title: Functional expression and characterization of an acidic actinoporin from sea anemone Sagartia rosea.
abstract: Src I is the first reported acidic actinoporin from sea anemone Sagartia rosea with a pI value of 4.8 and comprises 13.9% alpha-helix, 65.1% beta-sheet, and 18.2% random coil. For structure-function studies, Src I was expressed in Escherichia coli as a cleavable fusion protein. Recombinant Src I exhibited obviously hemolytic activity, but the fusion protein Trx-Src I almost lost its hemolytic activity, suggesting the importance of the N-terminal amphiphilic alpha-helix for its functional activity. The cytotoxic effects of Src I depending on the toxin concentration and incubation time were also observed on cultured cells. Among five cell lines: NIH/3T3, U251, NSCLC, BEL-7402, and BGC-823, NSCLC was the most sensitive cells with ID(50) 2.8 microg/ml and BGC-823 was the least sensitive cells with ID(50) 7.4 microg/ml. After incubated with lipid SUVs, such as SM-SUVs and SM/PC-SUVs, the hemolytic activity of Src I was inhibited to some extent. When incubated with calcein-entrapped lipid LUVs, such as SM-LUVs, SM/PC-LUVs, and SM/PG-LUVs, Src I induced release of entrapped calcein. According to the interaction with lipid vesicles, we proposed that it was the membrane matrix made up of phospholipids, not a particular phospholipid that facilitates Src I to react properly.
authors.affiliation: The Open Laboratory for Marine Functional Genomics of National High-Tech Development, Department of Biochemistry, College of Life Sciences, Sun Yat-sen (Zhongshan) University, Guangzhou 510275, People's Republic of China.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Jiang
authors.forename: Huiping
authors.initials: H
authors.lastname: Chen
authors.forename: Wenli
authors.initials: W
authors.lastname: Yang
authors.forename: Yun
authors.initials: Y
authors.lastname: Liu
authors.forename: Wei
authors.initials: W
authors.lastname: Liu
authors.forename: Jianwen
authors.initials: J
authors.lastname: Wei
authors.forename: Hongbin
authors.initials: H
authors.lastname: Tu
authors.forename: Xiaojin
authors.initials: X
authors.lastname: Xie
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.forename: Anlong
authors.initials: A
authors.lastname: Xu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D003064:Cnidarian Venoms / Q000096:biosynthesis / Q000737:chemistry* / Q000494:pharmacology / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006460:Hemolysin Proteins / Q000096:biosynthesis / Q000737:chemistry; D006461:Hemolysis / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D008563:Membrane Lipids / Q000737:chemistry*; D008969:Molecular Sequence Data; D018272:Porins; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology / Q000633:toxicity*; D012615:Sea Anemones / Q000737:chemistry / Q000378:metabolism*
chemical_list: D003064:Cnidarian Venoms; D006460:Hemolysin Proteins; D008563:Membrane Lipids; D018272:Porins; D011993:Recombinant Fusion Proteins; C482066:Src1 protein, Sagartia rosea