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Specific Epitopes of Domains II and III of Bacillus thuringiensis Cry1Ab Toxin Involved in the Sequential Interaction with Cadherin and Aminopeptidase-N Receptors in Manduca sexta

2006; Elsevier BV; Volume: 281; Issue: 45 Linguagem: Inglês

10.1074/jbc.m604721200

ISSN

1083-351X

Autores

Isabel Gómez, Iván Arenas, Itzel Benitez, Juan Miranda‐Ríos, Baltazar Becerril, Ricardo Grande, Juan C. Almagro, Alejandra Bravo, Mário Soberón,

Tópico(s)

Insect and Pesticide Research

Resumo

The Bacillus thuringiensis Cry toxins are specific to different insects. In Manduca sexta cadherin (Bt-R1) and aminopeptidase-N (APN) proteins are recognized as Cry1A receptors. Previous work showed that Cry1Ab binds to Bt-R1 promoting the formation of a pre-pore oligomer that binds to APN leading to membrane insertion. In this work we characterized the binding epitopes involved in the sequential interaction of Cry1Ab with Bt-R1 and APN. A Cry1Ab immune M13 phage repertoire was constructed using antibody gene transcripts of bone marrow or spleen from a rabbit immunized with Cry1Ab. We identified antibodies that recognize domain II loop 3 (scFvL3-3) or β16–β22 (scFvM22) in domain III. Enzyme-linked immunosorbent assay and toxin overlay binding competition assays in the presence of scFvL3-3, scFvM22, or synthetic peptides showed that domain II loop 3 is an important epitope for interaction with Bt-R1 receptor, whereas domain III β16 is involved in the interaction with APN. Both scFvL3-3 and scFvM22 lowered the toxicity of Cry1Ab to M. sexta larvae indicating that interaction with both receptors is important for in vivo toxicity. scFvL3-3 and anti-loop2 scFv (scFv73) promoted the formation of the pre-pore oligomer in contrast to scFvM22. In addition, scFvL3-3 and scFv73 preferentially recognized the monomeric toxin rather than the pre-pore suggesting a conformational change in domain II loops upon oligomerization. These results indicate for the first time that both receptor molecules participate in Cry1Ab toxin action in vivo: first the monomeric toxin binds to Bt-R1 through loops 2 and 3 of domain II promoting the formation of the pre-pore inducing some structural changes, then the pre-pore interacts with APN through β-16 of domain III promoting membrane insertion and cell death. The Bacillus thuringiensis Cry toxins are specific to different insects. In Manduca sexta cadherin (Bt-R1) and aminopeptidase-N (APN) proteins are recognized as Cry1A receptors. Previous work showed that Cry1Ab binds to Bt-R1 promoting the formation of a pre-pore oligomer that binds to APN leading to membrane insertion. In this work we characterized the binding epitopes involved in the sequential interaction of Cry1Ab with Bt-R1 and APN. A Cry1Ab immune M13 phage repertoire was constructed using antibody gene transcripts of bone marrow or spleen from a rabbit immunized with Cry1Ab. We identified antibodies that recognize domain II loop 3 (scFvL3-3) or β16–β22 (scFvM22) in domain III. Enzyme-linked immunosorbent assay and toxin overlay binding competition assays in the presence of scFvL3-3, scFvM22, or synthetic peptides showed that domain II loop 3 is an important epitope for interaction with Bt-R1 receptor, whereas domain III β16 is involved in the interaction with APN. Both scFvL3-3 and scFvM22 lowered the toxicity of Cry1Ab to M. sexta larvae indicating that interaction with both receptors is important for in vivo toxicity. scFvL3-3 and anti-loop2 scFv (scFv73) promoted the formation of the pre-pore oligomer in contrast to scFvM22. In addition, scFvL3-3 and scFv73 preferentially recognized the monomeric toxin rather than the pre-pore suggesting a conformational change in domain II loops upon oligomerization. These results indicate for the first time that both receptor molecules participate in Cry1Ab toxin action in vivo: first the monomeric toxin binds to Bt-R1 through loops 2 and 3 of domain II promoting the formation of the pre-pore inducing some structural changes, then the pre-pore interacts with APN through β-16 of domain III promoting membrane insertion and cell death. Crystal proteins (Cry) 2The abbreviations used are: Cry, crystal protein; BBMV, brush border membrane vesicles; Bt, Bacillus thuringiensis; ELISA, enzyme-liked immunosorbent assay; scFv, single-chain Fv antibody fragments; APN, aminopeptidase-N; Bt-R1, cadherin; GPI, glycosylphosphatidylinositol; Ab, antibody; PBS, phosphate-buffered saline; VL, variable light chain; HL, heavy chain. 2The abbreviations used are: Cry, crystal protein; BBMV, brush border membrane vesicles; Bt, Bacillus thuringiensis; ELISA, enzyme-liked immunosorbent assay; scFv, single-chain Fv antibody fragments; APN, aminopeptidase-N; Bt-R1, cadherin; GPI, glycosylphosphatidylinositol; Ab, antibody; PBS, phosphate-buffered saline; VL, variable light chain; HL, heavy chain. are widely used as insecticides in agriculture, forestry, and vector transmission due to their high specificity and their safety for the environment. Cry proteins are produced as protoxins of 70–130 kDa that are toxic to larval forms of several insects of different orders as well as to other invertebrates (1Bravo A. Soberón M. Gill S.S. Gilbert L.I. Iatrou K. Gill S.S. Comprehensive Molecular Insect Science. Vol. 6. Elsevier, Amsterdam2005: 175-206Google Scholar). Proteolytic activation of protoxin by midgut proteases produces Cry toxin fragments of 60–65 kDa. Cry toxins then bind to the cell surface where they undergo largescale irreversible conformational changes to convert them into an oligomeric form capable of inserting into the membrane, causing osmotic lysis of midgut cells and ultimately insect death (1Bravo A. Soberón M. Gill S.S. Gilbert L.I. Iatrou K. Gill S.S. Comprehensive Molecular Insect Science. Vol. 6. Elsevier, Amsterdam2005: 175-206Google Scholar). Receptor binding has been studied extensively as a key step determining insect specificity, toxicity, and resistance of Cry toxins (1Bravo A. Soberón M. Gill S.S. Gilbert L.I. Iatrou K. Gill S.S. Comprehensive Molecular Insect Science. Vol. 6. Elsevier, Amsterdam2005: 175-206Google Scholar, 2Ferré J. Van Rie J. Annu. Rev. Entomol. 2002; 47: 501-533Crossref PubMed Scopus (745) Google Scholar). In the case of the lepidopteran insect Manduca sexta, at least two Cry1A-binding proteins, a cadherin-like protein (Bt-R1) and a glycosylphosphatidylinositol (GPI)-anchored aminopeptidase-N (APN), have been described as receptors of Cry1A toxins (3Vadlamudi R.K. Weber E. Ji I. Ji T.H. Bulla L.A. J. Biol. Chem. 1995; 270: 5490-5494Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 4Knight P. Crickmore N. Ellar D.J. Mol. Microbiol. 1994; 11: 429-436Crossref PubMed Scopus (358) Google Scholar). Previously, we provided evidence showing that binding of monomeric Cry1Ab toxin to Bt-R1 promotes an additional proteolytic cleavage in the N-terminal end of the toxin (helix α1) facilitating the formation of a pre-pore oligomeric structure that is competent in membrane insertion and that oligomer formation is important for toxicity (5Soberón M. Perez R.V. Nuñez-Valdéz M.E. Lorence A. Gómez I. Sánchez J. Bravo A. FEMS Microbiol. Lett. 2000; 191: 221-225Crossref PubMed Scopus (28) Google Scholar, 6Gómez I. Sánchez J. Miranda R. Bravo A. Soberón M. FEBS Lett. 2002; 513: 242-246Crossref PubMed Scopus (202) Google Scholar). The pre-pore oligomer has a higher affinity to APN (7Zhuang M. Oltean D.I. Gómez I. Pullikuth A.K. Soberón M. Bravo A. Gill S.S. J. Biol. Chem. 2002; 277: 13863-13872Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 8Bravo A. Gómez I. Conde J. Muñoz-Garay C. Sánchez J. Zhuang M. Gill S.S. Soberón M. Biochem. Biopphys. Acta. 2004; 1667: 38-46Crossref PubMed Scopus (333) Google Scholar). The oligomeric Cry1A structure then binds to the APN receptor leading to its insertion into membrane lipid rafts (7Zhuang M. Oltean D.I. Gómez I. Pullikuth A.K. Soberón M. Bravo A. Gill S.S. J. Biol. Chem. 2002; 277: 13863-13872Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 8Bravo A. Gómez I. Conde J. Muñoz-Garay C. Sánchez J. Zhuang M. Gill S.S. Soberón M. Biochem. Biopphys. Acta. 2004; 1667: 38-46Crossref PubMed Scopus (333) Google Scholar) implying a sequential binding mechanism of Cry1A toxins with Bt-R1 and APN receptor molecules (8Bravo A. Gómez I. Conde J. Muñoz-Garay C. Sánchez J. Zhuang M. Gill S.S. Soberón M. Biochem. Biopphys. Acta. 2004; 1667: 38-46Crossref PubMed Scopus (333) Google Scholar). However, a different mechanism of action of Cry toxins based on the study of the effect of Cry1Ab toxin to cultured Trichoplusi ni H5 insect cells expressing M. sexta Bt-R1 (9Zhang X. Candas M. Griko N.B. Rose-Young L. Bulla Jr., L.A. Cell Death Differ. 2005; 12: 1407-1416Crossref PubMed Scopus (134) Google Scholar, 10Zhang X. Candas M. Griko N.B. Taussig R. Bulla Jr., L.A. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 9897-9902Crossref PubMed Scopus (292) Google Scholar) was recently proposed. It was proposed that the toxicity of Cry1Ab is mainly due to the interaction of monomeric Cy1Ab toxin with Bt-R1 by activating a Mg+2-dependent adenylyl cyclase/protein kinase A signaling pathway that leads to apoptosis and not to pore formation induced by insertion of oligomeric Cry1Ab into the membrane (9Zhang X. Candas M. Griko N.B. Rose-Young L. Bulla Jr., L.A. Cell Death Differ. 2005; 12: 1407-1416Crossref PubMed Scopus (134) Google Scholar, 10Zhang X. Candas M. Griko N.B. Taussig R. Bulla Jr., L.A. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 9897-9902Crossref PubMed Scopus (292) Google Scholar). Therefore, additional experimental evidence is needed to discriminate between the two models of mode of action of Cry toxins, in particular evidence that determines the role of the APN receptor in toxicity of Cry1A toxins will be valuable. To date, the tertiary structures of six different Cry proteins, Cry1Aa, Cry2Aa, Cry3Aa, Cry3Bb, Cry4Aa, and Cry4Ba, have been determined by x-ray crystallography (11Grochulski P. Masson L. Borisova S. Pusztai-Carey M. Schwartz J.L. Brousseau R. Cygler M. J. Mol. Biol. 1995; 254: 447-464Crossref PubMed Scopus (457) Google Scholar, 12Morse R.J. Yamamoto T. Stroud R.M. Structure (Camb.). 2001; 9: 409-417Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 13Li J. Carroll J. Ellar D.J. Nature. 1991; 353: 815-821Crossref PubMed Scopus (641) Google Scholar, 14Galitsky N. Cody V. Wojtczak A. Ghosh D. Luft J.R. Pangborn W. English L. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 1101-1109Crossref PubMed Scopus (158) Google Scholar, 15Boonserm P. Mo M. Angsuthanasombat C. Lescar J. J. Bacteriol. 2006; 188: 3391-3401Crossref PubMed Scopus (135) Google Scholar, 16Boonserm P. Davis P. Ellar D.J. Li J. J. Mol. Biol. 2005; 348: 363-382Crossref PubMed Scopus (186) Google Scholar). All these structures display a high degree of similarity with a three-domain organization, suggesting a similar mode of action of the Cry three-domain protein family. The N-terminal domain (domain I) is a bundle of seven α-helices in which the central helix-α5 is hydrophobic and is encircled by six other amphipathic helices, this helical domain is responsible for membrane insertion and pore formation (11Grochulski P. Masson L. Borisova S. Pusztai-Carey M. Schwartz J.L. Brousseau R. Cygler M. J. Mol. Biol. 1995; 254: 447-464Crossref PubMed Scopus (457) Google Scholar, 12Morse R.J. Yamamoto T. Stroud R.M. Structure (Camb.). 2001; 9: 409-417Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 13Li J. Carroll J. Ellar D.J. Nature. 1991; 353: 815-821Crossref PubMed Scopus (641) Google Scholar, 14Galitsky N. Cody V. Wojtczak A. Ghosh D. Luft J.R. Pangborn W. English L. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 1101-1109Crossref PubMed Scopus (158) Google Scholar, 15Boonserm P. Mo M. Angsuthanasombat C. Lescar J. J. Bacteriol. 2006; 188: 3391-3401Crossref PubMed Scopus (135) Google Scholar). Domain II consists of three anti-parallel β-sheets with exposed loop regions, and domain III is a β-sandwich (11Grochulski P. Masson L. Borisova S. Pusztai-Carey M. Schwartz J.L. Brousseau R. Cygler M. J. Mol. Biol. 1995; 254: 447-464Crossref PubMed Scopus (457) Google Scholar, 12Morse R.J. Yamamoto T. Stroud R.M. Structure (Camb.). 2001; 9: 409-417Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 13Li J. Carroll J. Ellar D.J. Nature. 1991; 353: 815-821Crossref PubMed Scopus (641) Google Scholar, 14Galitsky N. Cody V. Wojtczak A. Ghosh D. Luft J.R. Pangborn W. English L. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 1101-1109Crossref PubMed Scopus (158) Google Scholar, 15Boonserm P. Mo M. Angsuthanasombat C. Lescar J. J. Bacteriol. 2006; 188: 3391-3401Crossref PubMed Scopus (135) Google Scholar). Exposed regions in domains II and III are involved in receptor binding (1Bravo A. Soberón M. Gill S.S. Gilbert L.I. Iatrou K. Gill S.S. Comprehensive Molecular Insect Science. Vol. 6. Elsevier, Amsterdam2005: 175-206Google Scholar). The Cry1Aa, Cry1Ab, and Cry1Ac proteins share more than 95% amino acid sequence identity as protoxins and are selectively toxic to some lepidopteran insect pests. These Cry1A toxins bind to the same receptor molecules in M. sexta (Bt-R1 and APN) (17Keeton T.P. Francis B.R. Maaty W.S. Bulla L.A. Appl. Envionm. Microbiol. 1998; 64: 2158-2165Crossref PubMed Google Scholar, 18Masson L. Lu Y.-J. Mazza A. Brosseau R. Adang M.J. J. Biol. Chem. 1995; 270: 20309-20315Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Domain I in the three toxins share more than 98% amino acid sequence identity. However, there are important differences in domains II and III of these toxins. Cry1Ab and Cry1Ac toxins share the same domain II in contrast to Cry1Aa that has a different domain II sharing only 69% identity. In particular, the loop regions involved in receptor interaction are different in Cry1Aa. In contrast, Cry1Aa and Cry1Ab share a very similar domain III, whereas domain III of Cry1Ac shares only 38% identity with both toxins. Because different lepidopteran insects show different sensitivity to these Cry1A toxins, it is likely that the differences in domains II and III influence the receptor binding affinities and the activity of these toxins. The characterization of the epitopes involved in interaction of Cry toxins with their receptors could give clues on the molecular basis of insect specificity and resistance. In previous work, using a synthetic phage-antibody library, we isolated an scFv antibody (scFv73) that binds to domain II loop 2 (β6–β7 loop) of Cry1A toxins and inhibited binding of Cry1A toxins to Bt-R1 but did not affect binding to APN (19Gómez I. Oltean D.I. Sanchez J. Bravo A. Gill S. Soberón M. J. Biol. Chem. 2001; 276: 28906-28912Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 20Gómez I. Miranda-Rios J. Rudiño-Piñera E. Oltean D.I. Gill S.S. Bravo A. Soberón M. J. Biol. Chem. 2002; 277: 30137-30143Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Sequence analysis of the CDR3H region of scFv73 led to the identification of an 8-amino acid epitope in Bt-R1 cadherin repeat 7 (CADR7, 869HITDTNNK876) involved in the binding to domain II loop 2 of Cry1A toxins (19Gómez I. Oltean D.I. Sanchez J. Bravo A. Gill S. Soberón M. J. Biol. Chem. 2001; 276: 28906-28912Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 20Gómez I. Miranda-Rios J. Rudiño-Piñera E. Oltean D.I. Gill S.S. Bravo A. Soberón M. J. Biol. Chem. 2002; 277: 30137-30143Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). A second binding epitope in Bt-R1 CADR11 (1331IPLPASILTVTV1342) that interacts with domain II loop α8(α8a–α8b loop) and loop 2 of Cry1Ab toxin (21Gómez I. Dean D.H. Bravo A. Soberón M. Biochemistry. 2003; 42: 10482-10489Crossref PubMed Scopus (94) Google Scholar) was recently described. Finally, a third region in CADR12 of Bt-R1 (amino acids 1363–1464) involved in Cry1Ab interaction and toxicity was identified (22Hua G. Jurat-Fuentes J.L. Adang M.J. J. Biol. Chem. 2004; 279: 28051-28056Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In the case of the Heliothis virescens cadherin, this binding region was narrowed to residues 1422–1440 by mutagenesis and shown to bind Cry1Ac domain II loop 3(β10–β11 loop) (23Xie R. Zhuang M. Ross L.S. Gómez I. Oltean D.I. Bravo A. Soberón M. Gill S.S. J. Biol. Chem. 2005; 280: 8416-8425Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Regarding interaction of Cry1A toxins with APN, Cry1Ac toxin binds to APN receptor by means of domain III that specifically recognizes N-acetylgalactosamine (GalNAc) moieties in contrast to Cry1Aa and Cry1Ab toxins that show no GalNAc binding capacities (18Masson L. Lu Y.-J. Mazza A. Brosseau R. Adang M.J. J. Biol. Chem. 1995; 270: 20309-20315Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Based on the use of monoclonal antibodies that competed binding of Cry1Aa with Bombix mori APN, the Cry1Aa-APN interacting epitopes were recently mapped in domain III β16 (508STLRVN513) and β22 (582VFTLSAHV589) residues, which are exposed and in close proximity in the three-dimensional structure (24Nakanishi K. Yaoi K. Nagino Y. Hara H. Kitami M. Atsumi S. Miura N. Sato R. FEBS Lett. 2002; 519: 215-220Crossref PubMed Scopus (85) Google Scholar, 25Atsumi S. Mizuno E. Hara H. Nakanishi K. Kitami M. Miura N. Tabunoki H. Watanabe A. Sato R. Appl. Environm. Microbiol. 2005; 71: 3966-3977Crossref PubMed Scopus (47) Google Scholar). Binding of Cry1Ab toxin to anti-loop 2 scFv73 antibody or to Bt-R1 CADR7 or CADR11 peptides facilitates the formation of the pre-pore oligomeric structure in vitro, showing that domain II interaction with Bt-R1 is an important step in the formation of the pre-pore oligomer before toxin inserts into the membrane (6Gómez I. Sánchez J. Miranda R. Bravo A. Soberón M. FEBS Lett. 2002; 513: 242-246Crossref PubMed Scopus (202) Google Scholar, 21Gómez I. Dean D.H. Bravo A. Soberón M. Biochemistry. 2003; 42: 10482-10489Crossref PubMed Scopus (94) Google Scholar). Although some binding epitopes in the toxin have been characterized, little is known about the mechanism by which the Cry1A toxins undergo a sequential interaction with the two receptors molecules. The characterization of the binding epitopes in the pre-pore oligomer and the role of these binding sites in the interaction with both Bt-R1 and APN is still missing. Furthermore, the characterization of possible structural changes in the toxin epitopes involved in receptor interaction could give clues on the mechanism of differential interaction of monomeric and pre-pore oligomeric structures with Bt-R1 and APN. Also, the study of the role of the interaction of the pre-pore with APN in Cry1Ab toxicity will be important to determine the role of pre-pore formation in toxicity. In this study, we constructed immune libraries for Cry1Ab toxin and selected specific monoclonal scFv fragments that recognize Cry1Ab domain II loop 3 or domain III β16–β22 epitopes and demonstrated that both scFv molecules can inhibit the toxicity to M. sexta larvae. An anti-loop 3 antibody inhibited binding of Cry1Ab to Bt-R1, whereas an anti-β16–β22 antibody inhibited interaction with APN. In vitro oligomer formation assays with the selected scFv antibodies showed that only binding of Cry1Ab domain II with Bt-R1 is involved in oligomer formation in contrast to binding of domain III with APN that did not facilitate the formation of the pre-pore. Overall, these results suggest that interaction of Cry1Ab with both receptor molecules is important for toxicity. Also these data contribute to our understanding of the mechanism involved in the sequential interaction of Cry1Ab toxin with both receptor molecules. Purification of Monomeric Cry Toxins—The acrystalliferous Bt strain 407cry– (26Lereclus D. Arantès O. Chaufax J. Lecadet M.-M. FEMS Microbiol. Lett. 1989; 60: 211-218Google Scholar) transformed with pHT409 plasmid harboring the cry1Aa gene (27Arantès O. Lereclus D. Gene (Amst.). 1991; 108: 115-119Crossref PubMed Scopus (352) Google Scholar) or pHT315-cry1Ab (19Gómez I. Oltean D.I. Sanchez J. Bravo A. Gill S. Soberón M. J. Biol. Chem. 2001; 276: 28906-28912Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) were used for Cry1Aa and Cry1Ab production, respectively. Cry1Ac was produced from wild-type Bt strain HD73. Bt strains were grown for 3 days at 29 °C in nutrient broth sporulation medium (28Lereclus D. Agaisse H. Gominet M. Chaufaux J. Bio/Technology. 1995; 13: 67-71Crossref PubMed Scopus (112) Google Scholar) supplemented with 10 μg/ml erythromycin for Cry1Aa and Cry1Ab. After sporulation, crystals were purified by sucrose gradients as reported (19Gómez I. Oltean D.I. Sanchez J. Bravo A. Gill S. Soberón M. J. Biol. Chem. 2001; 276: 28906-28912Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The Cry1A protoxins were solubilized and proteolytically activated as reported (19Gómez I. Oltean D.I. Sanchez J. Bravo A. Gill S. Soberón M. J. Biol. Chem. 2001; 276: 28906-28912Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Oligomer Formation Assay—For activation, 1–2 μg of Cry1Ab protoxin was incubated with scFv molecules in the presence of M. sexta midgut juice as previously reported (6Gómez I. Sánchez J. Miranda R. Bravo A. Soberón M. FEBS Lett. 2002; 513: 242-246Crossref PubMed Scopus (202) Google Scholar). Purification of the activated toxins was done by size exclusion chromatography with Superdex 200 HR 10/30 (Amersham Biosciences) FPLC size exclusion as described (29Rausell C. Muñoz-Garay C. Miranda-CassoLuengo R. Gómez I. Rudiño-Piñera E. Soberón M. Bravo A. Biochemistry. 2004; 43: 166-174Crossref PubMed Scopus (50) Google Scholar). The oligomeric structure was detected by Western blot assays using Cry1Ab-polyclonal antibodies as reported (6Gómez I. Sánchez J. Miranda R. Bravo A. Soberón M. FEBS Lett. 2002; 513: 242-246Crossref PubMed Scopus (202) Google Scholar). Solubilization of GPI-anchored Proteins—M. sexta brush border membrane vesicles (BBMV) were treated with phospholipase C from Bacillus cereus (Sigma) as previously reported in Ref. 30Lorence A. Darszon A. Bravo A. FEBS Lett. 1997; 414: 303-307Crossref PubMed Scopus (57) Google Scholar. Membranes were recovered by ultracentrifugation (90,000 x g for 20 min), and the supernatant was analyzed for aminopeptidase activity. APN enzymatic activity was measured using 1 mg/ml leucine-p-nitroanilide (Sigma) as substrate. BBMV proteins (5 μg) were mixed with APN buffer (0.2 m Tris-HCl, pH 8, 0.25 m NaCl) containing 1 mm leucine-p-nitroanilide. APN enzymatic activity was monitored as change in the absorbance at 405 nm for 10 min at room temperature. Rabbit Immunization—A New Zealand White rabbit was immunized subcutaneously with a mixture of oligomeric and monomeric Cry1Ab toxin structures obtained after proteolytical activation of Cry1Ab in the presence of scFv73. The rabbit was boosted three times with 1 mg of Cry1Ab toxin structure mixture, mixed with incomplete adjuvant, at 15-day intervals. The bone marrow and spleen were dissected 40 days after the primary immunization. Phage Display Library Construction—Total RNA was prepared from spleen tissue and bone marrow as described (31Hawlisch H. Meyer zu Vilsendorf A. Bautsch W. Klos A. Kohl J. J. Immunol. Methods. 2000; 236: 117-131Crossref PubMed Scopus (17) Google Scholar). Total RNA and random primer were used for first strand cDNA synthesis using a kit (Roche Applied Sciences), according to the manufacturer's instructions. From cDNA, heavy and light chain DNA fragments were amplified separately and recombined by three subsequent PCR, essentially as described (31Hawlisch H. Meyer zu Vilsendorf A. Bautsch W. Klos A. Kohl J. J. Immunol. Methods. 2000; 236: 117-131Crossref PubMed Scopus (17) Google Scholar), except that PCR1 and PCR2 were done with Vent DNA polymerase (New England BioLabs, Beverly, MA). Primer sequences for amplification of VL and VH antibody regions have been described before (31Hawlisch H. Meyer zu Vilsendorf A. Bautsch W. Klos A. Kohl J. J. Immunol. Methods. 2000; 236: 117-131Crossref PubMed Scopus (17) Google Scholar). However, primers HH13, HH14, and HH15 sequences were corrected; HH13, 5′-GGCGGATCAGGAGGCGGAGGTTCTGGAGGTGGGAGTGMCCTCGATMTGACCCAGACTCCAGC-3′; HH14, 5′-GGCGGATCAGGAGGCGGAGGTTCTGGAGGTGGGAGTGMCCTCGTGMTGACCCAGACTCCAGC-3′; HH15, 5′-GGCGGATCAGGAGGCGGAGGTTCTGGAGGAGGTGGGAGTGMCCTCGTGMTGACCCAGACTCCATC-3′, where M = A/C. To construct the scFv libraries, scFv PCR products and phagemid vectors pCANTAB 5E and pSyn2 were digested with restriction enzymes SfiI and NotI (New England BioLabs, Beverly, MA), gel purified, and ligated. The ligation products were purified by extraction with phenol/chloroform and ethanol precipitation. The purified DNA was electroporated into TG1 Escherichia coli cells. Each library was grown on TYE agar plates, supplemented with ampicillin (100 μg/ml) and glucose (1% w/w). After overnight incubation at 37 °C, grown colonies were scraped off the agar plates, mixed with glycerol (20%), and stored at –70 °C. Libraries of 2.0 × 106 members were obtained. Selection and Characterization of Phage Displayed Antibodies—Preparation of phage particles for panning was done as described (19Gómez I. Oltean D.I. Sanchez J. Bravo A. Gill S. Soberón M. J. Biol. Chem. 2001; 276: 28906-28912Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Briefly, Maxisorp immunotubes (Nunc, Denmark) were coated with Cry1Ab and blocked (19Gómez I. Oltean D.I. Sanchez J. Bravo A. Gill S. Soberón M. J. Biol. Chem. 2001; 276: 28906-28912Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The tubes were incubated with phage antibodies (1011 phage particles in MPBS skim milk 2% in PBS) and washed 10 times to remove unbound phage Abs (19Gómez I. Oltean D.I. Sanchez J. Bravo A. Gill S. Soberón M. J. Biol. Chem. 2001; 276: 28906-28912Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The binders were eluted with triethylamine, and the eluant neutralized and mixed with 8.5 ml of exponentially growing TG1 cells to allow infection as described (19Gómez I. Oltean D.I. Sanchez J. Bravo A. Gill S. Soberón M. J. Biol. Chem. 2001; 276: 28906-28912Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Cells were resuspended in 1 ml of medium, and plated on three TYE agar plates (145-mm diameter) as described (19Gómez I. Oltean D.I. Sanchez J. Bravo A. Gill S. Soberón M. J. Biol. Chem. 2001; 276: 28906-28912Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). After overnight growth at 37 °C, bacteria were harvested and phage were rescued to produce phagemids for the next selection cycle. This panning cycle was done three times, after which polyclonal phage Abs from each round of selection were tested for binding activity in a phage ELISA. In the case of the panning of anti-domain III Cry1Ab antibodies, the last panning round was performed in the presence of 25 μg/ml soluble Cry1Ac toxin. Panning Selection Using Biotinylated Antigens—Streptavidin microtiter plates (Pierce) were coated with biotinylated Cry1Ab (10 nm) or biotin-loop 3 synthetic peptide (1 μm). Cry1Ab was biotinylated using biotinyl-N-hydroxysuccinimide ester (Amersham Biosciences) according to the manufacturer's instructions. Panning was carried out essentially as described above, except that volumes were adapted for microtiter plates. Synthetic peptides (Table 1) were purchased from Invitrogen.TABLE 1Synthetic peptide sequencesNameSequenceDescriptionBiotin-loop 3Biotin-SGSSG-FRSGFSNSSVSCry1Ab residues 436-379 fused to biotin-SGSSG peptideLoop2LYRRPFNIGINNQQCry1Ab residues 366-379Loop3FRSGFSNSSVSIIRCry1Ab residues 436-449DIII-1GQISTLRVNITACry1Ab residues 506-517DIII-2VFTLSAHVFNCry1Ab residues 583-592 Open table in a new tab Phage or scFv ELISA—For phage ELISA, the microtiter plates were coated with 2.5 μg of Cry1Ab toxin in 100 μl of carbonate buffer (50 mm, pH 9.6) per well overnight at 4 °C. For scFv ELISA the microtiter plates were coated with 100 ng of Cry1Ab monomer or 100 ng of oligomer in 100 μl of carbonate buffer per well overnight at 4 °C. The plates were washed 3 times with PBS and blocked with 200 μl/well of MPBS skim milk 2% in PBS for 2 h at 37 °C. For phage ELISA, 100 μl of phage Abs (1 × 108 plaque-forming units) were added and incubated for 90 min at 25 °C. After washing, 100 μl of horseradish peroxidase-conjugated sheep anti-M13 antibody (1:1000 in MPBS skim milk 2% in PBS) was added and incubated as before. For scFv ELISA, 200 nm pure scFv molecules were added to the Cry1Ab monomeric- or oligomeric-coated wells and incubated for 90 min at 25 °C. scFv antibodies were detected with horseradish peroxidase-conjugated anti-His antibody (Qiagen) (1:5000 dilution). After washing 3 times with TPBS and 3 times with PBS, ortho-phenylenediamine (Sigma) (0.5 mg/ml, 30% H2O2) was used as substrate for detection. Reaction was stopped with 100 μl of 1 m H2SO4 and measured at 490 nm using a microplate reader. For competition assays, 1 μg of synthetic peptides were added during the incubation with the selected scFv phages. Nucleotide Sequence Determination and Fingerprint Analysis—To determine the diversity of the original libraries and clones after panning selection, we randomly picked infected TG1 colonies and amplified their scFv inserts with the PCR 5′ primer PSYN1 (ATACCTATTGCCTACGGC) and 3′ primer PSYN2 (TTACAACAGTCTATGCGG), or primers JK2 and AW1 (31Hawlisch H. Meyer zu Vilsendorf A. Bautsch W. Klos A. Kohl J. J. Immunol. Methods. 2000; 236: 117-131Crossref PubMed Scopus (17) Google Scholar). To obtain DNA fingerprint of the insert sequences, the PCR products were digested with AluI (New England Biosciences) and resolved on 8% acrylamide gels. Expression and Purification of Soluble scFv Antibodies—The positive phage clones were subcloned into expression vector pET22b (Novagen). Recombinants were transformed into E. coli BL21(DE3) and induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside. After incubation at 30 °C overnight, the proteins in the periplasm were collected usi

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