Characterization of a high-affinity binding site for the pea albumin 1b
entomotoxin in the weevil
Sitophilus
Fre
´
de
´
ric Gressent, Isabelle Rahioui and Yvan Rahbe
´
UMR 0203 INRA/INSA de Lyon, BF2I (Biologie Fonctionnelle, Insectes et Interactions), INSA Ba
ˆ
timent Louis Pasteur,
Villeurbanne, France
The toxicity of the pea albumin 1b (PA1b), a 37 amino-acid
peptide extracted from pea seeds, for cereal weevils (Sito-
philus oryzae, Sitophilus granarius and Sitophilus zeamais)
was recently discovered. The mechanism of action of this
new entomotoxin is still unknown and potentially involves a
target protein in the insect tissues. This work describes the
characterization of a high-affinity binding site for PA1b in
a microsomal fraction of Sitophilus spp. extracts. Purified
PA1b was labeled to a high specific radioactivity
(c. 900 CiÆmmol
)1
)using
125
I, and the iodinated ligand was
found to be biologically active. Binding of this ligand to the
microsomal fraction of S. oryzae extract was found to be
saturable and reversible, with an affinity (K
d

different insect species. The Bacillus thuringiensis toxins [1],
proteins belonging to the lectin family [2], or enzymes
inhibitors [3], are in use or being tested, but to date these
molecules are not adapted to grain protection against
weevils.
Therefore, the recent finding of a plant peptide lethal for
these insects has enlarged the possibilities for cereal grain
protection [4]. This peptide was purified and sequenced
from Pisum sativum seeds and, being previously known as a
seed albumin [5], it was named PA1b for pea albumin 1b.
PA1b is the result of the post-translational cleavage of the
albumin proprotein PA1, also releasing a second peptide
(PA1a). PA1b consists of 37 amino acids, with six cysteines
involved in three disulfide bonds which confer the toxin its
high stability. Biological activity is conserved when boiled,
and the peptide is resistant to digestion by trypsin [6] or by
bovine rumen fluid [7].
Although no primary sequence homologies could be
found with other proteins in the available databases, it
was recently established by NMR studies and molecular
modeling that PA1b belongs to the cystine-knot family [8].
Characterized only recently as a structural family [9],
cystine-knot peptides have now been identified from a
variety of sources (plants, fungi, animal venoms, insects),
and show very diverse biological activities (elicitor AVR9,
trypsin inhibitor, antimicrobial, antiviral or antifungal
activities, and many channel blockers [10]). A subfamily of
these, the cyclic cystine-knot peptides, also named cyclo-
tides, were investigated for their potential as new anti-
biotics or anti-HIV properties. PA1b was the first

sensitivity to the toxin, since resistant strains do exist
naturally: the screening of up to 90 Sitophilus spp. strains for
the susceptibility to PA1b has revealed that three strains, all
belonging to the S. oryzae species, contained individuals
able to feed on pea seeds [12] and resistant to the purified
toxin. Other pest insects (the pea aphid Acyrthosiphon pisum
and the flour moth Ephestia kuehniella) were found to be
susceptible to the toxin, but the fruit fly Drosophila melano-
gaster was shown to be resistant [4]. Within the S. oryzae
species, a genetic analysis of the resistance demonstrated
that this trait was driven by a single recessive autosomal
gene [12]. This result suggested that a single weevil gene
product could be responsible for the susceptibility or
resistance towards the toxin. On this basis, the mechanism
of action of the peptide was likely to involve a discrete
molecular target at the insect side. Therefore our work
focused on the characterization of a PA1b binding site in
Sitophilus spp. extracts, using a
125
I labeled 3741 Da isoform
of the toxin (peptide sequence in Fig. 1 [4]). Given the
biological features presented here, this binding site could
potentially be regarded as the molecular target of the PA1b
toxicity in weevils.
Experimental procedures
Biological material
Cereal weevils (S. oryzae, zeamais and granarius,
Coleoptera, Curculionidae) were reared on wheat seeds at
27.5 °C 70% relative humidity. Nine strains were used,
differing in their genetic ability to thrive on pea seeds and

2
;10l
M
E-64), and extracted six times for 5 s
using an UltraTurrax blender. The slurry was centrifuged
for 10 min at 3000 g. The supernatant was further centri-
fuged to give a second pellet at 10 000 g (10 min) and a
third one at 45 000 g for 45 min (microsomal fraction). The
resulting fractions, sedimenting at 3000, 10 000 or 45 000 g,
were resuspended in binding buffer (20 m
M
Tris buffer
pH 7.0 containing 10 l
M
E-64) with 30% glycerol and
stored at )80 °C until use.
Purification of the toxin
One batch of purified toxin isoform, showing a molecular
mass of 3741 Da by mass spectrometry, and a pea albumin
extract (named SRA1) were provided by J. Gueguen and
E. Ferrasson (Laboratoire de Biochimie et Technologie des
Prote
´
ines, Nantes, France). From SRA1, a mixture of PA1b
isoforms was obtained by a 45-min incubation at )20 °Cin
acetone/water (80 : 20) followed by a 10-min centrifugation
at 12 000 g. The supernatant was dried under vacuum, and
more than 95% of the resulting powder consisted of a
mixture of PA1b isoforms (as checked by HPLC analysis).
PA1b or modified PA1b were purified or analyzed by

I-labelled PA1b ligand on labeling
day ranged from 890 to 1120 CiÆmmol
)1
. The labeled toxin
was stored in 60% methanol at )20 °C and used within a
month after labeling.
For iodination of the peptide using nonlabeled iodine, the
protocol was as described above, except that 200 lgofthe
3741 Da isoform, 0.3 mg of nonlabeled NaI and four times
50 lg of chloramine-T were used in a 100-lL volume.
Reduction and alkylation of the toxin
Reduction of the 3741 Da toxin (2 mgÆmL
)1
) was carried
out in Tris/HCl buffer (pH 8, 0.1
M
) in the presence of
Fig. 1. Peptide sequence [4] and disulfide bridges [8] of the 3741 Da
PA1b isoform.
2430 F. Gressent et al. (Eur. J. Biochem. 270) Ó FEBS 2003
20 m
M
dithiothreitol. The mixture was boiled for 10 min,
allowing a complete reduction of the peptide. After cooling
at room temperature, one volume of 0.2
M
iodoacetamide
was added and the mixture was incubated in the dark for
1 h. Alkylation of the cysteine residues was quantitative,
and the alkylated peptide was then purified by reverse-phase

Results
The iodinated toxin displays a high specific
radioactivity and is biologically active
The 3741 Da isoform of PA1b contains a single tyrosine
residue in position 31. Assays of iodination were performed
using IodoBeads and chloramine-T. The yield of
125
I
incorporated on the toxin was rather low (less than 5%)
with IodoBeads, while a 40–50% incorporation was
achieved using chloramine-T. The need for a high specific
radioactivity for binding studies, and the fact that native
PA1b and labeled PA1b cannot be separated by HPLC, led
us to use stoichiometric amount of toxin and iodine. Under
these conditions, the specific radioactivity of the
125
I-labelled
PA1b, calculated by the ratio of the radioactivity measured
by gamma counting and the amount of peptide evaluated by
absorbance at 210 nm during HPLC analysis, was about
942 CiÆmmol
)1
. This value is similar to the theoretical value
of 890 CiÆmmol
)1
for a 40%
125
I incorporation (40% of
the 2220 CiÆmmol
)1125

125
I-labelled PA1b ligand.
The specific binding activity was the highest in the 45 000 g
fraction (microsomal fraction), which presented an enrich-
ment of threefold on a protein basis. However, in terms of
total activities, the binding was found with similar values in
the 10 000 and 45 000 g fractions. The nonspecific binding
component was about 20% of the total binding activity in
all three fractions. The specific binding value increases with
the increase of microsomal proteins, until it reaches a
plateau for 25–30% of the radioligand bound (about
10–15 lg of microsomal proteins, Fig. 2). The microsomal
Table 1. Binding activities of 1.1 n
M
125
I-labelled PA1b to particulate
fractions prepared by differential centrifugation of S. oryzae WAA42
extracts (5 lg of proteins).
Particulate fraction
3000 g 10 000 g 45 000 g
Total protein (mg) 18.1 12.4 6.9
Total binding activity (c.p.m. · 10
6
) 24.5 45.2 49.9
Non-specific binding activity
(% of total binding)
22.3 18.9 18.3
Specific binding activity
(c.p.m.Ælg protein
)1

were preincubated at 40 °C and was practically undetect-
able at 60 °C (respectively 95 and 7% as compared to the
control, data not shown).
The binding of the
S. oryzae
microsomal fraction
to
125
I-labelled PA1b is reversible, saturable,
and displays a high affinity
Binding of the
125
I-labelled PA1b to the microsomal fraction
proceeded in a time-dependent manner and an apparent
equilibrium was reached after a 90-min incubation at room
temperature. Addition of excess unlabeled ligand after
equilibrium had been reached led to the loss of specifically
bound radioligand, and this displacement was complete
within 60 min (Fig. 3). The rate constants for association
(k
on
) was 2.03 · 10
5
M
)1
Æs
)1
, and for dissociation (k
off
)was

(Fig. 4). The Hill number was 0.98, suggesting the presence
of a single class of binding site. Moreover, a competition
experiment using the homologous nonradioactive
127
I
2
-
labelled PA1b as competitor confirmed the results obtained
by the saturation experiment on the 45 000 g fraction
(K
i
¼ 2.8 n
M
, B
max
¼ 39 pmol per mg of protein).
A single PA1b binding site is detectable in membrane
fractions of the
S. oryzae
extract, and is specific
for the native peptide
Competition experiments were performed on the three
subfractions of S. oryzae extracts using the 3741 Da PA1b
as the competitor. The results showed that the native
peptide displayed the same affinity as the iodinated toxin on
the 45 000 g fraction (K
i
¼ 2.2 n
M
; B

Binding activity (%)
Control
b
100
Proteinase K (0.2 lg) 24
Proteinase K (2 lg) 7
Denaturated proteinase K (0.2 lg)
c
107
Proteinase K (0.2 lg) + antiproteases
d
104
a
Microsomal fraction aliquots (2 lg of proteins) were incubated
with effectors for 10 min at 37 °C before measuring the binding
activity using 0.4 n
M
of
125
I-labelled PA1b.
b
Incubation in binding
buffer without antiproteases only.
c
Protease was denaturated by
boiling for 10 min.
d
Phenylmethylsulfonyl fluoride (1 m
M
),4l

susceptible or resistant weevil strains (data not shown).
The binding activity correlates with the susceptibility
or resistance of the
Sitophilus
spp. strains
The specific binding activity of the microsomal fraction of
different weevil strains, of the pea aphid A. pisum and of the
fruit fly D. melanogaster was determined. Figure 5 shows
that the five susceptible weevil strains tested (S. oryzae
WAA42, Be
´
nin and Bouriz, S. granarius Brayard and
S. zeamais LS) displayed a high specific binding activity,
between 1700 and 2200 c.p.m. per lgofprotein.By
contrast, no binding activity could be detected when
incubating the
125
I-labelled PA1b with the microsomal
fraction proteins of extracts of the four strains of S. oryzae
resistant to the toxin (China, Mex1, ISOR3 and GV), even
when increasing 25-fold the amount of microsomal proteins.
The pea aphid (susceptible to the toxin) was able to bind the
125
I-labelled PA1b, to a lesser extent than the susceptible
weevil strains. Surprisingly, the microsomal fraction of an
extract of the resistant insect D. melanogaster also displayed
a strong binding capacity.
The determination of the characteristics of the binding
site of these different strains or species is presented in
Table 3. The binding activities of extracts from the five

experiments. Following incubation, the radioactivity was
dissociated from the binding site and recovered at more
than 98% by a 60% methanol extraction. The HPLC
analysis of this fraction revealed a single radioactive peak at
a retention time corresponding to PA1b (data not shown).
Thus, the radioligand was not degraded during incubation
with extracts from either the susceptible or the resistant
strain.
Discussion
The recent finding of the insecticidal properties of the
PA1b peptide(s) [4] has opened new possibilities for cereal
grain protection against weevils. However, the occurrence
of naturally resistant strains of S. oryzae is an important
problem, and could limit the usefulness of this peptide for
plant protection. Although the mechanism of action of the
toxin is still totally unknown, genetical analysis of the
resistance within the S. oryzae species, implicating a single
recessive gene [12], suggested that the toxicity might involve
a specific receptor on the insect side. On this basis, the
search for a PA1b binding protein may be the first step
towards the cloning of a potential receptor in susceptible
insects.
In this work, we characterized the binding of PA1b to a
proteinaceous component of a particulate fraction of
S. oryzae extracts. The binding was saturable and reversible,
and the binding site exhibits a high affinity for the native
3741 Da toxin (K
d
¼ 2.4 n
M

S. granarius Brayard 6.3 ± 1.6 40
S. zeamais LS 9 ± 1.6 34
A. pisum 58 ± 10 40
D. melanogaster 16 ± 3.3 40
Fig. 5. Specific binding of the
125
I-labelled PA1b ligand on the micro-
somal fraction extracts from different insect species or strains. Bars
indicate specific binding of the
125
I-labelled PA1b (0.4 n
M
)(±SEM)
to 2 lg proteins from the microsomal fraction of S. oryzae strain
WAA42 (1), Be
´
nin (2), Bouriz (3), GV (4), Mex1 (6), ISOR3 (8) and
China (10), S. zeamais LS (12), S. granarius Brayard (13), A. pisum
(14) and D. melanogaster (15). Bars labeled 5, 7, 9 and 11 correspond to
specific binding displayed by 50 lg of microsomal proteins extracted
from the resistant S. oryzae strains GV, Mex1, ISOR3 and China,
respectively.
Ó FEBS 2003 A binding site for the PA1b entomotoxin (Eur. J. Biochem. 270) 2433
binding implicated only one toxin molecule per binding
site). PA1b seems to bind to a single protein, as only one
binding site is detectable in the microsomal fraction, and the
binding activity in the 3000 and 10 000 g fractions is
probably due to the same protein as the binding in the three
subfractions displayed the same characteristics. In terms of
ligand specificity, we presently do not have available site

the four S. oryzae resistant strains, including ISOR3, a
three generation backcross isoline to WAA42 [12]. This
result strongly suggests that the high-affinity binding site
is the molecular target of the peptide in Sitophilus,and
that it may play a major role in the toxicity process. As
the labeled ligand was not degraded during the binding
experiment, the absence of binding activity on the
resistant strains demonstrates that the resistance mechan-
ism within the Sitophilus genus involves either a modifi-
cation or the absence of the molecular target. Indeed, the
absence of binding in resistant strains could be due to
the absence of the target protein, or to a mutation within
the site of the binding to PA1b. Actually, target mutation
resistance is widely used by insects to resist a wide range
of toxins, and was reported for different insecticide
targets, as for the GABA receptor, the sodium channels
or the acetylcholinesterases [14].
The presence and density of the binding site in all insect
species tested, and among three insect orders (e.g.
Coleoptera, Diptera and Hemiptera) suggests that this
protein is widely represented and conserved in insects, and
that PA1b could share a similar mechanism of action in
all susceptible species. However, the presence of the
binding site in the resistant species D. melanogaster,with
characteristics roughly similar to those of the Sitophilus
susceptible strains, suggests that a different mechanism of
insensitivity exists in this species. This mechanism seems
not to implicate the binding site, and could take place
either upstream or downstream of the perception step. We
could for example speculate that certain insects are able to

trypsin, a-amylase and carboxypeptidase) [18]. PA1b is of
plant source, but all inhibitory assays conducted until now
has failed to detect any effect on enzymatic activities,
including papain, trypsin and chymotrypsin-like activities
[4]. Although PA1b could have some low similarity with
BBI peptides, the absence of consensus residues in the active
site results in probable lack of any antiprotease activity [5].
Then, the purification of the binding site, and the cloning of
the corresponding gene in susceptible and resistant strains
will probably help to answer these questions, and will
provide us with the molecular tools to monitor the outburst
of resistant populations, and eventually to engineer a pea-
related toxin into a peptide active on both resistant and
susceptible weevils.
Acknowledgements
We thank Francesca Cardinale for carefully reviewing this manuscript.
We thank J. Gueguen and E. Ferrasson for providing us the 3741 Da
toxin isoform and the PA1b isoform mixture.
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