CDNA cloning and expression of Contractin A, a phospholipase A2-like protein from the globiferous pedicellariae of the venomous sea urchin Toxopneustes pileolus. (15th December 2015)

• Record Type: Journal Article
• Authors: Hatakeyama, Tomomitsu; Higashi, Erika; Nakagawa, Hideyuki
• Is Part Of: Toxicon. Volume 108(2015)
• Page Start: 46
• Page End: 52
• Publication Date: 2015-12-15
• Subjects: Sea urchin -- Toxopneustes pileolus -- cDNA cloning -- Phospholipase A2 -- Liposome -- Carboxyfluorescein; CF carboxyfluorescein -- EDTA ethylenediamine tetraacetate -- PLA2 phospholipase A2 -- RACE rapid amplification of cDNA ends -- SUL-I sea urchin (Toxopneustes pileolus) lectin-I -- TBS Tris-buffered saline; Toxins -- Periodicals; Venom -- Periodicals; 615.9
• DOI: 10.1016/j.toxicon.2015.09.040
• Languages: English
• ISSNs: 0041-0101

Abstract

Abstract: 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 Ca 2+ -dependent release of carboxyfluorescein from the liposomes, suggesting that Contractin A has phospholipase A2 activity, which may be closely associated with its biological activities. Highlights: 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 Ca 2+ -dependent lipolytic activity.