title: cDNA cloning and expression of Contractin A, a phospholipase A2-like protein from the globiferous pedicellariae of the venomous sea urchin Toxopneustes pileolus.
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.
authors.affiliation: Biomolecular Chemistry Laboratory, Graduate School of Engineering, Nagasaki University, Bunkyo-machi 1-14, Nagasaki 852-8521, Japan. Electronic address: thata@nagasaki-u.ac.jp.
authors.forename: Tomomitsu
authors.initials: T
authors.lastname: Hatakeyama
authors.affiliation: Biomolecular Chemistry Laboratory, Graduate School of Engineering, Nagasaki University, Bunkyo-machi 1-14, Nagasaki 852-8521, Japan.
authors.forename: Erika
authors.initials: E
authors.lastname: Higashi
authors.affiliation: Division of Environmental Symbiosis, Graduate School of Integrated Arts and Sciences, Tokushima University, Tokushima 770-8502, Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Nakagawa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005452:Fluoresceins / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008387:Marine Toxins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D054467:Phospholipases A2 / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012617:Sea Urchins / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein
chemical_list: D018076:DNA, Complementary; D005452:Fluoresceins; D008081:Liposomes; D008387:Marine Toxins; D011993:Recombinant Fusion Proteins; C066979:contractin A; C024098:6-carboxyfluorescein; D054467:Phospholipases A2; D002118:Calcium
keywords: Carboxyfluorescein; Liposome; Phospholipase A(2); Sea urchin; Toxopneustes pileolus; cDNA cloning