The globiferous pedicellariae of the venomous sea urchin Toxopneustes pileolus contains several biologically active proteins. We have cloned the cDNA of one of the toxin components, SUL-I, which is a rhamnose-binding lectin (RBL) that acts as a mitogen through binding to carbohydrate chains on target cells. Recombinant SUL-I (rSUL-I) was produced in Escherichia coli cells, and its carbohydrate-binding specificity was examined with the glycoconjugate microarray analysis, which suggested that potential target carbohydrate structures are galactose-terminated N-glycans. rSUL-I exhibited mitogenic activity for murine splenocyte cells and toxicity against Vero cells. The three-dimensional structure of the rSUL-I/l-rhamnose complex was determined by X-ray crystallographic analysis at a 1.8 Å resolution. The overall structure of rSUL-I is composed of three distinctive domains with a folding structure similar to those of CSL3, a RBL from chum salmon (Oncorhynchus keta) eggs. The bound l-rhamnose molecules are mainly recognized by rSUL-I through hydrogen bonds between its 2-, 3-, and 4-hydroxy groups and Asp, Asn, and Glu residues in the binding sites, while Tyr and Ser residues participate in the recognition mechanism. It was also inferred that SUL-I may form a dimer in solution based on the molecular size estimated via dynamic light scattering as well as possible contact regions in its crystal structure.

The globiferous pedicellariae of the venomous sea urchin Toxopneustes pileolus contain several 25 biologically active proteins. Among these, a galactose-binding lectin SUL-I isolated from the venom 26in the large globiferous pedicellariae shows several activities such as mitogenic, chemotactic, and 27 cytotoxic activities through binding to the carbohydrate chains on the cells. We cloned cDNA 28 encoding SUL-I by reverse transcription-PCR using the degenerate primers designed on the basis of with the carbohydrate-recognition domains of the rhamnose-binding lectins, which have been mostly 34 found in fish eggs. While rSUL-I exhibited binding activity for several galactose-related sugars, the 35 highest affinity was found for L-rhamnose among carbohydrates tested, confirming that SUL-I is a 36 rhamnose-binding lectin. rSUL-I also showed hemagglutinating activity toward rabbit erythrocytes, 37 indicating the existence of more than one carbohydrate-binding site to cross-link the carbohydrate 38 chains on the cell surface, which may be closely related to its biological activities. 39 40