(Received for publication, January 31, 1995; and in revised form, May 22, 1995)
From the
To examine the binding of Bacillus thuringiensis -endotoxins, CryIAa, CryIAb, and CryIAc, to Lymantria
dispar (gypsy moth) brush border membrane vesicles (BBMV),
saturation kinetic analyses were conducted according to a two-step
interaction scheme
The order of toxicity of the
-endotoxins, as measured by the dose required for a 50% inhibition
of weight gain (ID
), was CryIAa (77.3 ng) > CryIAb (157
ng) > CryIAc (187 ng). While both the maximum extent of binding, B
, and the half-maximum insertion rate
concentration, K
, was observed to be indirectly
related to toxicity, the rate constant of irreversible binding, k
, was found to be directly correlated to
toxicity.
Bacillus thuringiensis synthesizes insecticidal
crystalline inclusions containing various -endotoxins during
sporulation. Due to the high specificity of the
-endotoxins
against target insects and its low persistence, B. thuringiensis has been an environmentally sound alternative in pest control for
decades. The mode of action of the
-endotoxin has now been
elucidated in considerable detail by extensive studies in
histopathology and biochemistry. Upon ingestion, the crystalline
inclusions of
-endotoxins are solubilized by the high pH
environment of the midgut of the insect. The solubilized protoxins are
converted to toxin by proteinases in midgut lumen. The toxins then bind
to specific binding proteins in the microvilli of columnar cells and
insert into the membrane of the microvilli. Pore formation by the
toxins in the membrane causes the leakage of ions and probably other
contents of the cell and finally lyses the
cell(1, 2) .
Binding of toxin to the microvillar
membrane is one of the most intensively studied steps in its mode of
action. An in vitro system with brush border membrane vesicles
(BBMV) ()established by Wolfersberger et al.(3) has made it possible to study binding at the molecular
level. Hofmann et al.(4) were the first to identify
specific binding sites of toxins in the BBMV of susceptible insects by
binding assays. Now the binding assay has become a standard tool in
studies of mechanism of specificity of wild type toxins and their
mutants(5, 6, 7, 8, 9, 10, 11, 12, 13) ,
as well as the mechanism of resistance development in
insects(14, 15, 16) .
Gypsy moth (Lymantria dispar) is a major forest pest in the United
States, as well as in other parts of the world. Members of the CryIA
class of B. thuringiensis -endotoxins show different
toxicities against gypsy moth. Interestingly, an inverse relationship
between toxicities against gypsy moth larvae and binding properties to
BBMV from the larvae has been observed with CryIAb and
CryIAc(13) . While CryIAb, a stronger toxin, showed lower
affinity in the binding assay, CryIAc, a weaker toxin, showed higher
affinity(13) . As with other published binding studies, the
binding properties of the toxins in this assay was analyzed by modified
Scatchard equations(17) .
So far, all published binding studies known to us have used the Scatchard equation or the Hill equation(18) , which analyzes binding parameters assuming a one-step reversible interaction ().
Here BS is the binding site on the membrane, T is the toxin, and BS*T is the dissociable complex formed by binding site and toxin. However, a large body of evidence indicates that the binding between toxin and BBMV quickly becomes irreversible(4, 6, 7, 19, 20) . Irreversibility of the toxin-BBMV interaction was observed in early studies (19) and has been considered briefly in most binding studies mentioned above. The irreversible binding might reflect insertion of toxin into brush border membrane(19, 20) . A two-step interaction would describe toxin-BBMV interaction better than .
Here BS-T is the toxin irreversibly bound to the membrane.
The observation of irreversible binding should immediately disqualify the application of the Scatchard equation or the Hill equation in analysis of binding parameters of toxin-BBMV interaction(21, 22) . However, the effect of irreversible interaction on the binding parameters has not been included in the calculations of binding parameters(4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) , even in a recent paper in which irreversibility of the toxin-BBMV interaction has been proposed as a determinant in toxicity of two toxins, CryIA(a) and CryIA(b), against Bombyx mori(20) .
The purpose of this study is to apply saturation kinetics of irreversible binding, rather than competition binding assays (4) to study binding parameters based on and use this method to explore the relationship between the toxicity of CryIA toxins against gypsy moth larvae and their binding properties to BBMV from the larvae.
For the time course of specific binding, 400 µg/ml BBMV was incubated with 0.1 nM or 10 nM labeled toxin in the presence or absence of 1,000-fold excess of unlabeled toxin for various times before centrifugation. The specific binding of the toxins to the BBMV was calculated by subtracting the nonspecific binding (the binding with excess of unlabeled toxins) from the total binding (the binding without unlabeled toxins).
For the time course of irreversible binding, 400 µg/ml BBMV was incubated with 0.1 or 10 nM labeled toxin for various times, after which 1 ml of binding buffer with at least 1,000-fold excess of cold toxin was added to the tube. The incubation was continued for 1 h before centrifugation. The nonspecific binding was determined from the tubes in which labeled toxin and 1,000-fold excess of unlabeled toxin were added to BBMV at the same time.
For the saturation assay of specific binding, various concentrations of BBMV were incubated with 0.1 nM labeled toxin in the presence or absence of 1,000-fold excess of unlabeled toxin for 3 h before centrifugation. The specific binding of toxins to BBMV was calculated by subtracting the nonspecific binding (the binding with excess of unlabeled toxins) from the total binding (the binding without addition of unlabeled toxins).
For the saturation assay of irreversible binding, the saturation assay of specific binding was performed allowing 3 h of incubation. Subsequently, binding buffer (1 ml) with or without 1000 fold excess of unlabeled toxin was added to the specific binding assay tubes and nonspecific binding assay tubes, respectively. Incubation was continued for another hour before centrifugation.
For
the saturation kinetics assay, irreversible binding time courses were
measured with 0.1 nM toxin and various concentrations of BBMV. k from these time courses were plotted against
concentration of binding sites in BBMV.
Here x is the amount of BBMV, and f is fitted
to the amount of irreversibly bound toxin; B is
equal to the slope of the curve, which is the maximum binding of given
amount of BBMV; a is the interception of the curve on the y axis, which is close to zero.
Here x is the time, f is fitted to the
concentration of bound toxin, and [T] is total specific
binding or total irreversible binding; k is the
observed first-order rate constant; a is the correction
factor, which is close to zero.
Here x is the concentration of the binding site; f is fitted to the observed first-order rate constant, k; k
is the irreversible
binding rate constant; K
is the concentration of
the binding sites giving half-maximum insertion rate of irreversible
binding.
Figure 1:
Time course of specific binding and
irreversible binding of CryIA toxins to L. dispar BBMV. a and d, CryIAa; b and e, CryIAb; c and f, CryIAc. a-c, 0.1 nM toxin
to 400 µg/ml BBMV; d-f, 10 nM toxin to 400
µg/ml BBMV. Opencircle, specific binding; opendiamond, irreversible binding; filleddiamond, reversible binding. Standard errors are
represented by errorbars. Single exponential fits
for irreversible binding curves in d-f give 0.06, 0.05,
and 0.04 min for rate constants, respectively. Two
exponential fits for specific binding curves in d-f give
1.4, 1.1, and 0.7 min
for the fast phase,
respectively, and 0.06, 0.05, and 0.04 min
for the
slow phase, respectively.
These data also indicate that
at low concentrations of toxin, the rate of irreversible binding (k = 0.03 min
for
CryIAa) is comparable with the rate of specific binding (k
= 0.03 min
for
CryIAa). Accumulation of reversible complex could not be detected by
chasing with cold toxins. Thus, initial binding is the rate-determining
step in the overall binding process when concentration of toxin is low.
At high concentrations of toxin, the kinetics of specific binding are
biphasic and fit to a fast rate constant of 1.4 min
and a slower rate constant of 0.06 min
(for
CryIAa). The kinetics of irreversible binding at high toxin
concentration, however, fit to a single exponential with a rate
constant of 0.06 min
(for CryIAa), which is the same
as the rate of the slower phase during specific binding. These kinetics
indicate that the reversible complex accumulates with a bimolecular
rate constant k
of about 1.4
10
M
min
(k
= k
/[T]) when toxin is in excess. Under
these condition, the irreversible binding is limited by the rate of
reversible complex changing to the irreversible complex.
Figure 2: Saturation binding of CryIA toxins to L. dispar BBMV. a, CryIAa; b, CryIAb; c, CryIAc. Opencircle, specific binding; opendiamond, irreversible binding. Standard errors are represented by errorbars. 0.1 nM labeled toxins were incubated with increasing concentration of BBMV.
The saturation
binding curves (Fig. 2) had two characteristics: a sharp
inflection point at saturation and linear portions of the curve both
before and after the inflection point. The same characteristic curve
can be found in most of previously reported saturation binding results (7, 8, 11, 15) . The stoichiometric
nature of the irreversible binding curve indicates that the slope of
the first straight line in the curve is equal to the irreversible B. Since specific binding consists of
irreversible and reversible binding components, the stoichiometric
nature of the specific binding curve indicates that the reversible
binding is tight under the conditions of the assay. The slope of the
first line in the specific binding would be close to specific B
. The B
calculated from
CryIAa, CryIAb, and CryIAc saturation curves (Fig. 2) are shown
in Table 2. A significant difference between specific B
and irreversible B
is
observed only in CryIAb. Since the toxicity of Cry toxins is assumed to
depend on their ability to insert into the membrane, the irreversible B
would be of primary concern. So
``B
'' in the later part of this paper
refers to the irreversible B
.
where k is the bimolecular association rate
constant for BS*T complex, k
is the
dissociation rate constant for BS*T complex, and k
is the rate constant for irreversible binding.
If binding of
toxin to BBMV is a two-step process as shown above, then the rate of
irreversible complex formation, k, is related to
BS concentration by the following
relationship(27) ,
which predicts that the plot of kversus [BS] should be saturable. The maximum
rate will be equal to k
and the BS concentration
at half the maximum rate is the K
, which is
equal to (k
+ k
)/k
. It represents
the stability of the reversible binding complex, when k
is comparable to k
, or k
k
. It is equal to K
, the dissociation constant of the reversible
binding complex, when the k
k
. On the other hand, if binding of toxin
was a one-step process, as shown in , then the rate, k
would increase linearly with BS concentration.
Saturable k was observed in time course of
irreversible binding assays of all three CryIA toxins with increasing
concentration of binding site, as shown in Fig. 3. CryIAa
appeared to have a saturated k
at a higher
concentration of binding site than CryIAb and CryIAc (Fig. 3).
The reaction constants, k
and K
, calculated from Fig. 3for all three
toxins are listed in Table 1.
Figure 3:
Saturation kinetics in the binding of
CryIA toxins to L. dispar BBMV. a, CryIAa; b, CryIAb; c, CryIAc. k of
irreversible binding of 0.1 nM labeled toxin to increasing
concentration of binding site was plotted against concentration of
binding site. Standard errors are represented by errorbars.
Channel or pore formation in the epithelium plasma membrane of the insect larvae midgut is believed to be critical to the mechanism of insecticidal activity of B. thuringiensis toxins. The first two steps in the channel formation process for an activated toxin are binding to the membrane and insertion into the membrane(1, 2) . Thus, characterization of these events is essential for a detailed understanding of the overall channel formation process. Specific binding sites for B. thuringiensis toxins have been successfully identified in many insect species by binding assays(4) . However, the role of binding studies now becomes more important and more complicated when the focus shifts to cloning and characterization of binding proteins (receptors) and extensive mutagenesis in all three domains of the toxins. For example, it may be necessary to determine whether a particular mutation affects only the initial binding step or the irreversible binding step.
Here
we propose a two-step kinetic model for interaction between B.
thuringiensis toxin and BBMV, as shown in , based on
the following observations. 1) A large body of evidence, including
results presented in this paper, has shown that the binding of B.
thuringiensis toxin to BBMV is mainly
irreversible(4, 6, 7, 19, 20) ;
2) accumulation of reversible binding complex at early times of
incubation (Fig. 1); 3) k of irreversible
binding was saturable at high concentration of BBMV (Fig. 2).
Neither a one-step reversible binding model nor a one-step irreversible
binding model could explain these observations.
This paper utilizes saturation kinetics to analyze binding data and to derive binding parameters based on the two-step interaction model between the toxins and BBMV. The method used by this paper enables us for the first time to study separately the first two steps in channel formation, initial binding and irreversible binding, by analyzing the affinity of the initial binding and the rate constant of irreversible binding.
We
observe that specific binding of toxin to BBMV involves two types of
binding sites: a site that could develop into an irreversible stage
(irreversible binding site) and a site that remains reversible
(reversible binding site) ( Fig. 1and Fig. 2, Table 2). Thus, it is important to compare the irreversible B values of different toxins rather than the
specific B
values, since irreversible binding is
thought to reflect insertion and, consequently, is more directly
related to toxicities.
To simplify the analysis of saturation kinetics, the reversible binding sites have not been considered in . Despite extensive studies, there are also many uncertain steps in the channel formation process. For example, it is not clear whether insertion is facilitated by the binding proteins or whether the binding proteins are recyclable after the toxins insert. Thus the should be considered as a minimum reasonable mechanism, which accounts for the experimental results.
Among the three wild type CryIA toxins, CryIAb and CryIAc show a similar binding affinity, which is 3 times higher than that of CryIAa (Table 1). However, CryIAa has a faster irreversible binding rate. The order of the irreversible binding rate constant of the three toxins is CryIAa>CryIAb>CryIAc, which is directly correlated to the order of toxicity of the three toxins (Table 1).
CryIAb has more
irreversible binding sites than CryIAc and almost twice of that of
CryIAa (Table 2). Several reports have shown that CryIAb toxin
and CryIAa toxin bind to a same binding protein in Western blots with
BBMV proteins from various species of
insects(28, 29) , including L. dispar. ()It would be tempting to speculate that the large
CryIAa-CryIAb-binding protein, which is 210 kDa as a monomer,
could have twice the amount of binding sites for CryIAb relative
to CryIAa.
Toxicity of a toxin can be represented by its efficacy and potency. Efficacy is determined by the number of binding sites for the toxin in the target larva. When sufficient toxins are administered to the larvae, maximum growth inhibition could be observed in all three toxins (data not shown). This indicates that a specific number of CryIAa or CryIAc binding sites is sufficient to induce a maximum efficacy in growth inhibition and an increase in the amount of binding sites of CryIAb would not increase its efficacy.
The potency of a
toxin is believed to depend on the mechanism of its action. For
example, if the effect of a drug is produced through the reversible
binding between the drug and its receptor, the affinity of binding
usually determines the potency of the drug. We have demonstrated that
the potency of the CryIA toxins is directly related to their insertion
rate constant, k. Quicker insertion brings a
stronger potency to a toxin. It would not be too difficult to imagine
that L. dispar larvae could have a self-defense mechanism when
it is attacked by CryIA toxins. For example, aminopeptidase N,
identified as CryIAc-binding protein in some insect
species(30, 31) , is attached to the plasma membrane
of the epithelium cell by a glycosyl phosphatidylinositol
anchor(32) . Conformational change of the aminopeptidase N upon
binding of the toxin could lead it to be susceptible to some membrane
bound phospholipases and cleaved from the membrane. Consequently, the
toxin could lose its activity in channel formation because of the
cleavage of the aminopeptidase N. Therefore, quicker insertion of a
toxin would leave less time for phospholipases to cleave its binding
protein, thus leading to a stronger potency.
It is worth noting that although the parameters derived from the Scatchard equation would be misleading, the data obtained from the competition assays, the most popular assay in previous literature, are still informative if the assay is conducted in an appropriate way. The competition assay could be represented by the following reaction ().
Here C is the competitor, BS*C is the reversible complex formed
between competitor and binding site, and BS-C is the irreversible
complex. Assume that the irreversible binding sites are predominant to
the reversible binding sites, and the incubation time is long enough
for all binding complexes at irreversible binding sites to become
irreversible. When the amount of binding sites is not greater than that
of labeled toxin, the decrease in the radioactivity detected with the
BBMV would be determined by the competition between rate of BS-T
formation and rate of BS-C formation. The rate of the irreversible
complex formation depends on both the concentration of reversible
complex and the rate constant for irreversible binding, k, for each toxin, rate = k
[BS*T]. The [BS*T]
depends on the stability of the reversible complex, K
(or K
). Thus, the competition assay reflects
the combination of the effect of k
and K
(or K
) in the whole
integration process of the toxin to membrane. A different competition
binding curve indicates a difference in the whole integration process.
However, it could not differentiate whether the difference comes from
the binding affinity (K
, or K
) or from the irreversible binding rate constant (k
). On the other hand, if there is no difference
between the competition binding curves, one may not assume that the two
toxins have the same binding affinity and the same irreversible binding
rate constant. A higher affinity might be offset by a smaller
irreversible binding rate constant. Indeed, Chen et al.(33) have identified such a mutant, CryIAa A92E. Keeping
this in mind, the competition assay is still a useful method to screen
mutants with different binding properties since it is a much simpler
assay than the method presented in this paper.
To quantitatively
represent the difference in competition binding curves, the
concentration of the competitor that causes 50% inhibition in specific
binding of labeled toxin (IC), would be useful in
comparison. However, the physiological meaning of the IC
should not be considered same as the K
in
the Scatchard equation; instead it reflects the combination of K
(or K
) and k
, as discussed above.
A second binding assay, the dissociation assay, has been more frequently used since researchers have became aware of the role of irreversible step in the determination of toxicity. The percentage of dissociation, or the amount of toxin that is chased off from specifically bound toxins, has been compared for different toxins(20) . However, because of the possible existence of a reversible binding site, the percentage of the dissociation is not very informative in understanding the mechanism of toxicity. To compare irreversible binding of two different toxins, a time course of irreversible binding as shown in this paper (Fig. 1) would be more informative than the dissociation binding assay. Not only would the absolute level of the irreversible binding be detected, the rate of irreversible binding would also be viewed through the time course of irreversible binding.
In this report we find a
direct relationship between toxicity of the CryIA toxins and the
irreversible binding rate constant, k, to L.
dispar BBMV. This indicates that this step, which we assume is
primarily the insertion of toxin into the apical membrane, is an
important step in determining the activity of a toxin. This observation
apparently solves the paradox posed by the paper of Wolfersberger (13) and is consistent with the observations of Wolfersberger (34) that inhibition of K
gradient-driven
amino acid transport by
-endotoxins CryIAb and CryIAc on L.
dispar BBMV is directly related to their larvacidal activity. This
of course does not exclude the importance of other events in
determining toxicity. Further investigation of post-insertion events,
such as oligomerization of inserted toxins in the membrane, will be
needed to elucidate the mechanism of channel formation of the toxin in
the membrane.