key: cord-0978761-mo3rqf68
authors: Ingle, Sanjay S.; Trivedi, Nidhi; Prasad, Rakesh; Kuruvilla, Jes; Rao, Koteshwara Kukatla; Chhatpar, H.S.
title: Aminopeptidase-N from the Helicoverpa armigera (Hubner) Brush Border Membrane Vesicles as a Receptor of Bacillus thuringiensis Cry1Ac δ-Endotoxin
date: 2001
journal: Curr Microbiol
DOI: 10.1007/s002840010297
sha: 5fad341a85e59e465164c3cd2af05e541d4ec2de
doc_id: 978761
cord_uid: mo3rqf68

Brush border membrane vesicles (BBMVs) were prepared from the 2(nd) instar larvae of Helicoverpa armigera. Binding of the activated Cry1Ac of Bacillus thuringiensis (Bt) toxin was shown by immunoblot. A 120-kDa protein was identified as a receptor for the Cry1Ac type δ-endotoxin. The aminopeptidase-N activity of BBMVs was measured as the hydrolysis of L-leucine p-nitroanilide. The specific activity was 35 units/mg protein. The BBMV preparation also showed low level of alkaline phosphatase activity. Zn(++) chelating agents 2,2′-dipyridyl and 1,10-phenanthroline inhibited aminopeptidase activity at 10 mM concentration, indicating the presence of zinc-dependent aminopeptidase in the brush border of H. armigera. The aminopeptidase activity was increased with increasing concentration of δ-endotoxin. The purified 120-kDa binding protein was N-terminally sequenced. The first 10-amino-acid sequence showed 60–77% similarity with human cysteine-rich secretory protein-1 precursor, inhibin alpha chain precursor. Salmonella flagellar hook protein and yeast carboxypeptidase S.

Bacillus thuringiensis produces insecticidal crystal proteins (ICPs) during sporulation. When these ICPs are ingested by the susceptible insect, they are first solubilized in the alkaline and reducing environment of the insect midgut and then processed by the midgut proteases to form the activated toxin. The activated toxin binds to receptors on the surface of the midgut epithelial cells of a susceptible insect and results in the rapid inhibition of K ϩ -dependent amino acid transport [18] . Changes in the permeability of the midgut membrane [9] and eventually lysis of the midgut cells result in death of the insect [15] . Brush border membrane vesicles are used to study the receptor binding and mode of action of Bt toxin in vitro [27] . It has been shown that B. thuringiensis toxins bind with high affinity to specific binding molecules present in the brush border membranes of susceptible insects [22] . In H. armigera the target of Bt insecticidal crystal protein is the brush border membrane of midgut cells. The bound toxin disrupts the midgut epithelial cells causing lysis [10] . Lepidoptcran insects have one or more toxin binding proteins. Cry lAc binding protein of 120 kDa and a Cry lAb binding protein of 205 kDa were shown in Manduca sexta [6, 23] . Heliothis virescens has toxin-binding proteins ranging from 45 to 170 kDa [4, 6, 12, 16] . The receptor for B. thuringiensis Cry lAc ␦-endotoxin in the brush border membrane of the lepidoptera Manduca sexta was shown to be the metalloprotease aminopeptidase-N [11] .

We report the brush border membrane receptor proteins of Helicoverpa armigera for B. thuringiensis Cry1Ac toxin. 120-kDa protein of BBmrs of H. armigera was observed as a major cry1Ac binding protein.

at 10,000 g for 10 min at 4°C. The activated toxin was used for binding studies.

Binding of Cry1Ac ␦-endotoxin to the brush border membrane proteins was detected by immunoblot. Brush border membrane protein resolved on SDS-PAGE (10%) [13] was transferred onto Immobilon-P polyvynilidene difluoride (PVDF) membrane for immunoblotting [21] . The PVDF membrane was incubated with activated Bt toxin, followed by washing and incubation with primary rabbit anti-Cry1Ac antibody. Anti-Cry1Ac antibodies were raised in the rabbit against purified Cry1Ac toxin. After washing and incubation with peroxidase-conjugated goat anti-rabbit antibody, the blot was developed by diaminobenzidene (Sigma) as a substrate.

Preparation of brush border membrane vesicles. Brush border membrane vesicles from the midgut of Helicoverpa armigera were prepared according to the method of Wolfersberger [28] . Midgut collected from the third-instar larvae of H. armigera was used to purify the brush border membrane aminopeptidase-N. The brush border membrane vesicles were solubilized in 20 mM Tris-HCl, pH 7.6 buffer with 1% (3-3(3-chloramidopropyl) dimethylammonia)-1-propane-sulphonate) (Sigma). The suspension was centrifuged to remove insoluble materials. The supernatant was concentrated and fractionated on Sephadex G-150 column equilibrated with 20 mM Tris-HCl, 0.1 mM DTT, 1mM MgCl 2 , and 50 mM KCl at pH 8.0. The fractions showing aminopeptidase activity were pooled and concentrated.

Enzyme assays. Aminopeptidase activity was assayed at 25°C in 250 mM Tris HCl, pH 7.8, 250 mM NaCl, with 1 mM L-leucine p-nitroanilide as a substrate. One unit of enzyme activity was defined as the amount enzyme catalyzing the hydrolysis of 1 mol of L-leucine p-nitroanilide per min at 25°C. Specific activity was expressed as units/mg protein [8] . Alkaline phosphatase activity was assayed at 25°C in 10 mM diethanolamine at pH 9.8, with 1.25 mM 4-nitrophenyl phosphate as the substrate. One unit of enzyme activity was defined as the amount of enzyme catalyzing the hydrolysis of 1 mol of 4-nitrophenyl phosphate per min at 25°C. Specific activity was expressed as units/mg protein [14] .

N-terminal amino acid sequencing. N-terminal amino acid sequence was determined from the purified receptor resolved on 10% SDS-PAGE and electroblotting. The Coomassie Brilliant blue-stained, 120-kDa band was excised from the membrane and washed several times in distilled water, dried, and subjected to sequencing. Sequencing was carried out using gas-phase protein sequencer from Shimadzu Model PPSQ-10 at the biomembrane lab, Indian Institute of Technology, Mumbai, India. This system consists of an Edman reaction unit, an on-line PTH-analyzer, and a CR-7A data processor.

Brush border membrane vesicles were isolated and purified from the midgut of Helicoverpa armigera to study the binding ability of Bt. toxin. To demonstrate which protein of the BBMV is a putative receptor for the binding of Bt toxin, we carried out immunoblotting. The Coomassie blue-stained gel showed various molecular weight protein bands ranging from 150, 120, 96, 80, 66 to 60 kDa. The BBMV proteins were separated on SDS-PAGE and transferred onto the membrane. The membrane was incubated with the activated Bt toxin, followed by anti-Cry1Ac antibody and developed. A protein band corresponding to 120 kDa was observed on the blot. To confirm that the 120-kDa protein is a putative receptor for Bt toxin, BBMV proteins were separated by gel filtration, and the fraction having aminopeptidase activity was concentrated and resolved on SDS-PAGE (10%). The duplicate sample was transferred to PVDF membrane. The Coomassie blue-stained gel showed a single band of 120 kDa. The immunoblot showed binding of Cry1Ac ␦-endotoxin to this protein.

Hence, a 120-kDa protein of the BBMV was identified as a receptor for the Cry1Ac type Bt toxin, as shown in Fig  1 and 2 . The enzymatic properties of this receptor protein were studied by the aminopeptidase assay. The specific activity of the purified protein was found to be 35 units/mg protein. The BBMV preparation showed alkaline phosphatase activity as well. The level of alkaline phosphatase activity was 5 units/mg protein. The purified protein did not show alkaline phosphatase activity [ Table  1 ]. The effect of Zn ϩϩ chelating agents on the activity of aminopeptidase from BBMV was studied. The Zn ϩϩ chelating agents 2,2-dipyridyl and 1,10-phenanthroline inhibited 42 and 51% aminopeptidase activity respectively at 1 mM concentration. At 10 mM concentration of these inhibitors, there was complete inhibition of aminopeptidase activity [ Table 2 ]. The 120-kDa protein has the characteristics of aminopeptidase-N. Aminopeptidase-N is a Zn ϩϩ -dependent dependent ectoenzyme existing in relatively high abundance in the brush border of many animals. Mammalian forms of intestinal aminopepti- dase-N occur as monomers of 120 -140 kDa proteins, which are associated with 95, 66, and 45 kDa breakdown products [17] . It was reported that toxin-binding protein acts by increasing the toxin concentration at the membrane surface microenvironment, resulting in pore formation at low concentration of toxin [5, 11] . The effect of Cry1Ac ␦-endotoxin on the aminopeptidase activity was studied by adding increasing concentrations of ␦-en-dotoxin in the assay system. The aminopeptidase activity was increased with increasing concentrations of ␦-endotoxin up to 60 g, but further concentrations lowered the enzyme activity to its original level. ␦-Endotoxin by itself does not have aminopeptidase activity. Those ␦-endotoxin which bind to the receptor enhance aminopeptidase activity. Inactive toxin did not show any increase in aminopeptidase activity [Fig. 3 ].

The 120-kDa protein showing aminopeptidase activity was electroblotted onto PVDF and subjected to N-terminal amino acid sequencing. The first ten amino acid residues found were GMYTHEGSDP. This amino acid sequence was used to search the Swiss-Prot protein database. The multiple alignments of the N-terminal sequence of H. armigera aminopeptidase-N (AMPN_HELAM) showed 60 -77% identity with four different N-terminal amino acid sequences, CRS1_HUMAN (cysteine-rich secretory protein-1 precursor), IHA_HUMAN (inhibin alpha chain precursor glycoprotein. Follitropin inhibitor), FLGE_SALTY (Salmonella typhimurium flagellar hook protein), and CBPS_Yeast (carboxypeptidase S precursor glycoprotein of Saccharomyces cerevisiae), as shown in Table 3 . A multiple aminopeptidase-N protein sequence alignment showed that the first 40 residues of the human, rat, pig, and mouse enzymes have the characteristics of a signal sequence, which may function as a membrane anchor [7, 19, 20] .

Aminopeptidase-N (EC.3.4.11.2) is a Zn ϩϩ -dependent enzyme existing in relatively high abundance in the brush border of many animals [3, 24, 25, 28] . Aminopeptidase catalyzes the removal of N-terminal neutral amino acids [8] . Aminopeptidase is a common component of lepidoptera larval midgut and is used as a marker enzyme to assess the purity of BBMVs from insect larval guts [26] . Correlation between toxicity and binding ability of BBMV to the Bt toxin was also reported [2] . The generally accepted theory for the mode of action of B. thuringiensis ␦-endotoxin postulates that, after binding to a receptor in the insect midgut, the toxin undergoes a conformational change and inserts into the membrane to form a pore [11] . We report the presence of a 120-kDa protein of H. armigera brush border as the receptor for the Cry1Ac ␦-endotoxin. G  M  Y  T  H  E  G  S  D  P  -P81731  CRS1 human  C  H  Y  C  H  E  G  N  D  P  75  P54107  IHA Human  G  G  F  T  H  R  G  S  D  P  60  P05111  FLGE SALTY  A  V  Y  T  H  D  S  S  D  P 

An analysis of Bacillus thuringiensis ␦-endotoxin action on insect midgut membrane permeability using a light scattering assay

Chemical modification of Bacillus thuringiensis activated ␦-endotoxin and its effect on toxicity and binding to Monduca sexta midgut membranes

Aminopeptidase-N is a major receptor for the entero-pathogenic coronavirus TGEV

␦-Endotoxin inhibits a phosphatase in midgut epithelial membrane of Heliothis virescens

Mode of action of ␦-endotoxins from Bacillus thuringiensis: a comparison with other bacterial toxins

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Signal sequences

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The receptor for Bacillus thuringiensis Cry1Ac ␦-endotoxin in the brush border membrane of the lepidopteran Manduca sexta is aminopeptidase-N

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A role for aminopeptidase-N in Na ϩ -dependent amino acid transport in bovine renal brush-border membranes

Bacillus thuringiensis toxin inhibits K ϩ -gradient-dependent amino acid transport across the brush border membrane of Pieris brassicae midgut cells

Identification and partial purification of a Bacillus thuringiensis Cry1c ␦-endotoxin binding protein from Spodoptera littoralis gut membrane

A mixture of Manduca sexta aminopeptidase-N and phosphatase enhances Bacillus thuringiensis insecticidal CryIA(c) toxin binding and 86 Rb ϩ -K ϩ efflux in vitro

Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications

Brush border membrane aminopeptidase-N in the midgut of the gypsy moth serves as the receptor for the CryIA(c) ␦-endotoxin of Bacillus thuringiensis

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Table 3. The alignment of the N-terminal amino acid sequence of H. armigera AP-N Source N-terminal amino acid sequence % Identity Accession No

Specificity of Bacillus thuringiensis ␦-endotoxin: importance of specific receptors on the brush border membrane of the midgut of target insects

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Preparation and partial characterization of amino acid transporting brush border membrane vesicles from the larval midgut of the cabbage butterfly (Pieris brassicae)

Aminopeptidase-N as Receptor of Cry1Ac ␦-Endotoxin

The authors are thankful to Dr. A.K. Lala, Biomembrane Laboratory, Indian Institute Of Technology, Pawai, Mumbai-400 076 for the amino acid sequencing.