Molecular characterization of four midgut aminopeptidase N isozymes from the cabbage looper, Trichoplusia ni

Four aminopeptidase N (APN) isoforms, TnAPN1, TnAPN2, TnAPN3 and TnAPN4, were identified from the cabbage looper, Trichoplusia ni, by cDNA cloning. The deduced amino acid sequences of the four APNs indicate that TnAPN1, TnAPN2, TnAPN3 and TnAPN4 are synthesized as pre-proteins of 110, 106, 114 and 108 kDa, respectively. Sequence features of the T. ni APNs include the presence of a signal peptide at their N-termini and a prepeptide at the C-termini for the GPI anchor, the zinc binding/gluzincin motif HEX2HX18E, the gluzincin aminopeptidase motif GAMENWG and the presence of glycosylation sites. After removal of the signal peptide and the C-terminal prepeptide, the predicted molecular weights of TnAPN1, TnAPN2, TnAPN3 and TnAPN4 are 106, 102, 110 and 104 kDa, respectively. Enzymatic activity assays of various larval tissues showed that aminopeptidase activities were mainly localized in the midgut and the specific enzyme activity per mg of midgut tissue proteins was constant in T. ni larvae regardless of the composition of dietary proteins and amino acids. Both enzyme activity assays and RT-PCR analyses for the expression of the APN genes in T. ni larval tissues demonstrated that APN genes were expressed in Malphigian tubules in addition to the midgut, which was the first observation that APNs were also synthesized in insect Malphigian tubules. The finding of APN gene expression and enzyme activity in the Malphigian tubules indicated the biochemical and functional similarity of the insect Malphigian tubules to the mammalian counterpart, the kidney, in which APNs are known to play important functions.     

棉铃虫幼虫中肠非糖基磷酯酰肌醇锚着氨肽酶N基因的克隆和测序

通过对棉铃虫Helicoverpa armigera (Hübner)幼虫中肠氨肽酶N的克隆和测序,鉴定了1个氨肽酶N基因APN1,其cDNA序列具有3 220个核苷酸,具有3 042 bp的开放阅读框,编码产生1 014个氨基酸的蛋白质。其推定的氨基酸序列具有氨肽酶N所共有的锌结合模体HEXXHX₁₈E和N末端20个氨基酸的疏水性信号序列,但C末端没有糖基磷酯酰肌醇（glycosylphosphatidylinositol,GPI）锚添加信号序列。该氨肽酶N的cDNA序列已提交GenBank,登录号为AY358034。     

Cloning of a Heliothis virescens 110 kDa aminopeptidase N and expression in Drosophila S2 cells

We previously identified a novel Heliothis virescens 110 kDa aminopeptidase N (APN) that binds Bacillus thuringiensis (Bt) Cry1Ac and Cry1Fa delta-endotoxins, and cloned an internal region of the 110 kDa APN gene (Banks et al., 2001). Here we describe the RACE-PCR cloning and sequence of a cDNA encoding 110 kDa APN. The 110 kDa APN gene was transiently co-expressed with green fluorescent protein (GFP) in Drosophila S2 cells using the pIZT expression vector. Enrichment of total membranes purified from S2 cells transfected with the 110 kDa APN gene had 3.3 fold increased APN enzymatic activity relative to enriched total membranes purified from S2 cells transfected with vector alone. Whereas the majority of S2 cells transfected with the 110 kDa APN gene bound rhodamine-labeled Cry1Ac toxin, no S2 cells transfected with vector alone bound rhodamine-labeled Cry1Ac toxin. This indicates that toxin binding to whole cells is APN mediated. However, flow cytometry and microscopy indicated that 110 kDa APN transfected S2 cells exposed to Cry1Ac or Cry1Fa toxin did not experience an increase in membrane permeability, indicating that APN transfected cells were resistant to toxin. This suggests while the H. virescens 110 kDa APN functions as a Bt toxin binding protein, it does not mediate cytotoxicity when expressed in S2 cells.     

Binding of Bacillus thuringiensis delta-endotoxins Cry1Ac and Cry1Ba to a 120-kDa aminopeptidase-N of Epiphyas postvittana purified from both brush border membrane vesicles and baculovirus-infected Sf9 cells

A 120-kDa protein was purified from brush border membrane vesicles of the tortricid moth Epiphyas postvittana (Walker) based both on its activity as an aminopeptidase and the ability to bind the Bacillus thuringiensis delta-endotoxin Cry1Ac. The purified enzyme had a pI of 5.6 and was a leucine aminopeptidase, with some isoleucine, phenylalanine and tryptophan aminopeptidase activity. Further characterisation showed that the protein was also able to bind Cry1Ba. During purification, the molecular weight of the protein decreased from 120 to 115 kDa due to the loss of a glycophosphatidinyl anchor. The protein was N-terminally sequenced and, using this information and conserved regions within other insect aminopeptidase-N (APN) sequences, redundant primers were designed to amplify the aminopeptidase coding sequence from E. postvittana midgut cDNA. The predicted protein sequence from the full-length cDNA was most closely related to the APN protein sequence from Heliothis virescens (61% identity) and shared other features of insect APNs including a Zn(2+) binding site motif and four conserved cysteines. The E. postvittana was expressed in Sf9 cells using baculovirus, yielding a protein of molecular weight 130 kDa, but with unchanged N-terminal sequence. Purified recombinant protein bound both Cry1Ac and Cry1Ba by ligand blot assays. However, despite the protein being expressed on the external surface of the Sf9 cells, it bound neither Cry1Ac nor Cry1Ba in vivo.     

A novel 96-kDa aminopeptidase localized on epithelial cell membranes of Bombyx mori midgut, which binds to Cry1Ac toxin of Bacillus thuringiensis

Proteins in the brush border membrane (BBM) of the midgut binding to the insecticidal Cry1Ac toxin from Bacillus thuringiensis were investigated to examine the lower sensitivity of Bombyx mori to Cry1Ac, and new aminopeptidase N that bound to Cry1Ac was discovered. DEAE chromatography of Triton X-100-soluble BBM proteins from the midgut revealed 96-kDa aminopeptidase that bound to Cry1Ac. The enzyme was purified to homogeneity and estimated to be a 96.4-kDa molecule on a silver-stained SDS-PAGE gel. However, the native protein was eluted as a single peak corresponding to approximately 190-kDa on gel filtration and gave a single band on native PAGE. The enzyme was determined to be an aminopeptidase N (APN96) from its substrate specificity. Antiserum to class 3 B. mori APN (BmAPN3) recognized APN96, but peptide mass fingerprinting revealed that 54% of the amino acids of matched peptides were identical to those of BmAPN3, suggesting that APN96 was a novel isoform of the APN3 family. On ligand blots, APN96 bound to Cry1Ac but not Cry1Aa or Cry1Ab, and the interaction was inhibited by GalNAc. K(D) of the APN96-Cry1Ac interaction was determined to be 1.83 +/- 0.95 microM. The lectin binding assay suggested that APN96 had an N-linked bi-antennal oligosaccharide or an O-linked mucin type one. The role of APN96 was discussed in relation to the insensitivity of B. mori to Cry1Ac.     

Analysis of glycan structures on the 120 kDa aminopeptidase N of Manduca sexta and their interactions with Bacillus thuringiensis Cry1Ac toxin

The Bacillus thuringiensis Cry1Ac toxin specifically binds to a 120 kDa aminopeptidase N (APN) receptor in Manduca sexta. The binding interaction is mediated by GalNAc, presumably covalently attached to the APN as part of an undefined glycan structure. Here we detail a simple, rapid and specific chemical deglycosylation technique, applicable to glycoproteins immobilized on Western blots. We used the technique to directly and unambiguously demonstrate that carbohydrates attached to 120 kDA APN are in fact binding epitopes for Cry1Ac toxin. This technique is generally applicable to all putative Cry toxin/receptor combinations. We analyzed the various glycans on the 120 kDA APN using carbohydrate compositional analysis and lectin binding. The data indicate that in the average APN molecule, 2 of 4 possible N-glycosylation sites are occupied with fucosylated paucimannose [Man(2-3)(Fuc(1-2)GlcNAc(2)-peptide] type N-glycans. Additionally, we identified 13 probable O-glycosylation sites, 10 of which are located in the Thr/Pro rich C-terminal "stalk" region of the protein. It is likely that 5-6 of the 13 sites are occupied, probably with simple [GalNAc-peptide] type O-glycans. This O-glycosylated C-terminal stalk, being GalNAc-rich, is the most likely binding site for Cry1Ac.     

Interaction of Gene-Cloned and Insect Cell-Expressed Aminopeptidase N of Spodoptera litura with Insecticidal Crystal Protein Cry1C

Insecticidal toxins produced by Bacillus thuringiensis interact with specific receptors located in the midguts of susceptible larvae, and the interaction is followed by a series of biochemical events that lead to the death of the insect. In order to elucidate the mechanism of action of B. thuringiensis toxins, receptor protein-encoding genes from many insect species have been cloned and characterized. In this paper we report the cloning, expression, and characterization of Cry toxin-interacting aminopeptidase N (APN) isolated from the midgut of a polyphagous pest, Spodoptera litura. The S. litura APN cDNA was expressed in the Sf21 insect cell line by using a baculovirus expression system. Immunofluorescence staining of the cells revealed that the expressed APN was located at the surface of Sf21 cells. Treatment of Sf21 cells expressing S. litura APN with phosphatidylinositol-specific phospholipase C demonstrated that the APN was anchored in the membrane by a glycosylphosphatidylinositol moiety. Interaction of the expressed receptor with different Cry toxins was examined by immunofluorescence toxin binding studies and ligand blot and immunoprecipitation analyses. By these experiments we showed that the bioactive toxin, Cry1C, binds to the recombinant APN, while the nonbioactive toxin, Cry1Ac, showed no interaction.     

Antisera-mediated in vivo reduction of Cry1Ac toxicity in Helicoverpa armigera

A functional assessment of Bacillus thuringiensis (Bt) toxin receptors in the midgut of lepidopteran insects will facilitate understanding of the toxin mode of action and provide effective strategies to counter the development of resistance. In this study, we produced anti-aminopeptidase (APN) and anti-cadherin sera with purified Cry1Ac toxin-binding APN or cadherin fragments from Heliocoverpa armigera. Antisera were evaluated for their effects on Cry1Ac toxicity through bioassays. Our results indicated that both the anti-APN and anti-cadherin sera reduced Cry1Ac toxicity in vivo, although cadherin antiserum reduced toxicity more than APN antiserum. These results suggest that both APN and cadherin are involved in Cry1Ac intoxication of H. armigera, evidence that the pore formation model may be representative of Cry1Ac toxin mode of action in this insect.     

Purification and characterization of aminopeptidase N from Spodoptera litura expressed in Sf21 insect cells

Insecticidal crystal proteins produced by strains of Bacillus thuringiensis cause larval death upon interaction with specific receptors located at the midgut epithelium of susceptible insects. Large quantities of easily purified aminopeptidase and cadherin-like Cry toxin receptors can facilitate the further study of Cry toxin binding and pore formation. Here, we report the solubilisation and purification of aminopeptidase N from Spodoptera litura (SlAPN). Recombinantly expressed and membrane anchored aminopeptidase N showed differential solubilisation with various ionic and nonionic detergents. The N-lauryl sarcosine (NLS)-solubilised SlAPN was purified to near homogeneity by anion exchange and gel filtration chromatography and refolded to its catalytically active form. The optimized purification regimen lead to >90% purification of the catalytically active SlAPN with 11% recovery and 9-folds purification. The interaction of purified SlAPN with biologically active Cry1C protein has been qualitatively and quantitatively characterized. By ligand blotting experiment, we demonstrated the linearity of interaction of the two purified proteins and lack of interaction of SlAPN with structurally divergent nontoxic Cry1Ac protein. The equilibrium dissociation constant (K(D)) of purified SlAPN for Cry1C was calculated by ELISA (90nM). Interaction of enzymatically inactive SlAPN with Cry1C and catalytic activity of APN-Cry1C complex suggested that the catalytic site and toxin-binding sites of SlAPN do not overlap.     

棉铃虫中肠钙粘蛋白、氨肽酶N及碱性磷酸酯酶的抗血清对Cry1Ac和Cry2Aa毒力的影响

【目的】Cry1A和Cry2A类Bt蛋白通过特异性地与昆虫中肠上的受体蛋白结合而发挥杀虫作用,现已广泛应用于转基因抗虫作物。本研究旨在进一步明确Cry2A类蛋白的作用机制和Cry1A受体蛋白在Cry2A发挥毒力中的作用。【方法】本研究首先提取了棉铃虫Helicoverpa armigera的BBMV,制备了钙粘蛋白(CAD)、氨肽酶N(APN)和碱性磷酸酯酶(ALP)3种受体蛋白的抗体和抗血清;然后,利用Western blot检测BBMV上这3种受体蛋白后,利用抗体封闭技术比较了敏感棉铃虫和Cry1Ac抗性棉铃虫(BtR)中3种受体蛋白的抗血清对Cry1Ac和Cry2Aa毒力的影响。【结果】对敏感品系棉铃虫,这3种已知的Cry1Ac受体蛋白抗血清显著地降低了Cry1Ac和Cry2Aa的毒力。其中APN抗血清对Cry1Ac毒力的影响最大,棉铃虫幼虫的死亡率降低了84.44%;ALP抗血清对Cry2Aa的毒力影响最大,棉铃虫幼虫死亡率比对照降低了71.04%。Cry1Ac对Cry1Ac抗性棉铃虫(BtR)的毒力显著降低,Cry2Aa的毒性也减弱。在Cry1Ac抗性棉铃虫(BtR)中,3种受体抗血清对Cry1Ac的影响比在敏感棉铃虫中的影响小,尤其是CAD和APN抗血清对Cry1Ac毒力的抑制率显著低于在敏感棉铃虫中的抑制作用;CAD和ALP抗血清对Cry2Aa毒力的影响与在敏感棉铃虫中的影响差异不显著,但APN抗血清可以显著降低Cry2Aa对Cry1Ac抗性棉铃虫(BtR)的毒力。【结论】棉铃虫CAD,APN和ALP不仅参与了Cry1Ac的杀虫过程,也对Cry2Aa毒力有一定的影响,而且这3种蛋白可能与棉铃虫对Cry1Ac和Cry2Aa产生抗性及交互抗性相关。     

小菜蛾中肠氨肽酶基因PxAPN5的克隆、原核表达及蛋白质同源建模分析

【目的】克隆和表达小菜蛾Plutella xylostella氨肽酶基因,并进行基因序列分析和同源建模分析。【方法】以小菜蛾中肠cDNA为模板克隆分析氨肽酶基因序列, 原核表达氨肽酶蛋白并进行酶活性测定, 应用配体印迹分析氨肽酶与Cry2Ab蛋白的结合, 通过蛋白质建模对突变位点进行分析。【结果】从小菜蛾中肠cDNA 扩增出氨肽酶基因, 该基因全长2 853 bp, 编码950个氨基酸, 预测蛋白分子量为107.3871 kDa, 等电点为5.24; 进化树分析显示, 克隆得到的氨肽酶基因属于APN家族5, 将其命名为PxAPN5（GenBank登录号: KM034756）。PxAPN5蛋白具有鳞翅目昆虫氨肽酶蛋白的保守性特征, 即含有N-糖基化位点、O-糖基化位点和GPI锚定位点, 具有“HEXXH”锌蛋白酶结构域和C端跨膜区域。在大肠杆菌Escherichia coli中原核表达PxAPN5, 表达产物经SDS-PAGE电泳, 在110 kDa附近出现特异性条带; 酶活性测试显示菌体破碎上清液具有氨肽酶活性, 比活力为1 047.2 U/g。配体印迹结果显示表达的PxAPN5能与Cry2Ab蛋白特异性结合。多序列比对结果表明, 与其他已报道的小菜蛾氨肽酶相比, PxAPN5氨基酸序列有3个保守性位点发生了突变,并通过蛋白质建模的方式表征突变位点。【结论】本研究成功克隆和表达了具有氨肽酶活性的小菜蛾氨肽酶, 并发现其能与Cry2Ab蛋白特异性结合; 通过蛋白质建模对氨肽酶突变位点的特征及功能进行了预测。 这些结果对小菜蛾氨肽酶的功能性研究提供了理论基础。     

Resistance to Bacillus thuringiensis Toxin Cry2Ab in Trichoplusia ni Is Conferred by a Novel Genetic Mechanism

The resistance to the Bacillus thuringiensis (Bt) toxin Cry2Ab in a greenhouse-originated Trichoplusia ni strain resistant to both Bt toxins Cry1Ac and Cry2Ab was characterized. Biological assays determined that the Cry2Ab resistance in the T. ni strain was a monogenic recessive trait independent of Cry1Ac resistance, and there existed no significant cross-resistance between Cry1Ac and Cry2Ab in T. ni. From the dual-toxin-resistant T. ni strain, a strain resistant to Cry2Ab only was isolated, and the Cry2Ab resistance trait was introgressed into a susceptible laboratory strain to facilitate comparative analysis of the Cry2Ab resistance with the susceptible T. ni strain. Results from biochemical analysis showed no significant difference between the Cry2Ab-resistant and -susceptible T. ni larvae in midgut proteases, including caseinolytic proteolytic activity and zymogram profile and serine protease activities, in midgut aminopeptidase and alkaline phosphatase activity, and in midgut esterases and hemolymph plasma melanization activity. For analysis of genetic linkage of Cry2Ab resistance with potential Cry toxin receptor genes, molecular markers for the midgut cadherin, alkaline phosphatase (ALP), and aminopeptidase N (APN) genes were identified between the original greenhouse-derived dual-toxin-resistant and the susceptible laboratory T. ni strains. Genetic linkage analysis showed that the Cry2Ab resistance in T. ni was not genetically associated with the midgut genes coding for the cadherin, ALP, and 6 APNs (APN1 to APN6) nor associated with the ABC transporter gene ABCC2. Therefore, the Cry2Ab resistance in T. ni is conferred by a novel but unknown genetic mechanism.     

Knockout of three aminopeptidase N genes does not affect susceptibility of Helicoverpa armigera larvae to Bacillus thuringiensis Cry1A and Cry2A toxins

Bacillus thuringiensis (Bt) insecticidal toxins have been globally utilized for control of agricultural insects through spraying or transgenic crops. Binding of Bt toxins to special receptors on midgut epithelial cells of target insects is a key step in the mode of action. Previous studies suggested aminopeptidase N1 (APN1) as a receptor or putative receptor in several lepidopteran insects including Helicoverpa armigera through evidence from RNA interefence‐based gene silencing approaches. In the current study we tested the role of APNs in the mode of action of Bt toxins using clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR‐associated protein 9‐mediated gene knockout. Three APN genes (HaAPN1, HaAPN2 and HaAPN5) were individually knocked out in a susceptible strain (SCD) of H. armigera to establish three homozygous knockout strains. Qualitative in vitro binding studies indicated binding of Cry1Ac or Cry2Ab to midgut brush border membrane vesicles was not obviously affected by APN knockout. Bioassay results showed that none of the three knockouts had significant changes in susceptibility to Cry1A or Cry2A toxins when compared with the SCD strain. This suggests that the three HaAPN genes we tested may not be critical in the mode of action of Cry1A or Cry2A toxins in H. armigera.     

Bacillus thuringiensis Cry1Ac and Cry1Fa delta-endotoxin binding to a novel 110 kDa aminopeptidase in Heliothis virescens is not N-acetylgalactosamine mediated.

We determined that Bacillus thuringiensis Cry1Ac and Cry1Fa delta-endotoxins recognize the same 110, 120 and 170 kDa aminopeptidase N (APN) molecules in brush border membrane vesicles (BBMV) from Heliothis virescens. The 110 kDa protein, not previously identified as an APN, contained a variant APN consensus sequence identical to that found in Helicoverpa punctigera APN 2. PCR amplification of H. virescens cDNA based on this sequence and a conserved APN motif yielded a 0.9 kb product that has 89% sequence homology with H. punctigera APN 2. Western blots revealed that the 110 kDa molecule was not recognized by soybean agglutinin, indicating the absence of GalNAc. A 125I labeled-Cry1Ac domain III mutant (509QNR(511)-AAA) that has an altered GalNAc binding pocket (Lee et al., Appl. Environ. Microbiol. 65 (1999) 4513) showed abolished binding to the 120 APN, reduced binding to the 170 kDa APN, and enhanced binding to the 110 kDa APN. Periodate treated H. virescens BBMV blots were also probed with 125I labeled-Cry1Ac and 509QNR(511)-AAA toxins. Both toxins still recognized the 110 kDa APN and a >210 kDa molecule which may be a cadherin-like protein. Additionally, 125I-(509)QNR(511)-AAA recognized periodate treated 170 kDa APN. Results indicate that the 110 kDa APN is distinct from other Cry1 toxin binding APNs and may be the first described Cry1Ac-binding APN that does not contain GalNAc.     

粉纹夜蛾类Cry1Ac受体氨肽酶N cDNA片段的克隆与分析

设计简并引物,采用RT-PCR方法对粉纹夜蛾Trichoplusia ni (Hübner)细胞系BTI-TN5B-4的氨肽酶N (aminopeptidase N, APN)基因cDNA片段进行了克隆和序列分析, 通过两对引物扩增出了两种氨肽酶N基因的cDNA片段, 大小分别为188 bp 和564 bp,分别命名为AS188（GenBank登录号: CD809324）和AS564（GenBank登录号: CD809326）。对这两个片段推导的氨基酸序列进行同源性分析, 结果表明两者与已报道的鳞翅目昆虫中肠的Cry1Ac 毒素受体氨肽酶N有较高的同源性。     

A 106-kDa aminopeptidase is a putative receptor for Bacillus thuringiensis Cry11Ba toxin in the mosquito Anopheles gambiae

Bacillus thuringiensis (Bt) insecticidal toxins bind to receptors on midgut epithelial cells of susceptible insects, and binding triggers biochemical events that lead to insect mortality. Recently, a 100-kDa aminopeptidase N (APN) was isolated from brush border membrane vesicles (BBMV) of Anopheles quadrimaculatus and shown to bind Cry11Ba toxin with surface plasmon resonance (SPR) detection [Abdullah et al. (2006) BMC Biochem. 7, 16]. In our study, a 106-kDa APN, called AgAPN2, released by phosphatidylinositol-specific phospholipase C (PI-PLC) from Anopheles gambiae BBMV was extracted by Cry11Ba bound to beads. The AgAPN2 cDNA was cloned, and analysis of the predicted AgAPN2 protein revealed a zinc-binding motif (HEIAH), three potential N-glycosylation sites, and a predicted glycosylphosphatidylinositol (GPI) anchor site. Immunohistochemistry localized AgAPN2 to the microvilli of the posterior midgut. A 70-kDa fragment of the 106-kDa APN was expressed in Escherichia coli. When purified, it competitively displaced 125I-Cry11Ba binding to An. gambiae BBMV and bound Cry11Ba on dot blot and microtiter plate binding assays with a calculated K d of 6.4 nM. Notably, this truncated peptide inhibited Cry11Ba toxicity to An. gambiae larvae. These results are evidence that the 106-kDa GPI-anchored APN is a specific binding protein, and a putative midgut receptor, for Bt Cry11Ba toxin.     

Expression of recombinant and mosaic Cry1Ac receptors from Helicoverpa armigera and their influences on the cytotoxicity of activated Cry1Ac to Spodoptera litura Sl-HP cells

Bacillus thuringiensis (Bt) toxin receptors play important roles in the killing of pests, and investigation on characterization of the receptors is essential for utilization of Bt and management of insect resistance. Here, recombinant and mosaic receptors of Bt Cry1Ac toxin from Helicoverpa armigera were expressed in Spodoptera litura Sl-HP cells and their influences on cytotoxicity of activated Cry1Ac toxin were investigated. When H. armigera aminopeptidase N1 (APN1), alkaline phosphatase 2 (ALP2) and cadherin fused with or without GFP tag were, respectively, expressed in Sl-HP cells, live cell-immunofluorescence staining detection revealed that the quantity of the toxin binding to cadherin or cadherin-GFP was much more than that binding to ALP2 and APN1 or their fusion proteins with GFP, and only the cadherin- or cadherin-GFP-expressing cells showed aberrant cell morphology after the treatment of the toxin at low concentrations. ALP2 and APN1 fused with or without GFP tag did not significantly enhance the cadherin-mediated cytotoxicity of the toxin. The mosaic ALP-TBR-GFP-GPI was located on cell membrane, but did not bind to the toxin. The mosaic truncated cadherin-GFP-GPI was not located on cell membrane even if the signal peptide was sustained. The concentrations of the toxin resulting in swelling of 50 % cells for noncadherin-expressing Sl-HP cells and cadherin-expressing Hi5 cells were 5.08 and 9.50 µg/ml within 1 h, respectively. Taken together, our data have indicated that the binding affinity of ALP2 and APN1 to activated Cry1Ac toxin is much weaker than that of cadherin and both ALP2 and APN1 do not enhance the cytotoxicity of the toxin even though cadherin is co-expressed, and the mosaic receptor of ALP2 inserted with cadherin toxin binding domain does not mediate cytotoxicity of the toxin. In addition, the noncadherin-expressing Sl-HP cells are more susceptible to activated Cry1Ac than the cadherin-expressing Hi5 cells.