Prepore formation is hypothesized to be an obligate step in the insertion of Cry1Ab toxin into insect brush border membrane vesicles. We examined the architecture of the putative prepore when isolated using the published protocols [1] [2]. Our results demonstrate that the putative prepore form of Cry1Ab is a combination of receptor proteins attached to the toxin, when purified. The results also suggest that this prepore form as prepared by the methods published is different from other membrane-extracted oligomeric forms of Cry toxins and prepore of other toxins in general. While most other known prepores are composed of multimers of a single protein, the Cry1Ab prepore, as generated, is a protein-receptor complex oligomer and monomers of Cry toxins.
Pore-forming toxins represent approximately 30% of bacterial toxins [
Most pore forming toxins including the Cry toxins are known to oligomerize during their mechanism of action. It is not clear whether the process begins with monomers of Cry toxins that dimerize and then add on monomers till a definite oligomeric shape is achieved or if monomers interact with preformed higher oligomers to form the channel structure. Bacterial toxins, in general, have been shown to form “prepore” oligomers that allow their insertion into target cell membranes. The term “prepore” refers to an intermediate state of these toxins in which the monomers of the toxin assemble to form a precursor that is competent to insert into these membranes. Many bacterial toxins that form prepore do undergo a substantial conformational change in the process, including a change in the secondary structure of the huge regions involved in the pore formation as in cytolysins [
Cry1Ab toxin from Bacillus thuringiensis has been the model for studying prepore formation and oligomerization of Cry toxins [
Bacillus thuringiensis sotto (for Cry1Aa), Bacillus thuringiensis 4Q7 (acrystalliferous strain for expression of Cry1Ab F371C mutant) and Bacillus thuringiensis HD1-19 (for Cry1Ab) strains were obtained from Bacillus Genetic Stock Center, The Ohio State University. Antibodies used in this study include anti-Cry1Ab polyclonal antibody generated against denatured Cry1Ab monomer protein and anti-Cry1Ab polyclonal antibody kindly provided by Dr. Alejandra Bravo of Universidad Nacional Autónoma de México (which is referred to in this manuscript as the anti-prepore antibody). Antibody to the cadherin (anti Bt-R1) was kindly provided by Dr. Lee A. Bulla Jr. from University of Texas at Dallas.
Prepore was produced by two separate methods that were published [
Cry1Ab prepore oligomers were prepared and purified using the two published methods [
In order to determine the identity of the proteins in the purified prepore complex, SDS-PAGE-resolved Cry1Ab prepore was subjected to LC-MS/MS analysis, performed at the W.M. Keck Facility, New Haven, CT. Briefly, purified prepore was run on SDS-PAGE gel and the entire lane from the gel, which showed cross-reactivity to the prepore antibody, was cut into 1 mm slices and digested with trypsin using in-gel digestion and the resulting samples were desalted in a 100 micron ID C18 column (Waters) in a gradient of 2% - 98% acetonitrile in the presence of 0.01% trifluoroacetic acid. Protein was considered identified if two or more peptides matched to the same protein accession numbers in the database (MASCOT analysis).
Although the purified form of the prepore had a mixture of oligomer and monomer forms, we nevertheless performed limited toxicity bioassays on them to measure any improvement over the purified monomer toxins as reported. Toxicity levels were determined by estimating the median lethal concentration (LC50) on first instar M. sexta larvae using the diet surface contamination assay [
Using the published protocols [
Protein samples verified for formation of the prepore form were purified in order to determine the regions of Cry1Ab present/absent in the prepore. Since SDS-PAGE of purified prepore showed multiple bands, we decided to characterize each band to determine toxin regions present in the prepore form and its variations if any, from the monomer. Proteins were identified from in-gel trypsin digests of the SDS-PAGE bands using LC MS/MS analysis of the digested bands. Proteins were identified from a MASCOT search with two criteria: two or more MS/MS spectra match the same protein in the database and that each of the matched peptides was ensured to be from trypsin digestion.
Prepore examined from the two methods showed presence of Cry1Ab toxin. However, the purified prepore in both the methods had additional proteins that were identified by the sensitive MS/MS process. The entire list of pertinent proteins identified is listed in
Of interest was the observation [
Even though we could not quantitate the amount of prepore form of Cry1Ab in the reaction mixture due to interference of monomers in all methods, we decided to perform a comparison of toxicity measurements of prepore as isolated by the published procedures to the pure monomer based on the premise that the conformational change would enhance the toxicity of Cry1Ab protein due to the presence of the active intermediate. Our studies using diet surface contamination assays with Manduca sexta first instar larvae showed overlapping LC50 confidence limits for prepore and monomer as listed in
The putative prepore formation of Cry1Ab toxin was reproduced using published protocols [
GI accession number of match | Protein identified | Maximum number of peptides identified |
---|---|---|
gi|40255 | Insecticidal crystal protein | 13 |
gi|143099 | Insecticidal crystal protein | 13 |
gi|20465244 | Cadherin from M. sexta | 5 |
gi|2499901 | APN-like protein (Membrane alanyl aminopeptidase precursor Cry1Ac receptor) | 32 |
gi|8488965 | Aminopeptidase 2 | 24 |
gi|20279109 | Aminopeptidase 3 | 18 |
GI accession number of match | Protein identified | Maximum number of peptides identified |
---|---|---|
gi|40255 | Insecticidal crystal protein | 21 |
gi|143099 | Insecticidal crystal protein | 16 |
gi|1902832 | Single chain (scFv) antibody Mol.wt. 26267 | 3 |
gi|106429 | Ig heavy chain V region (alpha-phOx15)-human fragment Mol.wt. 13696 | 2 |
Sequence of Peptides identified | Region of Toxin | Residue positions on toxin |
---|---|---|
DVSVFGQR | Domain I (alpha 5) | D174-R181 |
TLSSTLYR | Domain II (beta) | T361-R368 |
LSHVSMFR | Domain II (beta) | L430-R437 |
WYNTGLER | Domain I (loop between alpha 6 & 7) | W210-R217 |
TSPGQISTL | Domain III | T502-R511 |
GSAQGIEGSIR | Domain II | G282-R292 |
GPGFTGGDILR | Domain III | G490-R500 |
VNITAPLSQR | Domain III | V512-R521 |
IVAQLGQGVYR | Domain II | I350-R360 |
WGFDAATINSR | Domain I (loop between alpha 5 & 6) | W182-R192 |
EWEADPTNPALR | Domain I (loop between alpha 3 & 4) | E116-R127 |
EIYTNPVLENFDGSFR | Domain II | E266-R281 |
IEFVPAEVTFEAEYDLER | Domain III end (C-term of active toxin) | I602-R619 |
LEGLSNLYQIYAESFR | Domain I | L100-R115 |
ELTLTVLDIVSLFPNYDSR | Domain I (alpha 7) | E235-R253 |
SAEFNNIIPSSQITQIPLTK | Domain III | S458-K477 |
SPHLMDILNSITIYTDAHR | Domain II | S293-R312 |
Cry1Ab monomer | 20.0 (7.5 - 31.7) |
---|---|
Cry1Ab oligomers formed in solution | 28.0 (10.0 - 46.5) |
Cry1Ab toxin extracted from membrane** | 25.0 (5.0 - 35.0) |
Cry1Aa prepore oligomer | 17.1 (6.2 - 35.3) |
Cry1Ab prepore oligomer | 26.2 (3.9 - 40.8) |
Cry1Ab F371C prepore oligomer | >2000 |
**Cry1Ab toxin in this group was obtained by dissolving the BBMV after insertion of toxin using 10 mM HEPES pH 7.5 buffer + 1%b-octyl glucoside detergent.
and other proteins, certainly ones that were associated with the toxin in the putative prepore form as prepared. Sedimentation velocity measurements of the purified oligomer indicated that the native forms of the toxin persisted in many sizes ranging from monomer to dodecamer as deciphered by the limits of nomogram analysis. To test if only a limited portion of the toxin was associated with the membrane in the prepore as predicted by models, we extracted the membrane inserted form of prepore complex obtained by using scFv73 peptide with a detergent. LC-MS analysis of the resulting tryptic digest indicated presence of peptides from all domains of the Cry1Ab in the extracted form. It also indicated presence of scFv73 peptide or the receptors depending on the method of prepore formation that was pursued. Toxicity measurements of the Superdex 200 column purified oligomer did not provide a LC50 that was any significantly higher than the monomer form.
We now discuss our observation that the putative prepore does not react against a polyclonal antibody formed against denatured Cry1Ab. The possibility that a conformational change would obviate binding of the polyclonal antibody exists but seems unlikely since the antibody binds to both native and denatured forms of the toxin. We suggest that the Cry1Ab toxin in the putative prepore is masked by the scFv73 peptide or receptors to the extent of blocking it from interaction to the anti-Cry1Ab antibody. In addition, the anti-prepore antibody, which does bind to the putative prepore is observed to bind to purified cadherin (data not shown).
All of these data in combination suggest that while putative prepore forms of Cry1Ab can be successfully generated, the resultant product as examined is not a pure toxin. Since very low amounts of toxin are effective at mediating toxicity, based on the hypothesis of the serial receptor binding model [
The current data also questions the nature of the membrane bound tetramer as proposed by the serial receptor binding model, which indicates that only two alpha helices of the toxin from each monomer are present within the membrane. The rest of the toxin, if not associated with the membrane, should be easily digested by proteases. Our tryptic digests of membrane extracted prepore isolates presented almost equal number of peptides from all three domains of active Cry1Ab toxin (5 from Domain I, 7 from Domain II and 5 from Domain III) and none from the C-terminal region of the protoxin form used in these prepore formation methods or any peptides matching alpha helix 1 of active toxin. In addition, there are receptor peptides that were bound to the toxin. Our previous studies also highlight an intact 60 kDa monomer toxin that lacks alpha helix 1 as the form that inserts in the membrane [
In summary, we performed a proteomic and biophysical analysis of the putative prepore of Cry1Ab to determine the size and the contents of the complex and have discovered that the toxin does exist as a higher order tetramer as the serial-receptor model hypothesizes but not as a pure toxin protein; it is complexed with receptor proteins and forms several higher order species besides the tetramer. We conclude that the putative prepore of Cry proteins is not a classic prepore as proposed for other protein toxins and should be referred to as a “complex oligomer” of Cry proteins and receptor proteins used to generate it. We did not detect alpha-helix 1 in the proteomic analysis of the complex oligomer, so it remains to directly determine if it is proteolytically removed upon interaction with the receptor or later upon insertion into the membrane. Our efforts toward resolving the structure of the prepore complex will address further details on the exact nature of the complex oligomer. From these studies, we question the exact role of a prepore generated using protocols described [
We thank Dr. Daniel Zeigler for providing strains of Bacillus thuringiensis required for the study, Dr. Lee A. Bulla Jr. for providing us with anti-Bt-R1 antibody that reacts with cadherin and Dr. Alejandra Bravo for providing us with anti-Cry1Ab polyclonal antibody that reacts with the prepore form. We also want to thank Tom Abbott and Kathryn Stone at the Keck Biotechnology Resource Laboratory, Yale School of Medicine for performing the LC-MS experiments and MASCOT data analyses related to this work and Dr. James Lear for running purified prepore samples on the Beckman XL-I for us. This work was funded by NIH R01A29092 to DHD and Michael J. Adang.