Isolation of a novel lectin from the dorsal spines of the devil stinger, Inimicus japonicus

Isolation of a novel lectin from the dorsal spines of the devil stinger, Inimicus japonicus Hideyuki Nakagawa . Kuniko Nagasaka . Hitomi Sakai . Kozue Edo . Mitsuko Shinohara . Kiyoshi Ohurari 0 1 0 M. Shinohara K. Ohura Department of Pharmacology, Osaka Dental University , Hirakata 573-1121 , Japan 1 K. Nagasaka Fukae Nagasaka Clinic , Etajima 737-2214 , Japan A novel lectin was purified from the dorsal spines of the devil stinger, Inimicus japonicus using a combination of affinity chromatography techniques. A single band was detected on a native PAGE gel with a relative molecular mass of 97 kDa. The N-terminal partial amino acid of the intact 75 kDa subunit of the 97 kDa lectin was found to be DHEDS. The agglutination of rabbit erythrocytes by the 97 kDa lectin was inhibited most effectively by methyl a-D-mannoside. The 97 kDa lectin stimulated mitogenesis in murine splenocytes. This is the first study to examine the dorsal lectin of I. japonicus and one of the very few studies on venom lectins from venomous scorpaeniform fish. These results suggest that the devil stinger, I. japonicus, may be a novel resource for biologically active substances. Devil stinger; Inimicus japonicus; Dorsal spine; Lectin; Murine splenocytes - A large number of venomous and poisonous animals exist in aquatic environments worldwide. More than 200 of the approximately 22,000 species of fish in the ocean are considered to be venomous (Halstead 1988; Russell 1996). Most of these venomous fish are non-migratory, slow moving, and mainly live in shallow waters in protected habitats (Maretic 1988). Venomous scorpaeniform fish include the lionfish and scorpionfish from the family Scorpaenidae, devil stinger and stonefish from the family Synanceiidae, and waspfish from the family Tetrarogidae (Kiriake et al. 2013). These fish possess 1117 dorsal, 2 pelvic, and 3 anal spines, with the venom secretory complex being located within the anterolateral grooves of these spines (Russell 1965; Halstead 1988; Haddad et al. 2003; Smith and Wheeler 2006; Andrich et al. 2010). The devil stinger Inimicus japonicus, which belongs to the family Synanceiidae, has 17 dorsal, 1 pelvic, and 2 anal spines, which contain venom glands that are covered by an integumentary sheath (Tange 1954). I. japonicus, a valuable demersal scorpaenid fish, is widely distributed along the coastal areas of eastern Asia at a depth of between 10 and 200 m (Wang et al. 2013). The body of Inimicus is covered in warts or skin lumps, with many skin tubercle glands similar to the stonefish. Envenomation occurs when people carelessly handle or step on these fish, and are stung by the dorsal spines. Envenomation appears immediately as intense, sharp, and persisting local pain, and swelling around the sting (Auerbach 1991; Yamamoto et al. 2010). Symptoms depend on the amount of venom injected. Systemic effects including dizziness, fever, and delirium have been reported (Auerbach 1991). However, only a limited number of studies have investigated the toxicity of I. japonicus. Therefore, we herein examined the dorsal venom of the devil stinger, I. japonicus using column chromatography and, for the first time, separated a novel lectin that induced mitogenic activity. Isolation of a dorsal lectin Inimicus japonicus (18 specimens, average size of 20 cm) were collected by local fishermen from the coast of Hiroshima Prefecture and Tokushima Prefecture, Japan in May 2003 (Fig. 1a, b). The collected fish were transported alive or frozen to our laboratory. The dorsal spines (a total of 17) of I. japonicus were cut from their base, and the dorsal venom protein was extracted with 0.15 M NaCl as reported previously (Nagasaka et al. 2009). Briefly, in the first step of purification, the venom protein was applied to a Phenyl Sepharose CL4B (GE Healthcare, Uppsala, Sweden) affinity chromatographic column (2 ml) equilibrated with 16 mM TrisHCl buffer containing 2 M NaCl (pH 7.4). The sample was rinsed and washed with the same buffer containing 0.01 M NaCl at a flow rate of 20 ml/h (Fig. 1a). The 2-ml elution fractions were collected and analyzed for absorption at 280 nm and agglutinating activity. Each of the unbound and bound fractions was pooled and Fig. 1 The devil stinger Inimicus japonicus as seen from above (a) and a specimen with erect dorsal spines (b) analyzed for electrophoresis. The final step of purification was achieved using a Concanavalin A-Sepharose 4B (Sigma-Aldrich, Missouri, USA) column (2 ml) equilibrated with 20 mM Tris-HCl buffer containing 0.4 M NaCl (pH 7.4). The unbound fraction (the PS-I fraction) was rinsed and washed with the same buffer, and eluted with the same buffer containing 100 mM methyl-a-D-mannoside in the buffer at a flow rate of 20 ml/h (Fig. 1b). Elution fractions (2 ml) were collected and analyzed for absorption at 280 nm and agglutinating activity. Each of the unbound (PS-I-ConA-I fraction) and bound (PS-I-ConA-II fraction) fractions was pooled and analyz (...truncated)