Wide distribution of cysteine-rich secretory proteins in snake venoms: Isolation and cloning of novel snake venom cysteine-rich secretory proteins☆
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Materials
Lyophilized venom of Ophiophagus hannah was purchased from the Japan Snake Institute (Gunma, Japan). Lyophilized venoms of Agkistrodon piscivorus piscivorus and Crotalus atrox were from Kentucky Reptile Zoo (Kentucky, USA) and Sigma (MO, USA), respectively. Superdex 75 pg, SP–Sepharose High Performance, heparin–Sepharose CL-6B, Q-Sepharose Fast Flow, and Mono S columns were from Amersham–Pharmacia Biotech. The Vydac Protein & Peptide C18 HPLC column and the COSMOSIL 5C18 AR-300 HPLC column were
Screening of snake venom CRISP family proteins
Previously, we isolated and obtained full-length clones for triflin, ablomin, latisemin, tigrin, pseudechetoxin, and pseudecin, which are CRISP family proteins from the venom of six different snakes [41], [42]. To search for additional CRISP family proteins in snake venoms, we screened 15 snake venoms for reactivity to anti-triflin antiserum (Table 1). As expected, there was reactivity to the venoms from which CRISP family proteins had previously been isolated. In addition, venom from A. p.
Discussion
Recently, we reported the cloning and characterization of six novel snake venom proteins that are homologous to CRISPs from mammals and to helothermine from the Mexican beaded lizard [41], [42]. Three of them, ablomin (A. blomhoffi, Viperidae), triflin (T. flavoviridis, Viperidae), and latisemin (L. semifasciata, Elapidae) inhibit depolarization-induced contraction of rat tail arterial smooth muscle, showing the properties of L-type Ca2+ channel blocking toxins, although tigrin (R. tigrinus
Acknowledgements
We would like to thank Dr. Hisashi Koike for helpful advice on cDNA cloning. We thank Drs. Michihisa Toriba and Atsushi Sakai (Japan Snake Institute, Gunma, Japan) and Dr. Masatoshi Nozaki (Habu Research Section, Okinawa Prefectural Institute of Health and Environment) for extracting the venom glands and Dr. Mitsuo Mita for helpful comments on the smooth muscle contraction assay. We also thank Satsuki Hori, Ayako Ohkubo, Koji Takani, and Nao Kamei for technical assistance.
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This work was supported in part by Scientific Research Grants-in-Aid from the Ministry of Education, Science and Culture of Japan (T.M.).