Rapid Purification and Molecular Modeling of Aait Peptides From

Archives of Insect Biochemistry and Physiology 38:53–65 (1998) Rapid Purification and Molecular Modeling of AaIT Peptides from Venom of Androctonus australis Yoshiaki Nakagawa1,2 Martin Sadilek,3 Elisabeth Lehmberg4,5 Rafael Herrmann,1,6,7 Revital Herrmann,1,6 Haim Moskowitz,1,6 Young Moo Lee,8 Beth Ann Thomas,1 Ryo Shimizu,9 Masataka Kuroda,9,10 A. Daniel Jones,4 and Bruce D. Hammock1,6* 1Department of Entomology, University of California, Davis, California 2Graduate School of Agriculture, Kyoto University, Kyoto, Japan 3Department of Chemistry, University of Washington, Seattle, Washington 4Facility for Advanced Instrumentation, University of California, Davis, California 5Berlex Biosciences, Richmond, California 6Department of Environmental Toxicology, University of California, Davis, California 7Du Pont Agricultural Products, Stine-Haskell, Newark, Delaware 8Protein Structure Laboratory, University of California, Davis, California 9Tanabe Seiyaku Co. Ltd., Kashima, Yodogawa-ku, Osaka, Japan 10Department of Bioinformatics, Biomolecular Engineering Research Institute, Furuedai, Suita, Osaka, Japan As recombinant viruses expressing scorpion toxins are mov- ing closer toward the market, it is important to obtain large amounts of pure toxin for biochemical characterization and the evaluation of biological activity in nontarget organisms. In the past, we purified a large amount of Androctonus australis anti-insect toxin (AaIT) present in the venom of A. australis with an analytical reversed-phase column by repeated runs of crude sample. We now report 20 times improved efficiency and speed of the purification by employing a preparative reversed-phase column. In just two consecutive HPLC steps, al- most 1 mg of AaIT was obtained from 70 mg crude venom. Furthermore, additional AaIT was obtained from side fractions in a second HPLC run. Recently discovered insect selective toxin, AaIT5, was isolated simultaneously from the same venom batch. It shows different biological toxicity symptoms than the Abbreviations used: AaIT, Androctonus australis anti-in- ionization time-of-flight mass spectrometry; MS, mass sect toxin; AaH1, AaH2, AaH3, AaH4, Androctonus aus- spectrometry; PDB, protein data bank; SCR, structurally tralis alpha anti-mammal toxin; BCA, bicinchoninic acid; conserved regions; TFA:, trifluoroacetic acid; Ts7, Tityus BjIT2, Buthotus judaicus depressant anti-insect toxin; serrulatus beta anti-mammal toxin. Css2, Centruroides suffusus suffusus beta anti-mammal toxin; CsEV3, variant 3 from Centruroides sculpturatus Contract grant sponsor: U.S. Department of Agriculture; Ewing; 3-D, three-dimensional; ESI-MS, electrospray ion- Contract grant number: 94-37302-0567; Contract grant spon- ization mass spectrometry; HFBA, heptafluorobutyric acid; sor: BARD; Contract grant number: IS-2139-92; Contract LqhIT2, Leiurus quinquestriatus hebraeus depressant anti- grant sponsor: U.C. Systemwide Biotechnology Program; insect toxin; LqhαIT, Leiurus quinquestriatus hebraeus Contract grant sponsor: NIEHS Center for Environmental alpha anti-insect toxin; Lqq3, Leiurus quinquestriatus Health; Contract grant number: P30ES05707 quinquestriatus anti-insect and anti-mammal toxin; Lqq5, Leiurus quinquestriatus quinquestriatus alpha anti-mam- *Correspondence to: Bruce D. Hammock, Department of En- mal toxin; LqqIT1, Leiurus quinquestriatus quinques- tomology, University of California, Davis, CA 95616; E-mail: triatus excitatory anti-insect toxin; LqqIT2, Leiurus [email protected] quinquestriatus quinquestriatus depressant anti-insect toxin; MALDI-TOF-MS, matrix assisted laser desorption/ Received 26 June 1997; accepted 25 March 1998 © 1998 Wiley-Liss, Inc. 54 Nakagawa et al. known excitatory and depressant insect toxins. AaIT5 gave 100% mortality with a dose of less than 1.3 mg against fourth- instar tobacco budworms Heliothis virescens 24 h after injec- tion. During the purification process, we implemented mass spectrometry in addition to bioassays to monitor the presence of AaIT and AaIT5 in the HPLC fractions. Mass spectrometric screening can unambiguously follow the purification process and can greatly facilitate and expedite the downstream purification of AaIT and AaIT5 eliminating the number of bioassays required. Further, electrospray ionization was compared with matrix-assisted desorption/ionization and evaluated as a method of choice for mass spectrometric characterization of fractions from the venom purification for it provided higher mass accuracy and relative quantitation capability. Molecular models were built for AaIT5, excitatory toxin AaIT4, and depressant toxin LqhIT2. Three-dimensional structure of AaIT5 was compared with structures of the other two toxins, suggest- ing that AaIT5 is similar to depressant toxins. Arch. Insect Biochem. Physiol. 38:53–65, 1998. © 1998 Wiley-Liss, Inc. Key words: AaIT; AaIT5; mass spectrometry; Heliothis virescens; HPLC; scorpion toxin; molecular modeling INTRODUCTION preparations (Debin et al., 1993). Anti-mammal toxins affecting sodium channels are subclassi- A number of peptide anti-mammal and anti- fied into α- and β-toxins (Jover et al., 1980; insect toxins have been purified from the venom Couraud et al., 1982), and anti-insect toxins into of various scorpion species and their primary se- excitatory, depressant, and α-insect toxins quences have been determined (Rochat et al., (Zlotkin et al., 1985, 1995). The venom of the scor- 1979; Zlotkin et al., 1991; Bontems et al., 1991; pion Androctonus australis has been extensively Crest et al., 1992; Debin et al., 1993; Becerril et studied, and the sequences of 11 toxins, includ- al., 1995; Nakagawa et al., 1997). For several tox- ing both mammal and insect toxins, have been ins, their three-dimensional (3-D) structures were determined (Rochat et al., 1972, Kopeyan et al., determined by X-ray diffraction (Housset et al., 1979; Martin et al, 1987; Darbon et al., 1982; 1994; Zhao et al., 1992) and nuclear magnetic Loret et al., 1990, 1991; Bougis et al., 1989; resonance (NMR) (Bontems et al., 1991; Johnson Mansuelle et al., 1992, Nakagawa et al., 1997). and Sugg, 1992; Inisan et al., 1995; Landon et Among the insect selective toxins, AaIT ob- al., 1997), and it has been accepted that the in- tained from the venom of A. australis is the most tegrity of the 3-D structure of peptides is essen- toxic peptide to some insects so far reported tial for their interaction with ionic channels (Zlotkin et al., 1971, 1994, 1995). It induces fast (Sabatier et al., 1987). The relationship between contraction paralysis at a very small dose. Its 3-D structure and the interaction with ion chan- toxicity to lepidopterous larvae is lower than nel is carefully discussed (Sabatier et al, 1993; its toxicity to dipterous and coleopterous insects Inisan et al, 1995; Landon et al., 1997). These scorpion toxins affect the sodium, potassium, chlo- (Herrmann et al., 1990, 1995). Because AaIT has ride, and calcium channels. Generally long chain high potency and strict insect selectivity, the gene (60–70 amino acids) peptides mainly have an ef- coding of this toxin was introduced into bacu- fect on sodium channels (Darbon et al., 1982; loviruses to be used as a biopesticide for lepi- Fontecilla-Camps et al., 1988; Fontecilla-Camps, dopterous insects (Maeda et al., 1991; Stewart et 1989; Loret et al., 1990), and short chain (less al, 1991; McCutchen et al., 1991, McCutchen and than 40 amino acids) mainly on the potassium Hammock, 1994). Baculoviruses expressing the channels (Chicchi et al., 1988; Zerrouk et al., toxin showed an increased speed of kill with the 1993; Legros et al., 1996). Chlorotoxin has been tobacco budworm Heliothis virescens (McCutchen demonstrated to act on reconstituted rat epithe- et al., 1991; McCutchen and Hammock, 1994), lial chloride channels from embryonic rat brain which results in a substantial reduction of feed- Purification and Modeling of Scorpion Toxins 55 ing damage (Hoover et al., 1995). We recently Perkin Elmer software Omega-4 running on a PC found a new insect toxin, AaIT5, in the venom of computer (Everex 386/20). Fractions were col- A. australis (Nakagawa et al., 1997). This toxin lected manually according to the absorption at manifests stronger toxicity against tobacco bud- 280 nm. worm, as compared with AaIT, and it may be an For the first HPLC purification, the com- improved tool for use in recombinant pest control bined supernatant (20 ml) was loaded repeatedly agents. As recombinant viruses expressing scorpion through 5-ml loop injector onto a preparative re- toxins move toward the market, it is important to versed-phase C4 column (22 × 250 mm; Vydac, obtain large amounts of highly pure toxin for evalu- Hesperia, CA) at a flow rate of 10 ml/min. The ation of toxicity in nontarget organisms and their gradient was 0–60% CH3CN/0.1% trifluoroacetic biochemical characterization including the primary acid (TFA) in 60 min. For the second purifica- structure. Thus, it is critical to have rapid and high tion, the fraction of interest was diluted with 0.1% yield purification procedures. heptafluorobutyric acid (HFBA)/water to lower We describe a significantly improved and the CH3CN concentration. The second step was more rapid purification procedure for AaIT and performed using an analytical reversed phase AaIT5 over the one reported previously (Naka- Vydac C4 column (4.6 × 250 mm) at a flow rate gawa et al., 1997). We used a preparative reverse- of 0.6 ml/min; 5-ml aliquots of samples were in- phase high-performance liquid chromatography

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