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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Abstract

Cysteine-rich secretory proteins (CRISPs) are found in epididymis and granules of mammals, and they are thought to function in sperm maturation and in the immune system. Recently, we isolated and obtained clones for novel snake venom proteins that are classified as CRISP family proteins. To elucidate the distribution of snake venom CRISP family proteins, we evaluated a wide range of venoms for immuno-cross-reactivity. Then we isolated, characterized, and cloned genes for three novel CRISP family proteins (piscivorin, ophanin, and catrin) from the venom of eastern cottonmouth (Agkistrodon piscivorus piscivorus), king cobra (Ophiophagus hannah), and western diamondback rattlesnake (Crotalus atrox). Our results show the wide distribution of snake venom CRISP family proteins among Viperidae and Elapidae from different continents, indicating that CRISP family proteins compose a new group of snake venom proteins.

Section snippets

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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