(Received for publication, June 27, 1995; and in revised form, September 7, 1995)
From the
Bacillus thuringiensis toxins are insecticidal to a
variety of insect species. The selectivity of the toxins produced by
these bacteria is dependent on both the toxin structure and the
receptor sites that are present in different insect species. One of
these toxins, CryIAc, is highly insecticidal to the noctuid pest Heliothis virescens. Using toxin overlay assay, a 120-kDa
glycoprotein was identified as a toxin-binding protein. This protein
was partially purified, its N-terminal sequence was determined, and the
full-length cDNA encoding this protein was isolated from a H.
virescens midgut library. The B. thuringiensis toxin-binding protein, BTBP, has high homology to
aminopeptidase N from eukaryotes and prokaryotes.
Bacillus thuringiensis, a Gram-positive bacterium,
produces insecticidal parasporal inclusions during
sporulation(1) . This insecticidal activity is dependent on a
unique set of inclusion proteins or -endotoxins produced by these B. thuringiensis strains(2) . Most of these
insecticidal proteins are usually produced as large protoxins, about
130 kDa, although smaller naturally truncated proteins are also
observed. When ingested by susceptible insect larvae, the parasporal
inclusions dissolve in the alkaline environment of insect midgut giving
rise to soluble protoxins that are activated by midgut
proteases(2, 3) . For example, the 130-kDa CryIAc
protoxin is activated to a 65-kDa toxin in the midgut of lepidopteran Heliothis virescens and Manduca sexta larvae and is
toxic to these insects.
These activated toxins then interact with
the apical membranes of insect midgut columnar
cells(4, 5) . Using preparations of columnar cell
brush-border membrane vesicles (BBMV) ()previous studies
have demonstrated that in susceptible insects, high affinity toxin
binding sites are correlated with the insecticidal activity of B.
thuringiensis toxins(6, 7) . Changes in toxin
structure, even minor, that are associated with decreased toxin binding
result in decreased insecticidal
activity(8, 9, 10) .
To identify the
precise toxin binding targets in insects, midgut cell membranes have
been electrophoretically separated and probed with radiolabeled toxins
in toxin overlay assays. Several CryIAc binding proteins ranging from
150 to 50 kDa were observed in H.
virescens(11, 12, 13, 14) . We
have identified the proteins involved in CryIAc toxicity to H.
virescens using toxin overlay assays (15) . A 120-kDa B. thuringiensis toxin-binding protein, BTBP, from
the midgut brush-border membrane that binds the CryIAc toxin was
partially purified, and its N-terminal sequence was determined. A cDNA
clone encoding this protein was isolated and characterized. The deduced
amino acid sequence shows that the BTBP
belongs to the
aminopeptidase N family of proteins.
This Pool II toxin binding fraction was then
applied to a Ricinis communis agglutinin (RCA) agarose column
in 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM MgSO, 1% CHAPS (w/v) containing
protease inhibitors at 4 °C overnight, with gentle mixing. The
preparation was then poured into a column, washed extensively, and
eluted with 200 mM lactose in the appropriate buffer, and the
eluted material was concentrated in a stirred cell using PM10 filters
(Amicon).
Dideoxy double-stranded sequencing of the cDNA insert was performed using Sequenase 2.0 as described by the manufacture (U. S. Biochemical).
Figure 1: Partial purification of the CryIAc binding protein from H. virescens midgut. Lane 1, BBMV; lane 2, CHAPS-solubilized BBMV; lanes 3 and 4, fractions I and II isolated from Mono Q column; lane 5, flow-through from RCA column; lane 6, RCA-bound fraction eluted with 0.2 M lactose. The gel is stained with Coomassie Blue, and each lane contains 10 µg of protein, except lane 6, which contains 2.5 µg of protein.
Toxin overlay assay (15) showed that the 120- and 105-kDa RCA-purified proteins bound iodinated CryIAc toxin (Fig. 2, lane 2) and that addition of excess unlabeled CryIAc toxin significantly decreased binding (Fig. 2, lane 3). Low level binding was also observed with a 140-kDa protein, but this binding was not as readily displaced with excess unlabeled CryIAc toxin. The 140-kDa protein is not visible in the SDS-polyacrylamide gel (Fig. 1, lane 6). The 170-kDa protein did not bind the CryIAc toxin. Similar results were obtained with Mono Q Pool II fractionated proteins, except the 170-kDa protein also bound the CryIAc toxin(15) .
Figure 2:
Analysis of CryIAc toxin binding to RCA
isolated fractions. Lane 1 contains 10 µg of RCA isolated
fraction stained with Coomassie Blue. Lanes 2 and 3 are autoradiographs; lane 2 is probed with 1 nMI-CryIAc, and lane 3 is probed with 1
nM
I-CryIAc and 500 nM cold
CryIAc.
Analysis of enzyme activity in the various fractions during
purification through the Mono Q anion-exchange chromatography shows
that alkaline phosphatase and aminopeptidases are enriched in the BBMV,
solubilized BBMV, and in Pool II (Table 1). The alkaline
phosphatase activity was enriched about 17 from that found in
crude homogenates, while the activities of leucine, lysine, and
phenylalanine aminopeptidases were enriched 10, 14 and 9
,
respectively. Leucine aminopeptidase, an enzyme used as a marker for
insect BBMV(22) , had the highest specific activity in the Mono
Q pooled fraction, Pool II.
The sequence YRLPT was conserved in all three insect CryIAc toxin-binding proteins, and the sequence YRLP was conserved in all of the proteins compared (Table 2). Oligonucleotide primers, forward and reverse, were made ensuring the sequence YRLPT was present at the 3` end of the primers. When used in conjunction with a primer to conserved regions of aminopeptidase N (AMN2R) in PCR screening, both the M. sexta (MS1F) and H. virescens (HV1F) primers gave the same size product, 0.85 kilobase. Reamplification of these PCR products with AMN1F and AMN1R gave products of the expected size, 170 bp. Amplification of one of the fractionated libraries, number 3, using the primers V1F and AMN1R, followed by reamplification using V2F and HV1R, showed the presence of prominent bands between 220 and 300 bp, suggesting the presence of cDNA sequence upstream of the N-terminal sequences obtained. Electroporation of fraction 3 plasmid DNA into DH10B cells and successive rounds of PCR screening (21) resulted in the isolation of two clones. Nucleotide sequencing of these two clones showed that one clone contained a sequence that matched that of the 120-kDa protein. This clone had an insert of 3419 bp, with a 3027-bp open reading frame encoding a 1009-amino acid protein (Fig. 3). The putative translation start site at nucleotide 35 contains a consensus Kozak sequence, AAGATGG (26) . A polyadenylation sequence, AATAAA(27) , at nucleotide 3385 precedes the poly(A) tail, which is 337 bp downstream of the termination codon.
Figure 3:
Deduced amino acid sequence of the CryIAc
binding protein from H. virescens midgut. The oligonucleotide
sequence of the entire 3471-bp cDNA was sequenced in both orientations.
The hydrophobic N and C termini are underlined. The N terminus
of the mature protein that was sequenced is underlined and in boldface. The putative N-glycosylation sites are double underlined. The putative metal binding sites are
indicated by , and the putative nucleophile is indicated by
. The putative GPI-anchor signal sequence is indicated by a dotted underline. Two hydrohobic domains are observed. The
first is at the N terminus, amino acids 1-19, and the second is
at the C terminus between amino acids
933-1009.
The protein, BTBP, has a
calculated molecular weight of 113,461 Da and a pI of 5.29. The
N-terminal sequence obtained from protein sequencing is between amino
acids 53 and 63 and indicates that the mature BTBP
isolated
from H. virescens BBMV has 52 amino acids cleaved from the N
terminus. Both the N and C termini are hydrophobic. Two potential N-glycosylation sites are observed at amino acid residues 581
and 906. A metal binding motif, HEXXH (28, 29) is observed at 374-378.
Figure 4:
Autoradiograph of inhibition of CryIAc
binding to RCA purified fraction by N-acetylgalactosamine. Lane 1, 10 µg of RCA-isolated fractions are probed with 1
nMI-CryIAc, and lane 2 is probed with
1 nM
I-CryIAc in the presence of 200 mM GalNAC.
In vitro transcription and translation of BTBP cDNA gives a
protein of about 113 kDa (Fig. 5, lane 3). This size,
as predicted from the deduced amino acid sequence, lends support to the
putative start site. The in vitro translated CryIAc binding
protein was immunoprecipitable with anti-H. virescens BBMV
antibodies (Fig. 5, lane 4), but not with preimmune
antibodies (lane 5). The smaller and weakly labeled products (lane 3) are derived either from incomplete transcription and
translation or are proteolytically cleaved BTBP
, since
these products all are immunoprecipitable only by anti-BBMV antibodies (lane 4) and not by preimmune antibodies (lane 5). No
products were obtained when only the vector was used for in vitro transcription and translation (Fig. 5, lanes 1 and 2). Immunolocalization studies show these antibodies react
with the midgut brush-border membrane of H. virescens (data
not shown), demonstrating that the cloned BTBP
is
apparently localized to the brush-border membrane of H. virescens midgut. The in vitro translated BTBP
protein
does not bind CryIAc toxin (data not shown) consistent with previous
data showing the CryIAc toxin binding to carbohydrate moieties of this
120-kDa protein.
Figure 5:
Autoradiograph of in vitro transcribed and translated CryIAc binding protein binding to
antibodies to BBMV. The total or the products immunoprecipitated by
rabbit anti-H. virescens BBMV antibodies were separated by
SDS-PAGE and then subjected to autoradiography. Lane 1, in
vitro transcribed and translated products of the plasmid vector
minus the BTBP cDNA labeled with
[
S]methionine. Lane 2, the product in lane 1 was immunoprecipitated with anti-BBMV antibodies. Lane 3, in vitro transcribed and translated products
of the BTBP
cDNA labeled with
[
S]methionine. Lane 4, the product in lane 3 was immunoprecipitated with anti-BBMV antibodies. Lane 5, the product in lane 3 was immunoprecipitated
with preimmune rabbit serum.
Protein purification, N-terminal sequencing, and
characterization of the cDNA isolated identify the CryIAc toxin-binding
protein from the midgut of H. virescens as an aminopeptidase
N-like protein (Fig. 6). An aminopeptidase N from M. sexta, that also binds the CryIAc toxin, has recently been
reported(30) . The H. virescens BTBP has
42 and 62% identity and similarity, respectively, to the M. sexta protein. Comparison of these two sequences suggests the M.
sexta CryIAc binding protein probably has an additional
10-25 amino acids upstream of the reported N-terminal
sequence(30) . Similar levels of identity, although slightly
lower, are observed with other eukaryote aminopeptidases N,
zinc-dependent metalloproteases(28, 29) . For example, H. virescens BTBP
has 32 and 52% identity and
similarity, respectively, to human aminopeptidase N.
Figure 6:
Comparison of H. virescens BTBP with known aminopeptidases. BTBP
sequence was compared with nucleotide sequences (Genbank, release
88.0) and with protein sequences (Swiss Protein, release 31.0). The
greatest similarity is observed with zinc-dependent metalloproteases,
which includes aminopeptidases N. Only some sequences are used for
comparison and include human(24) , Caenorhabditis
elegans(54) , mouse(51) , Saccharomyces(55) , and Lactobacilli(56) . The M. sexta sequence used
was as published recently(30) .
The BTBP N terminus is quite divergent from that of other aminopeptidases.
The first 15 amino acids are highly hydrophobic (2.6) and probably
contain an appropriate signal sequence that facilitates membrane
targeting(31, 32) . However, unlike mammalian
aminopeptidases, where the mature protein undergoes limited truncation
at the N terminus(33) , the first 52 amino acid residues of H. virescens BTBP
are cleaved. This can be
explained, in part, by the different membrane anchors used by H.
virescens BTBP
, and those used by aminopeptidase N in
mammals. In the latter, aminopeptidase N is membrane-bound apparently
by a hydrophobic N terminus(33) , and trypsin treatment results
in cleavage of the membrane anchor, with the resulting soluble protein
beginning at amino acid residue 40(33) . In insects,
aminopeptidase N is apparently membrane bound at the C terminus. The
soluble form of the insect leucine aminopeptidase is obtained by
treatment with phosphatidylinositol-specific phospholipase
C(34, 35) . Immunoprecipitation of BTBP
by
BBMV-specific antibodies suggests that this protein is localized to the
midgut cell brush border of H. virescens. Leucine
aminopeptidase activity is often used as a marker for insect midgut
columnar cell brush border(22, 36) .
Unlike the
other aminopeptidases, the H. virescens BTBP has a
long hydrophobic C terminus. In two insect species, Bombyx mori and M. sexta, the midgut aminopeptidase N activity is
membrane-linked via a glycosylphosphatidylinositol (GPI)
anchor(34, 35) . The presence of a long hydrophobic C
terminus preceded by hydrophilic residues suggests that the BTBP
is similarly linked by a GPI anchor. A putative sequence
signaling the addition of GPI anchors(37) , DSA, is observed.
The aspartic acid at 987 is a likely site for attachment of the GPI
anchor, with cleavage of the hydrophobic tail between Asp
and Ser
. The presence of a GPI anchor in H.
virescens, however, needs to be established, and alternative
mechanisms of BTBP
membrane anchoring, however, cannot be
excluded. N- and C-terminal proteolytic cleavage will result in a
105-kDa protein containing 935 amino acid residues. The BTBP
isolated from H. virescens midgut, however, is of 120
kDa, consequently the difference in the molecular masses of these two
proteins results from carbohydrate or other posttranslational
modifications. The M. sexta CryIAc binding protein similarly
has a putative GPI signal sequence, a hydrophobic N terminus, and
identical molecular masses for the processed and isolated
proteins(30) .
The hydrophobic tail and the GPI-linked
anchor could play a crucial role in insects resistant to B.
thuringiensis toxins. Elevation of endogenous
phosphatidylinositol-specific phospholipase C, or B. thuringiensis phosphatidylinositol-specific phospholipase C could result in
decreased levels of membrane-bound BTBP or aminopeptidase N
causing increased tolerance and/or resistance to the CryIAc toxin.
Alternatively down-regulation of BTBP
expression in insect
midgut will result in the loss of toxin binding sites, and decreased
CryIAc toxicity. However, H. virescens resistance to the
CryIAc toxin does not appear to be correlated with decreased toxin
binding(38) , and consequently alternative mechanisms of toxin
resistance are likely. On the other hand, in Plutella xylostella and Plodia interpunctella, resistance to the CryIAb
toxins is correlated with the availability of toxin binding
sites(39, 40, 41) .
A CryIAb toxin-binding
protein has recently been cloned and shown to be a cadherin-like
210-kDa membrane glycoprotein(42) . Unlike the CryIAb-binding
protein, which apparently has an intracellular component, BTBP is entirely extracellular and is probably GPI-anchored. Hence it
is unlikely to directly participate in any intracellular changes that
have been observed with B. thuringiensis intoxication(43) , although it could affect external
mediators of signal transduction.
The precise mechanism of B.
thuringiensis toxicity is not known. The generally accepted model
is that following toxin binding to a receptor protein or macromolecule,
the toxin undergoes a conformational change that facilitates toxin
insertion into the apical cell membrane of insect midgut columnar
cells. This initial binding is then followed by oligomerization of the
bound toxin(3, 44, 45) . The cation ion
selective pore formed by this oligomer causes a disruption of osmotic
balance in the midgut epithelial
layer(46, 47, 48) . This disruption of midgut
function ultimately leads to the insect's death (3) .
Potentially the 120-kDa toxin-binding proteins isolated here and that
in M. sexta(23, 25) , since they have an
ability to bind the CryIAc toxin, function to localize the toxin to the
columnar cell membrane in insect midguts. Indeed phospholipid vesicles
containing a 120-kDa aminopeptidase N from M. sexta enhanced
Rb permeability when challenged with the CryIAc
toxin(25) . The CryIAc binding in M. sexta, like that
in H. virescens, is partially blocked by N-acetylgalactosamine, suggesting that the binding occurs on
the carbohydrate moieties of this protein. Two putative N-glycosylation sites are observed in the H. virescens BTBP
protein. Both sites occur in predicted turns in
protein structure (49) and are likely glycosylated. Moreover,
just as in M. sexta(30) , the H. virescens BTBP
protein sequence preceding the GPI signal
sequence is rich in Ser/Thr residues, which could also provide O-linked glycosylated residues required for CryIAc binding.
Furthermore, the addition of a GPI anchor provides additional
carbohydrate moieties for toxin interaction. The nature of these
carbohydrate moieties, at the N- and O-glycosylation
sites, and at the GPI anchor, is not known. Their characterization
should provide a better understanding of the selectivity of B.
thuringiensis toxins.
If B. thuringiensis CryIAc toxin activity is mediated predominantly by the carbohydrate moieties, it is likely that the differing responses observed between insects is due to heterogeneity in glycosylation. This heterogeneity in glycosylation could in part explain the rapid recovery of susceptibility from previously resistant insects(41) . A variety of glycoproteins could function as toxin-binding proteins, hence facilitating toxin interaction with the midgut epithelium.
Aminopeptidases N are not only widely distributed in a number of mammalian cell types, but they also appear to play various roles in these cell types(50) . In mice they are involved in hematopoeisis (51) and in the degradation of extracellular matrix and tumor cell invasion (52) . In intestinal epithelia, aminopeptidase N is involved in peptidyl bond cleavage releasing amino acids that are transported across the brush-border membrane(50) . Their high level expression in a variety of cell types facilitates the entry of certain coronaviruses in humans (53) . Hence the interaction observed here with the CryIAc toxin similarly enables B. thuringiensis to be insecticidal.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U35096[GenBank].