Department of Biological Sciences, Purdue
University, West Lafayette, Indiana 47907
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INTRODUCTION |
Bacillus thuringiensis,
primarily during sporulation, produces large intracellular inclusions
which are comprised of a mixture of protoxins (
-endotoxins) (1,
12, 29). Following ingestion by insect larvae, the inclusions are
solubilized, and the protoxins are converted to toxins of about 60 kDa.
Initially, the toxins bind reversibly to receptors on the surfaces of
larval midgut cells. At least two such receptors have been identified
(15, 28, 31), and there may even be more than one toxin
binding site on one of these, aminopeptidase N (25). There
is some correlation between the specificity of this binding and
toxicity (32, 33), but there are exceptions (14, 24,
35).
Subsequently, the toxin inserts into the membrane in an irreversible
step which, at least in some cases, appears to be more specific
(14, 20). The factors involved in this toxin insertion step
are not known. A receptor complex purified from Manduca
sexta brush border membrane vesicles (BBMV) (including the
aminopeptidase N receptor) formed functional ion channels (28,
30), as did a receptor complex from Heliothis
virescens (21). These ion channels are presumably
formed in the membrane by an oligomerization of toxin monomers (9,
16), but the nature of this process (i.e., whether it occurs at
the membrane surface or within the membrane, the number of toxin
monomers involved, and whether there are any interactions with membrane
components) is not known.
There is a report that the cytolytic protein (CytA) produced by
Bacillus thuringiensis subsp. israelensis
oligomerizes in membrane vesicles (5). This protein is
structurally and functionally very different from the -endotoxins
(11, 18, 19). -Endotoxins are processed from the amino
ends of the protoxins and are comprised of three structural domains
(11, 18). Domain I consists of seven amphipathic 
-helices
and is believed to be the portion of the toxin which inserts into the
membrane to form the ion channel. Evidence for this contention is based
on the high frequency of losses of toxicity due to mutations in certain
helices, especially the very hydrophobic 4-loop-5 region
(17, 29, 37). There are also studies of the binding of
synthetic peptides to these helices which show that only helices 4
and 5 insert into the membrane, while the other helices appear to be
localized at the membrane surface (7-10). The kinetics of
binding of peptide helix 5 indicates cooperative interactions,
suggesting oligomerization (7-10). A mutation in the 5
synthetic peptide, which is known to reduce toxicity, also resulted in
a decrease of binding to and insertion into the membrane (7,
8). In addition, the 5 peptide formed ion channels in
phospholipid vesicles (8). Domains II and III of the
-endotoxins are comprised of 
sheets, and selected regions,
especially certain loops, are important in toxin binding and
specificity (29).
While these studies are very helpful for defining the functional
regions of these toxins, little is known about the steps between the
initial, reversible binding to the receptor and the ultimate formation
of an ion channel. We have exploited the availability of mutant toxins
with known defects in reversible or irreversible binding and in
toxicity to help define some of the steps which follow toxin binding.
We have found that, after binding, about 90% of the toxin molecule is
protected from protease, and much of the toxin is present as a large
aggregate, due to oligomerization and/or interaction with membrane
components. The relevance of these processes to the mode of action of
the toxin is discussed.
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MATERIALS AND METHODS |
Toxin purification and vesicle preparation.
Clones of the
Bacillus thuringiensis subsp. kurstaki HD1
cry1Ab and cry1Ac genes and mutant
cry1Ac genes were electroporated into the acrystalliferous
strain B. thuringiensis CryB (2, 37). The Cry1Ac
protoxin was also purified from B. thuringiensis subsp.
kurstaki HD73. Cells were grown and sporulated at 30°C on
G-Tris agar plates containing 25 µg of erythromycin ml
1
(2). The spores plus inclusions were harvested in 1 M
KCl-0.005 M EDTA, pH 8.0, and washed three times with deionized water.
The inclusions were purified on Renografin gradients (2),
and the protoxins were solubilized in 0.03 M
Na2CO3-1% -mercaptoethanol, pH 9.6, by
three extractions of 20 min each at 37°C. The protoxins were then
dialyzed at 4°C against a 2,000× volume (relative to the extract) of
0.01 M Tris, pH 8.5, and digested with tolylsulfonyl phenylalanyl
chloromethyl ketone-treated trypsin (1:50) (Sigma) at 37°C for 90 min, followed by a second addition of trypsin and a further 90-min
incubation. The digests were dialyzed at 4°C in 50-cut dialysis
tubing (Spectropore) against two 1,000-volume changes of 0.01 M
NaHCO3-0.25 M NaCl, pH 9.5. Final purification was done
with a Mono Q cartridge (Pharmacia) eluting with a NaCl step gradient
from 0.2 to 0.4 M in 0.02 M Tris, pH 8.7. The toxins were eluted at 0.3 to 0.4 M NaCl and, following dialysis against 0.03 M
NaHCO3-0.25 M NaCl, pH 9.5, were stored at 
70°C
(23). All of the toxins used were present as monomers in
solution except for wild-type Cry1Ac. This purified toxin contained
variable amounts of what appeared to be dimers and trimers (see Fig. 3
to 5).
Mutant Cry1Ac toxins A164D, Q163P, L167F, A164P, H168R, and S170C all
had mutations in helix 5 and have been described (37) (Table 1). Among these, the S170C toxin
retained full activity and the H168R toxin was slightly more active,
whereas all of the others were at least 100 times less active than the
wild type in three test insects (37). The mutation W210C is
in helix 6, and the resulting mutant toxin is fully toxic
(2). All of these mutant toxins bound to an M. sexta larval midgut vesicle protein in immunoblots or to BBMV
(2, 37), so the initial receptor binding was not markedly
altered. Mutant toxins A92D and R93F have substitutions in the loop
connecting helices 2 and 3 and are nontoxic (37). They
both bind to the receptor in immunoblots, but the A92D toxin did not
insert into membrane vesicles (R93F was not tested) (4, 13).
BBMV were prepared from the midguts of fifth-instar larvae of M. sexta and H. virescens, essentially as described
previously (2, 27). The protein contents of the vesicles
were determined with the bicinchoninic acid reagent (Pierce Chemical
Co.), and suspensions were aliquoted and stored at 70°C. M. sexta eggs were obtained from either Purdue University or Carolina
Biological Supplies. H. virescens eggs were supplied by the
U.S. Department of Agriculture, Stoneville, Miss.
Vesicle binding and protease K digestion.
Fifty to one
hundred nanograms of purified toxin was incubated with vesicles (10 to
20 µg of protein) in 50 µl of 0.1 M NaHCO3-0.1 M NaCl,
pH 9.5, at 27°C for 30 min. The suspensions were pelleted in a
microcentrifuge (8 min) and washed twice with 0.5 ml (in each wash) of
the incubation buffer, followed by a wash with either 1 ml of 0.01 M
Tris, pH 7.8, or 1 ml of 0.03 M NaHCO3-0.25 M NaCl, pH 9.5 (identical results). The pellets were suspended either in 20 µl of
0.05 M Tris-0.5% sodium dodecyl sulfate (SDS), pH 8.5, with
incubation at 65°C for 5 to 15 min, or in 0.05 M Tris-1% octyl--D-glucopyranoside (Calbiochem), pH 8.5. The
former protocol was based on one used in studies of
Staphylococcus -hemolysin oligomerization
(34), where the oligomers were stable at 65°C in SDS but
dissociated at 70°C. The -endotoxins retained their activity at
65°C but not at 70°C in 0.5% SDS, so the former temperature was
selected for extraction. A mutant toxin, A92D, which did not bind
irreversibly to vesicles (4, 37) was used to control adventitious toxin binding.
After the 65°C incubation or octylglucoside extraction, the
suspensions underwent SDS-6% polyacrylamide gel electrophoresis (PAGE). Following electrophoresis and transfer to polyvinylidene difluoride (PVDF) membranes, the membranes were treated with various antibodies (2, 27): Cry1Ac rabbit polyclonal, Cry1Ab
monoclonal, or anti-gypsy moth aminopeptidase N antibody, kindly
provided by D. Dean.
For protease digestion, BBMV plus toxin was incubated as described
above, and the washed vesicles were suspended in 0.01 M MOPS
[3(N-morpholino)propanesulfonic acid]-5 mM
CaCl2, pH 7.0. Protease K (Sigma) at 0.2 to 0.5 µg
ml1 was added, and the suspensions were incubated at
37°C for 45 min. Controls were 50 to 100 ng of toxin plus protease K
with no vesicles and the vesicle-toxin complex incubated without
protease K. These samples were boiled in loading buffer and
fractionated by SDS-8% PAGE. Following transfer to PVDF
(26), the two closely migrating bands of ca. 65 and 60 kDa
in the vesicle-protease K lane were cut out, and 18 to 20 residues of
each were sequenced. Gels were also stained with Coomassie blue, and
photographs of the gels were scanned in a General Dynamics ImageQuant.
| |
RESULTS |
Toxin bound to BBMV is largely protected from protease K.
As
shown in Fig. 1, >95% of the Cry1Ac
toxin bound to M. sexta BBMV was protected from complete
digestion by 0.1 to 0.5 µg of protease K and was present in two
bands, one of which was the same size as the intact toxin and the other
slightly smaller. In contrast, >70% of the toxin in solution was
completely digested by 0.2 to 0.5 µg of protease K. The lack of
digestion of the toxin bound to BBMV was not due to the inactivation of
protease K, since after the incubation, the supernatant was capable of
digesting toxin (17).
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FIG. 1.
Digestion of the Cry1Ac toxin either in solution (lanes
1 to 4) or bound to M. sexta BBMV (lanes 5 to 8) with
various concentrations of protease K at 37°C for 45 min. Following
incubation, the samples were boiled in loading buffer for 3 min.
Fractionation was achieved by SDS-10% PAGE, and staining was done
with Coomassie blue. Lanes 1 and 5, treatment with 0.1 µg; lanes 2 and 6, treatment with 0.2 µg; lanes 3 and 7, treatment with 0.5 µg;
lanes 4 and 8, no treatment. The stained bands were quantitated in a
General Dynamics ImageQuant with the following percentages of control
values: lane 1, 80%; lane 2, 28%; lane 3, 25%; and lanes 5 to 7, 95 to 100%.
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The two bands protected from complete protease K digestion by BBMV were
excised and sequenced. The lower band gave the sequence LVDIIWGIFG-S-DAFLV (where the dashes indicate the lack of definitive identification of a residue), indicating cleavage at about residue 59, which is just after helix 1 and perhaps into helix 2A
(11). The upper band sequence was TGYTPIDSLSLTQFLL,
which starts at about residue 29, as was expected for the intact toxin.
Cleavage by protease K had reduced the mass of the toxin by about 3,100 Da, and there was no evidence of further degradation with longer incubation of the vesicle-toxin complex. Similar results were obtained
with the Cry1Ab toxin (17). Pronase (type VI protease) (Sigma) was also tested, but it was more difficult to find conditions under which the addition of this protease resulted in nearly complete digestion of the toxin in solution without also digesting the BBMV-bound toxin (and probably membrane proteins as well).
Higher-molecular-weight forms of toxin extracted from BBMV.
Following incubation of the Cry1Ab or Cry1Ac toxins with M. sexta BBMV, the toxins were solubilized by incubation at 65°C, as described in Materials and Methods, and were resolved by SDS-6% PAGE. In both cases, the major toxin antigen extracted from the vesicles had a molecular mass of 190 to 200 kDa with the virtual absence of toxin monomers (Fig. 2 and
3). In solution, the Cry1Ac toxin
contained some oligomers of the same sizes as those extracted from BBMV
(Fig. 3, lane 2, and Fig. 4 and
5), suggesting that vesicle binding
enhanced oligomerization. There were no such aggregates present in the
Cry1Ab toxin preparations (Fig. 2, lane 2) or among all of the Cry1Ac
mutant toxins. Yet the active mutants (S170C and W210C) could also be
extracted from BBMV as ca. 200-kDa aggregates (Fig. 2). The presence of
some aggregated toxin in the initial preparation was not essential,
therefore, for this oligomerization.
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FIG. 2.
Immunoblot of toxin antigens extracted from M. sexta BBMV (see Materials and Methods). Lane 1, extraction of the
Cry1Ab antigen from BBMV; lane 2, 50 ng of Cry1Ab; lanes 3 and 6, extraction of the Cry1Ac S170C toxin from BBMV; lane 4, extraction of
the Cry1Ac A164D toxin from BBMV; lane 5, extraction of the Cry1Ac
W210C toxin from BBMV; lane 7, standards of -galactosidase and
bovine serum albumin; lane 8 (from top to bottom), myosin,
-galactosidase, phosphorylase b, and bovine serum albumin
standards.
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FIG. 3.
Immunoblot of toxin antigens extracted from M. sexta BBMV resolved by SDS-6% PAGE. Lane 1, extraction of the
Cry1Ac antigen from BBMV; lane 2, 50 ng of Cry1Ac toxin; lane 3, extraction of the Cry1Ac A164P toxin from BBMV; lane 4, 30 ng of Cry1Ac
A164P toxin; lane 5, extraction of the Cry1Ac L167F toxin from BBMV;
lane 6, 50 ng of Cry1Ac L167F toxin; lane 7, extraction of the Cry1Ac
A92D toxin from BBMV; lane 8, extraction of the Cry1Ac R93F toxin from
BBMV; lane 9, extraction of the Cry1Ab toxin from BBMV; lane 10, 50 ng
of Cry1Ab toxin.
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FIG. 4.
Immunoblot of Cry1Ac and H168R, following incubation
with M. sexta BBMV. (A) Lanes 1 to 3, 20 µg of BBMV
incubated with 20, 40, and 60 ng of Cry1Ac toxin, respectively; lane 4, 60 ng of Cry1Ac toxin; lanes 5 to 7, 20 µg of BBMV incubated with 20, 40, and 60 ng of H168R toxin, respectively; lane 8, 60 ng of H168R
toxin; lane 9, standards as in Fig. 2 plus ovalbumin (45 kDa) and
carbonic anhydrase (29 kDa). (B) Toxin samples (20 ng) were incubated
with 20 µg of BBMV and then extracted for different times at 65°C.
Lane 1, Cry1Ac toxin extracted at 65°C for 15 min; lane 2, Cry1Ac
toxin extracted at 65°C for 5 min; lane 3, 20 ng of Cry1Ac toxin;
lane 4, H168R toxin extracted at 65°C for 15 min; lane 5, H168R toxin
extracted at 65°C for 5 min; lane 6, 20 ng of H168R toxin. Markers on
the right indicate (from top to bottom) positions of myosin,
-galactosidase, and bovine serum albumin.
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FIG. 5.
Immunoblot of toxin antigens and aminopeptidase N
extracted from M. sexta BBMV and resolved by SDS-6% PAGE.
Lane 1, incubation of 50 ng of Cry1Ac toxin with BBMV; lane 2, 50 ng of
Cry1Ac toxin; lane 3, incubation of the Cry1Ac W210C toxin with BBMV;
lane 4, 50 ng of Cry1Ac W210C toxin; lane 5, 50 ng of Cry1C toxin; lane
6, same as lane 1 but treated with aminopeptidase N antibody; lane 7, M. sexta BBMV extract treated with aminopeptidase N
antibody; lane 8 (from top to bottom), thyroglobulin, myosin,
-galactosidase, and bovine serum albumin standards.
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In contrast, mutant Cry1Ac toxins with low or no activity (in three
insect species), such as A164D, A164P, L167F, A92D, and R93F, did not
remain associated with vesicles in any form (Fig. 2 and 3). The first
three of these toxins have mutations in helix 5, and the latter two
have mutations in a loop connecting helices 2 and 3 (Table 1).
The A92D toxin, which has no detectable toxicity, can bind reversibly
to BBMV but cannot insert into the membrane (4, 13).
The one exception to a total correlation between activity and the
formation of a ca. 200-kDa oligomer was the Cry1Ac mutant toxin H168R
(Fig. 4). This toxin is at least as active as the wild type
(37), and yet no oligomers were found. Instead, a relatively
large amount of the monomer could be extracted from BBMV after washing.
Trimers may have formed, but they were unstable under the extraction
conditions used. There was no ca. 200-kDa band even when heating at
65°C was reduced to 5 min (Fig. 4B). Instead, there was a band of
>200 kDa, which was also present with the wild-type Cry1Ac toxin and
when other active toxins were incubated with BBMV (17). The
possible significance of this larger aggregate is discussed below.
Toxin was not bound to aminopeptidase N in BBMV.
Extracts of
BBMV incubated with either the wild-type Cry1Ac toxin or the fully
active mutant Cry1Ac toxin W210C were electrophoresed, blotted to PVDF,
and treated either with anti-Cry1Ac antibody or with antibody to
aminopeptidase N (Fig. 5). Aggregates of ca. 200 kDa were present for
the two toxins. The aminopeptidase N antibody reacted with a major band
of ca. 120 kDa, the expected size of this enzyme, as well as with a
minor band of >200 kDa and some bands of <120 kDa, but the antibody
reacted to no band of the same size as the toxin aggregate. The profile
was the same for BBMV incubated with or without toxin, with no
detectable shifts in the size of aminopeptidase N after incubation with toxin.
Binding of the Cry1Ac toxin to BBMV from different insects.
BBMV were prepared from late-instar larvae of M. sexta and
H. virescens and incubated with the Cry1Ac toxin (Fig.
6). In all cases, there were bands of ca.
130 and 200 kDa as well as monomers. At both concentrations of toxin,
the 200-kDa band extracted from H. virescens BBMV was about
twice as intense as the band from M. sexta BBMV. The total
amount of Cry1Ac antigen recovered from the BBMV at higher toxin
concentrations (Fig. 6, lanes 2 and 4) is somewhat greater (50 and
20%, respectively) than the amount of toxin added (lane 5). The Cry1Ac
toxin is about three to four times more active in H. virescens than in M. sexta (12).
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FIG. 6.
Immunoblot of the Cry1Ac toxin antigen extracted from
BBMV prepared from M. sexta and H. virescens.
Lane 1, 10 µg of M. sexta BBMV plus 50 ng of Cry1Ac toxin;
lane 2, 20 µg of M. sexta BBMV plus 50 ng of Cry1Ac toxin;
lane 3, 10 µg of H. virescens BBMV plus 50 ng of Cry1Ac
toxin; lane 4, 20 µg of H. virescens BBMV plus 50 ng of
Cry1Ac toxin; lane 5, 50 ng of Cry1Ac toxin.
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DISCUSSION |
Following incubation with BBMV, most of the Cry1Ac toxin molecule
was protected from digestion by protease K. In an earlier study, it was
found that only intact Cry1B toxin was extracted from Pieris
brassicae BBMV following a prolonged incubation (36). In this study, the extracted toxin was intact, and only the ca. 30 N-terminal amino acids, which primarily comprise helix 1
(11), were susceptible to protease K. Interestingly, the
synthetic 1 peptide was the only one of the seven amphipathic
-helical peptides which did not bind to synthetic vesicles
(10). The rest of the toxin was protected, perhaps with a
surface location for all parts except 4-loop-5, which is very
hydrophobic (11). Mutations in these particular helices, but
not in the loop, resulted in loss of toxicity (17, 29, 37),
whereas mutations in presumed surface helices 2, 3, and 6 had
no effect (2, 37). The 4 and 5 synthetic peptides were
the only ones to insert into phospholipid vesicles, and the kinetics of
insertion for peptide 5 indicated aggregation within the membrane
(8-10). There was no evidence of cleavage within domain I
following binding to BBMV, as has been reported for the CryIIIA toxin
(3).
Under the particular conditions used (i.e., either mild heating or
solubilization with octylglucoside [17]), toxin bound to the membrane was extracted primarily as an aggregate or oligomer of
about 200 kDa. In some cases, a band of ca. 130 kDa was also present
(Fig. 4 and 6), but this was variable. No toxin aggregates were found
after incubation of the Cry1Ac toxin with BBMV pretreated with
octylglucoside or with the lipid fraction extracted from BBMV
(6) or by addition of deoxycholate at concentrations which formed micelles (17). Oligomerization appears to require
more than just a hydrophobic environment and very likely requires an intact membrane.
Based on size, the toxin is present either as a trimer or as toxin
monomers (or dimers) bound to some component of BBMV. The latter is
unlikely to be aminopeptidase N, which can function as a receptor for
initial, reversible toxin binding (15, 28) and perhaps for
toxin insertion (28, 30). There is evidence for a BBMV
component affecting the properties of the ion channel (22,
30), so such an interaction is possible. Since the size of the
ca. 200-kDa aggregate is identical to that of a small fraction of the
Cry1Ac toxin found in solution (Fig. 3 to 5), it is likely that the
aggregates are toxin trimers. These soluble aggregates may reflect the
oligomerization required to form channels, or they may be stable
intermediates, either in the formation of the pore or in its
degradation during extraction.
There was a correlation between the extent of formation of aggregates
by the Cry1Ac toxin and its effectiveness in two insect species (Fig.
6). There was also a good correlation between the presence of these
toxin aggregates and the activity of the Cry1Ac mutant toxins. Two such
mutants with full activity formed aggregates, whereas five others with
low or no toxicity due to mutations either in helix 5 or in the loop
between helices 2 and 3 failed to do so. The one exception was
the very active Cry1Ac toxin mutant H168R, which bound well to the
membrane, but no band of ca. 200 kDa was found (Fig. 4). The tighter
binding of the monomer to BBMV must be due to the H168R substitution in
the hydrophobic face of helix 5 (37), which also appears
to alter the stability of oligomers, at least the relatively stable
molecule of ca. 200 kDa. If this oligomer is important to toxin
function, it must form transiently in this mutant. Alternatively, the
aggregate of >200 kDa seen with both the wild-type and H168R mutant
toxins (Fig. 4B) could be more indicative of toxin function. If so, the ca. 200-kDa band may be a stable intermediate or a degradation by-product.
Recently, it has been found that toxins inactive due to mutations in
helix 4 form aggregates of 200 kDa following incubation with BBMV
(17). Overall, the results are consistent with helix 5
being critical for aggregation (perhaps among other functions), whereas
helix 4 would be essential for the formation of a functional channel.
It is not known whether toxin aggregation occurs at the surface of the
membrane following binding to the receptor or only after portions of
the toxin insert into the membrane. All of the nontoxic Cry1Ac mutants
bind to the receptor (2, 37), but those lacking a positive
charge in the loop between helices 2 and 3 do not insert
irreversibly (4, 13), and those with mutations in helix 5
do not aggregate. The loop between helices 2 and 3 may be part of
the face of the toxin which interacts with the phospholipids in the
membrane (13), whereas helix 5 appears to be pivotal
for aggregation. In solution, this helix is surrounded by the other
helices of domain I (11) and thus is probably not accessible
for interaction until it inserts into the membrane as part of the
hydrophobic helix 4-loop-5 segment of the toxin.
This research was supported by a grant from the Binational
Agricultural Research and Development Fund (Bard project IS-2629-95).
The Purdue Laboratory for Macromolecular Structure did the protein
sequencing. M. sexta larvae were kindly provided by the Department of Entomology at Purdue. Michael Wolfersberger kindly provided critical comments.
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Abstract
Introduction
