Rapid and efficient identification of cysteine-rich peptides by random screening of a venom gland cDNA library from the hexathelid spider Macrothele gigas H. Satake a , E. Villegas a,1 , N. Oshiro b , K. Terada b , T. Shinada c , G. Corzo a, * a Suntory Institute for Bioorganic Research, 1-1-1 Wakayamadai, Shimamoto-Cho, Mishima-Gun, Osaka 618-8503, Japan b Okinawa Prefectural Institute of Health and Environment, 2085 Ozato, Ozato-Son, Okinawa 901-1202, Japan c Graduate School of Science, Osaka City University, Sugimoto, Osaka, Japan Received 20 January 2004; revised 23 April 2004; accepted 11 May 2004 Available online 17 June 2004 Abstract We identified novel 10 multi-cysteine peptides, namely Magi 7 –16, from the spider Macrothele gigas by simple random cDNA screening of the venom gland. Mass analysis of the crude venom detected the mass numbers of the cross-linked forms of all peptides, confirming their presence in the venom. Magi 11, a C-terminus amidated peptide, was chemically synthesized and was indistinguishable from the native peptide proving the feasibility of the method for peptide identification. Moreover, toxicological assays showed diverse lethal or paralytic activities of these peptide toxins on mice and/or insects. q 2004 Elsevier Ltd. All rights reserved. Keywords: Spider; Macrothele gigas; Peptide; Toxin; cDNA; Ion channel 1. Introduction Spider venom glands produce diverse peptide toxins possessing various biological activities (Corzo and Escoubas, 2003; Escoubas et al., 2000). One of the most prevalent spider peptide toxin characteristics is that they are potent and unique ion channel blockers or modulators (Corzo and Escoubas, 2003; Escoubas et al., 2000). These peptide toxins comprise from 25 to 70 amino acid residues primary structure tightly folded through multiple intramolecular disulfide bridges (Corzo and Escoubas, 2003; Escoubas et al., 2000). The specific binding affinities, prominent bioactivities, and ease of chemical or biological synthesis of short spider multi-cysteine-containing peptide toxins indicate that studies on such peptide toxins will contribute a great deal to development of not only useful molecular probes for studies on ion channels but also leading compounds of novel insecticides and clinical agents (Bourinet et al., 2001; Corzo et al., 2000; Ford et al., 2002; Middleton et al., 2002; Newcomb et al., 1998; Swartz and MacKinnon, 1995). Sequence determination and precise pharmacological analysis of peptide toxins from spiders of interest are frequently failed or restricted because of difficulties in obtaining plenty venom material. To overcome these problems, screening of venom gland cDNAs is a promising method with regard to efficiency of characterization of potential peptide toxins. Recently, we purified six peptide neurotoxins from the venom of the hexathelid spider Macrothele gigas, and revealed their pharmacological effects on insects and mice, which were exerted by their site-specific binding affinities to sodium channels (Corzo et al., 2003). Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) 0041-0101/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2004.05.012 Toxicon 44 (2004) 149–156 www.elsevier.com/locate/toxicon 1 Present address: Centro de Investigacion en Biotecnologia UAEM, Av. Universidad 2001, Cuernavaca, Morelos, 62210, Mexico. * Corresponding author. Address: Institute of Biotechnology- UNAM, Department of Molecular Medicine and Bioprocesses Av. Universidad 2001, Cuernavaca, Morelos, 62210, Mexico. Tel.: þ 52-777-317-1209; fax: þ52-777-317-2388. E-mail address: corzo@ibt.unam.mx (G. Corzo).