Bacillus thuringiensis Cry toxins bound specifically to various proteins via domain III, which had a galactose-binding domain-like fold.

Cry toxins have been reported to bind not only to receptors on insect cells but also to several unrelated proteins. In this study, we investigated the binding properties of Bacillus thuringiensis Cry toxins, focusing on domain III, a Cry toxin region with a structure that of the galactose-binding domain-like. Cry1Aa, Cry1Ac, and Cry8Ca specifically bound to several proteins unrelated to insect midgut cells. Cry1Aa binding to Cry toxin-binding proteins was inhibited by a monoclonal antibody, 2C2, indicating that Cry1Aa binds to these Cry toxin-binding proteins through domain III. Cry1Aa binding to Bombyx mori aminopeptidase N and other Cry toxin-binding proteins was inhibited by carbonic anhydrase, a Cry toxin-binding protein. The binding regions of carbonic anhydrase and Bombyx mori aminopeptidase N were narrowed to regions of less than 20 amino acids that did not have any similarity, suggesting that Cry toxin domain III has a binding pocket for multiple proteins.

Localization of the serine protease homolog BmSPH-1 in nodules of E. coli-injected Bombyx mori larvae and functional analysis of its role in nodule melanization.

The molecular mechanisms underlying nodule formation and melanization, an important pathogen defense mechanism in insects, are poorly understood. In this study, we investigated the role of BmSPH-1, a catalytically inactive Bombyx mori serine protease homolog, in nodule melanization induced by injection of Escherichia coli cells into the B. mori larval hemocoel. Addition of the melanization substrate L-3,4-dihydroxyphenylalanine (DOPA) to newly formed nodules prompted nodule melanization, confirming that nodules contain activated prophenoloxidase needed for melanization. Immunoprecipitation and immunoblot studies demonstrated that BmSPH-1 interacts with BmLBP, a C-type lectin that binds Gram-negative bacteria, and that BmSPH-1 is present in a truncated, putatively activated form at the E. coli cell surface in nodules. Pretreatment of larvae with anti-BmSPH-1 serum inhibited nodule melanization in E. coli-injected larvae. These results suggest that BmSPH-1 regulates nodule melanization and is recruited into nodules from the hemolymph by BmLBP.

Analysis of the region for receptor binding and triggering of oligomerization on Bacillus thuringiensis Cry1Aa toxin.

The determination of the receptor-binding region of Cry toxins produced by Bacillus thuringiensis is expected to facilitate an improvement in their insecticidal ability through protein engineering. We analyzed the region on Cry1Aa molecules involved in interactions with the cadherin-like protein receptor BtR175 using cysteine-substituted mutant toxins and several synthetic peptides corresponding to the loops in domain 2. In addition, the region necessary to trigger oligomerization was analyzed using these mutant toxins. The mutant toxins were modified by two types of molecule, i.e. digested fragments of the Cry1Aa precursor with an average molecular mass of 2 kDa and 5-iodoacetamidofluorescein, which has a molecular mass of 515 kDa. We examined whether these modifications interfere with the toxin-BtR175 interaction as a result of steric hindrance. 5-Iodoacetamidofluorescein modification of R311C, N376C and G442C revealed steric hindrance effects, indicating that R311 on loop 1, N376 on loop 2 and G442 on loop 3 are on the contact face of the toxin-BtR175 interface when Cry1Aa binds to BtR175. Loop 2 is thought to interact with BtR175 directly, as a peptide corresponding to the N-terminal half of loop 2, (365)LYRRIILG(372), has the potential to bind to BtR175 fragments. Meanwhile, mutant toxins with cysteine substitutions in loops 1 and 2 were oligomerized by the binding of digested fragments in the activation process without receptor interaction, and the wild-type toxin formed oligomers by interaction with BtR175 fragments. These observations suggest that loops 1 and 2 form both a binding region and a sensor region, which triggers toxin oligomer formation. Structured digital abstract: * MINT-7259673, MINT-7259722, MINT-7259737, MINT-7259757, MINT-7259774, MINT-7259791, MINT-7259808, MINT-7259685, MINT-7259707, MINT-7259830: btr175 (uniprotkb:Q9XY09) binds (MI:0407) to cry1Aa (uniprotkb:P0A366) by surface plasmon resonance (MI:0107).

Identification and comparative analysis of three novel C-type lectins from the silkworm with functional implications in pathogen recognition.

C-type lectins can act as pattern recognition receptors (PRRs) in innate immunity. Previously, we identified two C-type lectins from silkworm (Bombyx mori), BmLBP and BmMBP, as PRRs. In the present study, we identified three homologs of these lectins by searching the silkworm genome database. These novel B. mori low-expression lectins were designated BmLEL-1, BmLEL-2, and BmLEL-3. Although Western-blot analysis failed to detect BmLEL-1, -2, or -3 in plasma, affinity precipitation of larval plasma with various microorganisms revealed that BmLEL-1 and -2 bind to rough and smooth strains of Gram-negative bacteria, respectively. BmLEL-1, -2, and -3 were found to be expressed in testis and ovary, where BmLEL-2 expression was up-regulated after bacteria infection. These results indicate that the novel C-type lectins might play a role in the innate immunity in these tissues as PRRs. Here, we discuss the roles and members of the C-type lectins as primary PRRs in B. mori cellular immunity.

Location of the Bombyx mori 175kDa cadherin-like protein-binding site on Bacillus thuringiensis Cry1Aa toxin.

To identify and gain a better understanding of the cadherin-like receptor-binding site on Bacillus thuringiensis Cry toxins, it is advantageous to use Cry1Aa toxin, because its 3D structure is known. Therefore, Cry1Aa toxin was used to examine the locations of cadherin-like protein-binding sites. Initial experiments examining the binding compatibility for Cry1Aa toxin of partial fragments of recombinant proteins of a 175kDa cadherin-like protein from Bombyx mori (BtR175) and another putative receptor for Cry1Aa toxin, amino peptidaseN1, from Bo.mori (BmAPN1), suggested that their binding sites are close to each other. Of the seven mAbs against Cry1Aa toxin, two mAbs were selected that block the binding site for BtR175 on Cry1Aa toxin: 2A11 and 2F9. Immunoblotting and alignment analyses of four Cry toxins revealed amino acids that included the epitope of mAb 2A11, and suggested that the area on Cry1Aa toxin blocked by the binding of mAb 2A11 is located in the region consisting of loops2 and 3. Two Cry1Aa toxin mutants were constructed by substituting a Cys on the area blocked by the binding of mAb 2A11, and the small blocking molecule, N-(9-acridinyl)maleimide, was introduced at each Cys substitution to determine the BtR175-binding site. Substitution of Tyr445 for Cys had a crippling effect on binding of Cry1Aa toxin to BtR175, suggesting that Tyr445 may be in or close to the BtR175-binding site. Monoclonal antibodies that blocked the binding site for BtR175 on Cry1Aa toxin inhibited the toxicity of Cry1Aa toxin against Bo.mori, indicating that binding of Cry1Aa toxin to BtR175 is essential for the action of Cry1Aa toxin on the insect.

A system for the directed evolution of the insecticidal protein from Bacillus thuringiensis.

Theoretically, the activity of AB-type toxin molecules such as the insecticidal toxin (Cry toxin) from B. thuringiensis, which have one active site and two binding site, is improved in parallel with the binding affinity to its receptor. In this experiment, we tried to devise a method for the directed evolution of Cry toxins to increase the binding affinity to the insect receptor. Using a commercial T7 phage-display system, we expressed Cry1Aa toxin on the phage surface as fusions with the capsid protein 10B. These recombinant phages bound to a cadherin-like protein that is one of the Cry1Aa toxin receptors in the model target insect Bombyx mori. The apparent affinity of Cry1Aa-expressing phage for the receptor was higher than that of Cry1Ab-expressing phage. Phages expressing Cry1Aa were isolated from a mixed suspension of phages expressing Cry1Ab and concentrated by up to 130,000-fold. Finally, random mutations were made in amino acid residues 369-375 in domain 2 of Cry1Aa toxin, the mutant toxins were expressed on phages, and the resulting phage library was screened with cadherin-like protein-coated beads. As a result, phages expressing abnormal or low-affinity mutant toxins were excluded, and phages with high-affinity mutant toxins were selected. These results indicate that a method combining T7 phage display with selection using cadherin-like protein-coated magnetic beads can be used to increase the activity of easily obtained, low-activity Cry toxins from bacteria.

Characterization of a novel C-type lectin, Bombyx mori multibinding protein, from the B. mori hemolymph: mechanism of wide-range microorganism recognition and role in immunity.

To investigate the system used by insects to recognize invading microorganisms, we examined proteins from the larval hemolymph of Bombyx mori that bind to the cell surface of microorganisms. Two hemolymph proteins that bound to the cell surfaces of Micrococcus luteus and Saccharomyces cerevisiae were shown to be identical. This protein bound to all 11 microorganisms examined-5 Gram-negative bacteria, 3 Gram-positive bacteria, and 3 yeasts-and was consequently designated B. mori multibinding protein (BmMBP). The sequence of the cDNA encoding BmMBP revealed that it was a C-type lectin with two dissimilar carbohydrate-recognition domains (CRD1 and CRD2) distantly related to known insect C-type lectins. CRD1 and CRD2 were prepared as recombinant proteins and their binding properties were investigated using inhibition assays. Each domain had wide, dissimilar binding spectra to sugars. These properties enable BmMBP to bind to two sites on a microorganism, facilitating high-affinity binding to many types of microorganisms. The dissociation constants of BmMBP with M. luteus cells and S. cerevisiae were 1.23 x 10(-8) and 1.00 x 10(-11) M, respectively. rBmMBP triggered the aggregation of hemocytes from B. mori larvae in vitro and microorganisms recognized by BmMBP were surrounded by aggregated hemocytes in vivo, forming a nodule, which is the typical cellular reaction in insect immune responses. These observations suggest that BmMBP functions as a trigger for the nodule reaction and that the multirecognition characteristic of BmMBP plays an important role in the early stages of infection by a variety of microorganisms.

Mechanism by which Bombyx mori hemocytes recognize microorganisms: direct and indirect recognition systems for PAMPs.

Hemocytes play an important role in cellular reactions in the immune system. Although the recognition of pathogens is thought to involve pattern-recognition proteins (PRPs) in insects, the exact mechanisms by which insect hemocytes recognize pathogens are not clear. This study examined the mechanism by which Bombyx mori hemocytes recognize microorganisms and pathogen-associated molecular patterns (PAMPs) using flow cytometry and fluorescence microscopy. Fluorescence-labeled bacterial or fungal cells were observed to bind to hemocytes and this binding was inhibited by adding lipoteichoic acid (LTA) or beta-1,3-glucan. Lipopolysaccharide (LPS) bound to hemocytes directly. These results suggest that hemocytes have a mechanism that recognizes LPS, LTA, and beta-1,3-glucan directly. Previously, we identified two types of C-type lectin (BmLBP and BmMBP) and showed that they recognize a variety of PAMPs leading to the induction of nodule formation. These lectins enhanced hemocyte binding to microorganisms and their direct binding to hemocytes suggests that hemocytes have a mechanism for recognizing microorganisms using lectin receptors.

Purification and cDNA cloning of a cecropin from the longicorn beetle, Acalolepta luxuriosa.

We have cloned and characterized a novel antibacterial peptide from the hemolymph of the coleopteran insect Acalolepta luxuriosa, of the superfamily Cerambyocidea. This peptide is active against Micrococcus luteus and Escherichia coli, and the amino acid sequence deduced by cloning of the cDNA identifies it as a coleopteran cecropin. Sequence comparisons and phylogenetic analyses performed using Clustal X suggest that this cecropin is evolutionarily intermediate between dipteran and lepidopteran cecropins. The results of MALDI-TOF mass spectrometry indicate that the mature form of this antibacterial peptide is 35 amino acid residues in length and has an amidated C-terminal isoleucine. This report is the first description of a cecropin from a coleopteran insect.

Location of the Bombyx mori aminopeptidase N type 1 binding site on Bacillus thuringiensis Cry1Aa toxin.

We analyzed the binding site on Cry1Aa toxin for the Cry1Aa receptor in Bombyx mori, 115-kDa aminopeptidase N type 1 (BmAPN1) (K. Nakanishi, K. Yaoi, Y. Nagino, H. Hara, M. Kitami, S. Atsumi, N. Miura, and R. Sato, FEBS Lett. 519:215-220, 2002), by using monoclonal antibodies (MAbs) that block binding between the binding site and the receptor. First, we produced a series of MAbs against Cry1Aa and obtained two MAbs, MAbs 2C2 and 1B10, that were capable of blocking the binding between Cry1Aa and BmAPN1 (blocking MAbs). The epitope of the Fab fragments of MAb 2C2 overlapped the BmAPN1 binding site, whereas the epitope of the Fab fragments of MAb 1B10 did not overlap but was located close to the binding site. Using three approaches for epitope mapping, we identified two candidate epitopes for the blocking MAbs on Cry1Aa. We constructed two Cry1Aa toxin mutants by substituting a cysteine on the toxin surface at each of the two candidate epitopes, and the small blocking molecule N-(9-acridinyl)maleimide (NAM) was introduced at each cysteine substitution to determine the true epitope. The Cry1Aa mutant with NAM bound to Cys582 did not bind either of the two blocking MAbs, suggesting that the true epitope for each of the blocking MAbs was located at the site containing Val582, which also consisted of 508STLRVN513 and 582VFTLSAHV589. These results indicated that the BmAPN1 binding site overlapped part of the region blocked by MAb 2C2 that was close to but excluded the actual epitope of MAb 2C2 on domain III of Cry1Aa toxin. We also discuss another area on Cry1Aa toxin as a new candidate site for BmAPN1 binding.

Purification and cDNA cloning of luxuriosin, a novel antibacterial peptide with Kunitz domain from the longicorn beetle, Acalolepta luxuriosa.

We have purified a novel antibacterial peptide from the hemolymph of the coleopteran insect Acalolepta luxuriosa, of the family Cerambyocidae, and named it luxuriosin. This peptide showed growth-inhibitory activity against Micrococcus luteus and germination- and/or growth-inhibitory activity against the conidia from rice blast fungus, Magnaporthe grisea. The amino acid sequence determined by cDNA cloning identified luxuriosin as a peptide of 88 amino acids with a theoretical molecular weight of 10,368.34, containing a Kunitz domain.

Aminopeptidase N isoforms from the midgut of Bombyx mori and Plutella xylostella -- their classification and the factors that determine their binding specificity to Bacillus thuringiensis Cry1A toxin.

Novel aminopeptidase N (APN) isoform cDNAs, BmAPN3 and PxAPN3, from the midguts of Bombyx mori and Plutella xylostella, respectively, were cloned, and a total of eight APN isoforms cloned from B. mori and P. xylostella were classified into four classes. Bacillus thuringiensis Cry1Aa and Cry1Ab toxins were found to bind to specific APN isoforms from the midguts of B. mori and P. xylostella, and binding occurred with fragments that corresponded to the BmAPN1 Cry1Aa toxin-binding region of each APN isoform. The results suggest that APN isoforms have a common toxin-binding region, and that the apparent specificity of Cry1Aa toxin binding to each intact APN isoform seen in SDS-PAGE is determined by factors such as expression level in conjunction with differences in binding affinity.