•cDNA of a lectin SUL-I in the venom of Toxopneustes pileolus was cloned.•The primary structure of SUL-I showed the homology with the rhamnose-binding lectins.•The recombinant SUL-I showed the binding affinity for l-rhamnose.•Homology model of SUL-I suggested the importance of the C-terminal region.The globiferous pedicellariae of the venomous sea urchin Toxopneustes pileolus contain several biologically active proteins. Among these, a galactose-binding lectin SUL-I isolated from the venom in the large globiferous pedicellariae shows several activities such as mitogenic, chemotactic, and cytotoxic activities through binding to the carbohydrate chains on the cells. We cloned cDNA encoding SUL-I by reverse transcription-PCR using the degenerate primers designed on the basis of the N-terminal amino acid sequence of the protein and expressed the recombinant SUL-I (rSUL-I) in Escherichia coli cells. The SUL-I gene contains an open reading frame of 927 nucleotides corresponding to 308 amino acid residues, including 24 residues of a putative signal sequence. The mature protein with 284 residues is composed of three homologous regions, each showing similarity with the carbohydrate-recognition domains of the rhamnose-binding lectins, which have been mostly found in fish eggs. While rSUL-I exhibited binding activity for several galactose-related sugars, the highest affinity was found for l-rhamnose among carbohydrates tested, confirming that SUL-I is a rhamnose-binding lectin. rSUL-I also showed hemagglutinating activity toward rabbit erythrocytes, indicating the existence of more than one carbohydrate-binding site to cross-link the carbohydrate chains on the cell surface, which may be closely related to its biological activities.

•cDNA of a venom protein Contractin A from the sea urchin Toxopneustes pileolus was cloned.•The amino acid sequence of Contractin A shares homology with secreted phospholipase A2.•Recombinant Contractin A exhibited Ca2+-dependent lipolytic activity.Venomous sea urchins contain various biologically active proteins that are toxic to predators. Contractin A is one such protein contained within the globiferous pedicellariae of the venomous sea urchin Toxopneustes pileolus. This protein exhibits several biological activities, such as smooth muscle contraction and mitogenic activity. N-terminal amino acid residues of Contractin A have been determined up to 37 residues from the purified protein. In this study, we cloned cDNA for Contractin A by reverse transcription-PCR using degenerate primers designed on the basis of its N-terminal amino acid sequence. Analysis of the cDNA sequence indicated that Contractin A is composed of 166 amino acid residues including 31 residues of a putative signal sequence, and has homology to the sequence of phospholipase A2 from various organisms. In this study, recombinant Contractin A was expressed in Escherichia coli cells, and the protein was subjected to an assay to determine lipid-degrading activity using carboxyfluorescein-containing liposomes. As a result, Contractin A was found to exhibit Ca2+-dependent release of carboxyfluorescein from the liposomes, suggesting that Contractin A has phospholipase A2 activity, which may be closely associated with its biological activities.