 186,
532-540.
27. Arias, F. J., Rojo, M. A,, Ferreras, J. M., Iglesias, R., Mufioz, R. &
Girbes, T. (1992) Preparation and optimization of a cell-free
translation system from Vicia sativa germ lacking ribosome-inactivating protein activity, J. Exp. Bof. 43, 729-737.
28. GirbCs, T., Cabrer, B. & Modolell, J. (1979) Preparation and assay
of purified Escherichia coli polysomes devoid of free ribosomal
subunits and endogenous GTPase activities, Methods Etzzymnl.
59, 353-362.
29. Rojo, M. A,, Arias, F. J., Iglesias, R., Ferreras, J. M., Mufioz, R. &
Girbts, T. (1993) Cucumis sativits cell-free translation system :
preparation, optimization and sensitivity to some antibiotics and
ribosome-inactivating proteins, Phys. Plant. 88, 549-5S6.
30. Pestka, S. (1977) Inhibitors of proteins synthesis in Molecular mechunims of pmfein synthesis (Weissbach, H. & Pestka, S., eds)
pp. 468-553, Academic Press, New York.
31. Ishibashi, N., Yamauchi, D. & Minamikawa, T. (1990) Stored
mRNA in cotyledons of Vigna Ltnguiculatu seeds: nucleotide sequence of cloned cDNA for a stored mRNA and induction of its
synthesis by precocious germination, Plant Mol. Biol. 15, 5964.
32. Stiekema, W. J., Heidekamp, F., Dirkse, W. G., van Beckum, J., de
Haan, P., ten Bosch, C. & Louwerse, J. D. (1988) Molecular cloning and analysis of four potato tuber mRNAs, Plant. Mol. Bid.
11, 255 -269.
33. Bruix, M., Gonzalez, C., Santoro, J., SoriAno, F., Rocher, A., Mtndez, E. & Rico, M. (1995) H-NMR studies on the structure of a
new thionin from barley endosperm, Biopolymer 36, 751 -763.