©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Expression of a Receptor for an Insecticidal Toxin of Bacillus thuringiensis(*)

(Received for publication, November 8, 1994)

Ratna K. Vadlamudi Eric Weber Inhae Ji Tae H. Ji Lee A. Bulla Jr. (§)

From the Department of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071-3944

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Environmentally friendly toxins of Bacillus thuringiensis are effective in controlling agriculturally and biomedically harmful insects. However, little is known about the insect receptor molecules that bind these toxins and the mechanism of insecticidal activity. We report here for the first time the cloning and expression of a cDNA that encodes a receptor (BT-R(1)) of the tobacco hornworm Manduca sexta for an insecticidal toxin of B. thuringiensis. The receptor is a 210-kDa membrane glycoprotein that specifically binds the cryIA(b) toxin of B. thuringiensis subsp. berliner and leads to death of the hornworm. BT-R(1) shares sequence similarity with the cadherin superfamily of proteins.


INTRODUCTION

Biopesticides based on the bacterium Bacillus thuringiensis currently are being used as safe alternatives to chemical insecticides. B. thuringiensis toxins are environmentally friendly because they kill only those insects susceptible to the toxins, whereas current synthetic chemical pesticides indiscriminately kill pest and beneficial insects alike and are considered to be major toxic pollutants of the environment. Insecticidal properties of B. thuringiensis are manifested in crystalline glycoprotein toxins (cry gene products) (1) that are produced during the sporulation cycle of this bacterium. The insects affected by B. thuringiensis include many agriculturally and biomedically detrimental pests in the orders Lepidoptera, Coleoptera, and Diptera. The primary action of B. thuringiensis toxins occurs in the brush border of insect midgut epithelial cells(2) . Specific binding of these toxins to midgut brush-border membrane vesicles has been reported(3, 4, 5) . A number of putative receptors have also been identified(6, 7, 8, 9) .

However, little is known about the molecular nature of the insect receptors that bind these toxins and the mechanism of insecticidal activity. Here, we report the cloning and expression of a cDNA that encodes a novel cadherin-like glycoprotein receptor present in the midgut of the tobacco hornworm Manduca sexta. The receptor binds the cryIA(b) toxin of B. thuringiensis subsp. berliner, leading to death of this particular lepidopteran insect. We have named this receptor molecule BT-R(1).


EXPERIMENTAL PROCEDURES

BT-R(1) Purification and Sequencing

Natural BT-R(1) of M. sexta was purified, as previously reported(6) , by immunoprecipitating toxinbinding protein complexes with toxin-specific antisera and separating the complexes by SDS-polyacrylamide gel electrophoresis, followed by electroelution. Purified BT-R(1) was subjected to cyanogen bromide digestion. The cyanogen bromide fragments were separated on a 17% high resolution Tricine(^1)/SDS-polyacrylamide gel (10) and transferred to Problott membranes (Applied Biosystems Inc.). Five distinct bands (see Fig. 1) were extracted, and peptide sequences were determined by Edman degradation (Applied Biosystems Inc.). The amino acid sequences obtained from microsequencing were (M)LDYEVPEFQSITIRVVATDNNDTRHVGVA, (M)XETYELIIHPFNYYA, (M)XXXHQLPLAQDIKNH, (M)F/PN/IVR/YVDI/G, and (M)NFF/HSVNR/DE.


Figure 1: BT-R(1) purification and cyanogen bromide digestion. A, natural BT-R(1) was purified following immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and electroelution(4) . Lane1, Coomassie Blue-stained gel of M. sexta brush-border membrane vesicle proteins (50 µg); lane2, 2.5 µg of purified BT-R(1) protein; lane3, ligand blot of purified protein with I-cryIA(b) toxin. B, purified BT-R(1) (10 µg) was subjected to cyanogen bromide digestion for 20 h at 25 °C, and the resulting products were resolved on a 17% high resolution Tricine/SDS-polyacrylamide gel. Arrowheads point to peptides 1-5 that were sequenced. Sizes of marker proteins are indicated in kilodaltons.



cDNA Cloning and Sequencing

An M. sexta cDNA library was constructed in gt10 using the Superscript Choice System according to the manufacturer's instructions (Life Technologies, Inc.). Synthetic oligonucleotides corresponding to peptides 1-3 were labeled with -P using polynucleotide kinase and were utilized to screen the cDNA library(11) . Approximately 4 times 10^5 recombinants were screened. A clone hybridizing to all three probes was plaque-purified and subcloned into pBluescript (Stratagene). Double-stranded cDNA in pBluescript was sequenced in both directions by the dideoxy chain termination method with Sequenase (U. S. Biochemical Corp.) according to the manufacturer's instructions.

Northern Blot Analysis

Total RNA from M. sexta midgut (10 µg) was separated on a 0.8% formaldehyde-agarose gel and blotted onto a nylon membrane (Amersham Corp.). The analysis was carried out according to Maniatis et al.(11) . The filter was hybridized with P-labeled, random-primed BT-R(1) cDNA (SacI fragment, 4.8 kilobases). Filter hybridization was carried out at 42 °C in 50% formamide, 5 times Denhardt's reagent, 5 times phosphate-buffered (25 mM KPO(4)) SSC, and 50 µg/ml salmon sperm DNA. The filter was washed two times with 1 times SSC plus 0.1% SDS and two times with O.25 times SSC plus 0.1% SDS at 42 °C. Each wash was for 20 min, and the filter was exposed to x-ray film for 24 h.

Expression of BT-R(1) cDNA in COS-7 and 293 Cells

The 4810-base pair cDNA that encodes the open reading frame of BT-R(1) was subcloned into the pcDNA3 vector (Invitrogen). COS-7 cells and human embryonic kidney 293 cells were transfected with the construct using a modified calcium phosphate method(11) . For transient expression assays, cells (COS-7 and 293) were harvested 60 h after transfection and washed with phosphate-buffered saline(12) . Cell membranes were prepared by differential centrifugation(13) . Control cells were transfected with the pcDNA3 vector without insert. For stable expression, 293 cells were subcultured 1 day after transfection into fresh medium containing G418 (400 µg/ml). Surviving cells were assayed for their ability to bind the toxin, and those cells expressing the highest level of receptors were selected.

Blotting and Binding Studies

Cell membranes (10 µg) were separated on a 7.5% SDS-polyacrylamide gel, blotted onto a nylon membrane, and blocked with Tris-buffered saline containing 5% nonfat dry milk powder, 5% glycerol, and 0.1% Tween 20. The nylon membrane then was incubated with I-cryIA(b) toxin (2 times 10^5 cpm/ml) for 2 h. The nylon membrane was washed four times (20 min each) with blocking buffer, dried, and exposed to x-ray film at -70 °C. For competition binding assays, 293 cells transiently expressing the BT-R(1) cDNA clone were incubated with I-cryIA(b) toxin (0.32 nM) in the presence of increasing concentrations (0-10M) of unlabeled cryIA(b) toxin. Nonspecific binding was measured as bound radioactivity in the presence of 1 µM unlabeled toxin. The toxin was radioiodinated as described previously(14) .

In Vitro Translation

pBluescript plasmid containing BT-R(1) cDNA was linearized and transcribed with T(3) polymerase (Pharmacia Biotech Inc.). In vitro translation was carried out according to the manufacturer's instructions with nuclease-treated rabbit reticulolysate (Life Technologies, Inc.). After 1 h of incubation at 30 °C, the reaction mixture was either combined with an equal volume of SDS buffer or lysed with 50 mM Tris buffer containing 1% Nonidet P-40 and 250 mM NaCl (pH 8.0) for immunoprecipitation. Water was substituted for mRNA in the control reaction (see Fig. 6, lane1). Immunoprecipitation was carried out with either preimmune serum or anti-BT-R(1) serum. Translation and immunoprecipitation products were electrophoresed on a 7.5% SDS-polyacrylamide gel, fixed, treated with ENHANCE (DuPont NEN), dried, and exposed to x-ray film for 12 h.


Figure 6: In vitro translation and N-glyconase digestion of BT-R(1). A, in vitro translation of the BT-R(1) cDNA clone. mRNA produced in vitro was translated in a rabbit reticulolysate system. S-Labeled proteins were separated on a 7.5% SDS-polyacrylamide gel and visualized by autoradiography. Lane1, translation products generated in the absence of mRNA; lane2, translation products generated with BT-R(1) mRNA; lane3, translation products immunoprecipitated with normal serum; lane4, translation products immunoprecipitated with anti-BT-R(1) serum. B, N-glycanase F treatment of BT-R(1). Purified BT-R(1) protein was digested with N-glycanase F (lane6) and without enzyme (lane5) as described under ``Experimental Procedures,'' and the digestion products were separated on a 7.5% SDS-polyacrylamide gel and stained with Coomassie Brilliant Blue. Positions of molecular mass markers are indicated in kilodaltons.



N-Glycanase F Treatment of BT-R(1)

Purified BT-R(1) protein was denatured by boiling in 1% SDS for 5 min, followed by the addition of Nonidet P-40 to a final concentration of 1% in the presence of 0.1% SDS. The preparation was incubated in sodium phosphate buffer (pH 8.5) at 37 °C with N-glycanase F (10 units/ml) and without enzyme for 10 h. Digestion products were separated on a 7.5% SDS-polyacrylamide gel and stained with Coomassie Brilliant Blue.


RESULTS AND DISCUSSION

Previously, we described the identification, purification, and characterization of natural BT-R(1), which specifically recognizes the cryIA(b) toxin of B. thuringiensis subsp. berliner(6) . The binding of the toxin to natural BT-R(1) present in brush-border membrane vesicles of M. sexta was specific and high, with a K(d) value of 0.71 nM. Immunoprecipitation and I-cryIA(b) toxin binding in a ligand blot revealed specific binding of the toxin to a 210-kDa protein (Fig. 1A, arrowheads in lanes2 and 3, respectively). Purified natural BT-R(1) was digested with cyanogen bromide (Fig. 1B), and five major peptides were sequenced. Degenerate oligonucleotides were synthesized, based on these peptide sequences (15) , and were used to screen an M. sexta midgut cDNA library. A single clone hybridized to three of the oligonucleotide probes and contained an insert of 5571 bases. It had an open reading frame of 4584 bases and 1528 amino acids. Amino acid sequences of the cyanogen bromide fragments of natural BT-R(1) matched perfectly within the deduced sequence of BT-R(1) (Fig. 2A). The deduced polypeptide is 172 kDa and has a pI of 4.5. The translation start site is flanked by the consensus translation initiation sequence (GAGATGG) of eukaryotic mRNAs(16) . A polyadenylation signal(17) , AATAAA, was observed at position 5561.


Figure 2: Deduced amino acid sequence of BT-R(1) and alignment of BT-R(1) repeats with published cadherin repeats. A, the putative signal sequence and the transmembrane domain are underlined with single and doubleboldfacesolidlines, respectively. Asterisks denote putative N-glycosylation sites. Cysteines are indicated in boldface. Amino acids determined by sequencing of cyanogen bromide fragments of BT-R(1) are underlined with thinsolidlines(19) . Arrows delineate boundaries between the repeats. The arrowhead designates the C terminus of repeat 11. B, extracellular repeats of BT-R(1) (BT-R(1)EC 1-11) are aligned with representative extracellular repeats of mouse P-cadherin (mPEC1), Drosophilafat extracellular repeat 18 (fatEC18), protocadherin (PC42EC2), and human intestinal peptide transporter extracellular repeat 1 (HPT-1 EC1). Conserved residues are in boldface.



Total RNA was prepared from midguts of M. sexta and was hybridized by Northern blotting with the antisense 4.8-kilobase SacI fragment of the BT-R(1) cDNA clone. The probe hybridized to a single 5.6-kilobase band that corresponds to the 5571-base-long BT-R(1) cDNA clone (Fig. 3). This result indicates that the BT-R(1) cDNA clone represents the full-length coding sequence of the BT-R(1) gene. To demonstrate that the protein encoded by the BT-R(1) cDNA is a membrane protein and is capable of binding cryIA(b) toxin, the BT-R(1) cDNA was subcloned into the mammalian expression vector pcDNA3 (Invitrogen), and the construct was transfected into COS-7 cells. Membranes isolated from the COS-7 transfectants were solubilized, electrophoresed, and ligand-blotted with I-cryIA(b) toxin. I-cryIA(b) toxin bound to a protein of 210 kDa (Fig. 4). It was labeled only in membranes prepared from M. sexta (Fig. 4, lane1) and from COS-7 cells transfected with the BT-R(1) cDNA construct (lane3). No such band was observed in membranes isolated from mock-transfected COS-7 cells (lane2). Human embryonic 293 cells were transfected with BT-R(1) cDNA and selected for stable expression. Ligand blotting of the transfected cell lysates showed expression of the 210-kDa protein, which recognizes the cryIA(b) toxin (lane5). No binding of toxin to control 293 cell lysates was observed (lane4). BT-R(1) was expressed on the surface of transfected human embryonic 293 cells and showed high affinity (K(d) = 1 nM) for the toxin (Fig. 5), as did natural BT-R(1)(4) .


Figure 3: Northern blot of total RNA isolated from M. sexta midgut. Total RNA was resolved on an agarose gel, blotted onto a nylon membrane, and hybridized with random-primed P-BT-R(1) cDNA (4.8-kilobase SacI fragment) as described under ``Experimental Procedures.'' Arrowheads indicate 28 S and 18 S RNAs.




Figure 4: Ligand blot analysis of BT-R(1) transfectants. A, ligand blot analysis of membranes from M. sexta and COS-7 transfectants. Membranes (10 µg) were resolved on a 7.5% SDS-polyacrylamide gel, transferred to a nylon membrane, and labeled with I-cryIA(b) toxin. Lane1, M. sexta brush-border membranes; lane2, mock transfectant; lane3, transfectant with the BT-R(1) cDNA clone. B, ligand blot analysis of 293 cells stably expressing BT-R(1) cDNA. Proteins from 293 cells were extracted with SDS sample buffer, separated by SDS-polyacrylamide gel electrophoresis, and labeled with I-cryIA(b) toxin. Lane4, protein extracts from control 293 cells; lane5, protein extracts from 293 cells stably expressing BT-R(1) cDNA.




Figure 5: Binding of I-cryIA(b) toxin to intact transfected human embryonic 293 cells expressing BT-R(1). The cells were transfected with the BT-R(1) cDNA in pcDNA3 and incubated with I-cryIA(b) toxin (0.32 nM) in the presence of increasing concentrations (0-10M) of unlabeled cryIA(b) toxin. Nonspecific binding was determined as bound radioactivity in the presence of 1 µM unlabeled toxin. The K value (1 nM) was determined by Scatchard analysis.



The size of the expressed 210-kDa protein is larger than 172 kDa, the estimated molecular mass of the cloned protein (Fig. 2). To determine whether the difference was due to glycosylation of the native protein, the BT-R(1) clone was translated in a rabbit reticulolysate system that does not support glycosylation. The resulting translated products were immunoprecipitated with polyclonal antibodies raised against natural BT-R(1). In vitro translation of the BT-R(1) cDNA clone generated two protein bands of 172 and 150 kDa as determined by SDS-polyacrylamide gel electrophoresis (Fig. 6, lane2). The two bands were immunoprecipitated specifically by anti BT-R(1) (lane4), but not by preimmune serum (lane3). The presence of the second translation product (150 kDa) probably was due to the initiation of translation from an internal methionine (18) at amino acid 242. The presence of a 172-kDa band and its immunoreactivity further confirm that this clone represents native BT-R(1), and the difference in size presumably is due to glycosylation. N-Glycanase F treatment reduced the molecular mass of native BT-R(1) from 210 to 190 kDa (lanes5 and 6). These results indicate N-glycosylation at some of the 16 consensus N-glycosylation sites in the protein (Fig. 2A). Treatment of BT-R(1) with O-glycanase and neuraminidase did not alter the mobility of the natural protein (data not shown).

We believe that the protein expressed from the BT-R(1) cDNA clone is the same as the natural protein found in the midgut of M. sexta because they both have identical amino acid compositions and sequences, molecular masses, and toxin binding specificity and affinity as well as similar pI values(6) . The amino acid sequence (Fig. 2) shows a putative signal peptide (19) of 20 amino acids and a transmembrane domain of 22 amino acids (20) beginning at position 1406. A 101-amino acid-long C terminus follows the transmembrane domain. Because the toxin binds to a 50-kDa extracellular fragment of BT-R(1)(6) , the smaller C-terminal region is likely to reside in the cytoplasm. This feature is consistent with the fact that there is only one consensus N-glycosylation site in the C-terminal cytoplasmic domain compared with 15 N-glycosylation sites in the Nterminal extracellular domain.

BT-R(1) shows 30-60% similarity and 20-40% identity to members of the cadherin superfamily of proteins (Fig. 2B) (21) . Cadherins are membrane glycoproteins and are believed to mediate calcium-dependent cell aggregation and sorting(22) . Recently, other cadherin-like molecules such as Drosophilafat tumor suppressor, human intestinal peptide transport protein, protocadherins, and T-cadherin have been described(23, 24, 25, 26) . Like cadherins, the extracellular domain of BT-R(1) is highly repetitive and contains 11 repeats (Fig. 2A). The length of the BT-R(1) repeats is similar to that of cadherins (110 amino acids), except for repeats 6, 7, and 9, which are 20-30 amino acids longer than cadherin repeats. The conserved motifs in the repeats of BT-R(1) (Fig. 2B) include AXDXD, DXE, and DXNDXXP and 1 Glu and 2 Gly residues (Fig. 2B). Motifs A/VXDXD and DXNDN are the consensus sequences for calcium binding(27) . The putative cytoplasmic domain of 101 amino acids is smaller than vertebrate cadherin cytoplasmic domains (160 amino acids) and shows no homology to any proteins in the data base. Also sequences flanking the conserved cadherin motifs share little homology with those of all the cadherins described so far. This particular structural arrangement along with the lack of a conserved cytoplasmic domain, characteristic of vertebrate cadherins, may contribute to a unique tertiary structure and physiological function for BT-R(1). Although the function of BT-R(1) is unknown, there is evidence suggesting that BT-R(1) may be involved in membrane transport(28, 29) . Possibly, its function is similar to that of the cadherin-like human intestinal peptide transport protein, which channels peptide antibiotics through epithelial cells that line the small intestine (25) .

B. thuringiensis toxins are thought to act primarily at epithelial cells in the midgut of sensitive insects(2) . It has been suggested that the toxins bind to a specific membrane receptor and then are inserted into the membrane to form a pore that alters membrane permeability. The ultimate consequence is lysis of the epithelial cells and death of the insect(30) . Using competition binding experiments, Van Rie et al.(4) concluded that M. sexta brush-border membranes have two cryIA toxin-binding sites, one recognized by all three toxins (cryIA(a), cryIA(b), and cryIA(c)) and another that recognizes only cryIA(b). In ligand blots of M. sexta brush-border membrane vesicle proteins, the 210-kDa receptor is capable of specifically recognizing all three cryIA toxins(6, 31) . This phenomenon indicates that the cloned BT-R(1) contains a common binding site for all cryIA toxins identified by Van Rie et al.(4) .

To our knowledge, this report is the first to describe the cloning and expression of an insect receptor for a B. thuringiensis toxin. Further characterization of BT-R(1) will lead to a better understanding of the molecular mode of action of B. thuringiensis toxins and to an appreciation of the mechanism of insect resistance to B. thuringiensis toxins. In general, such knowledge should facilitate the rational design of environmentally friendly biopesticides for current insect pests as well as for emerging mutant insects resistant to B. thuringiensis toxins.


FOOTNOTES

*
This work was supported in part by Research Agreement 58-319R-3-011 from the Office of International Cooperation and Development, United States Department of Agriculture, and Cooperative Agreement 58-5410-1-135 from the Arthropod-borne Animal Disease Laboratory, Agricultural Research Service, United States Department of Agriculture (to L. A. B.), and by Grant HD-18702 from the National Institutes of Health (to T. H. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

(^1)
The abbreviation used is: Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.


ACKNOWLEDGEMENTS

We express our appreciation to E. L. Belden, R. V. Lewis, and P. E. Thorsness for valuable advice and assistance during the course of this research and to I. L. Kaiser, K. W. Miller, and M. M. Stayton for reviewing the manuscript before submission.


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