(Received for publication, November 8, 1994)
From the
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) 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
shares sequence similarity with the cadherin
superfamily of proteins.
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.
Figure 1:
BT-R purification and
cyanogen bromide digestion. A, natural BT-R
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
protein; lane3, ligand blot of purified protein with
I-cryIA(b) toxin. B, purified
BT-R
(
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.
Figure 6:
In vitro translation and N-glyconase digestion of BT-R. A, in
vitro translation of the BT-R
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
mRNA; lane3, translation products
immunoprecipitated with normal serum; lane4,
translation products immunoprecipitated with anti-BT-R
serum. B, N-glycanase F treatment of
BT-R
. Purified BT-R
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.
Previously, we described the identification, purification,
and characterization of natural BT-R, which specifically
recognizes the cryIA(b) toxin of B. thuringiensis subsp. berliner(6) . The binding of the toxin to
natural BT-R
present in brush-border membrane vesicles of M. sexta was specific and high, with a K
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
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
matched perfectly within the
deduced sequence of BT-R
(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 and alignment of BT-R
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
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
(BT-R
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 cDNA clone. The probe hybridized to a
single 5.6-kilobase band that corresponds to the 5571-base-long
BT-R
cDNA clone (Fig. 3). This result indicates that
the BT-R
cDNA clone represents the full-length coding
sequence of the BT-R
gene. To demonstrate that the protein
encoded by the BT-R
cDNA is a membrane protein and is
capable of binding cryIA(b) toxin, the BT-R
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
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
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
was expressed on the surface of transfected human
embryonic 293 cells and showed high affinity (K
= 1 nM) for the toxin (Fig. 5), as did
natural BT-R
(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
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 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
cDNA clone. B, ligand blot analysis of 293
cells stably expressing BT-R
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
cDNA.
Figure 5:
Binding of I-cryIA(b) toxin to intact transfected human embryonic 293 cells expressing
BT-R
. The cells were transfected with the BT-R
cDNA in pcDNA3 and incubated with
I-cryIA(b) toxin (0.32 nM) in the presence of increasing
concentrations (0-10
M) 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 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
. In vitro translation of the BT-R
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
(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
, and the
difference in size presumably is due to glycosylation. N-Glycanase F treatment reduced the molecular mass of native
BT-R
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
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 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
(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 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
is highly
repetitive and contains 11 repeats (Fig. 2A). The
length of the BT-R
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
(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
. Although the function of BT-R
is
unknown, there is evidence suggesting that BT-R
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 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 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.