Structural analysis of heteroheptamer transmembrane pore supramolecule of staphylococcal gamma-hemolysin

• Project/Area Number: 15590380
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Bacteriology (including Mycology)
• Research Institution: Tohoku University
• Principal Investigator: TOMITA Toshio Tohoku University, Graduate School of Agricultural Science, Associate Professor, 大学院・農学研究科, 助教授 (00126129)
• Co-Investigator(Kenkyū-buntansha): KAMIO Yoshiyuki Tohoku University, Graduate School of Agricultural Science, Professor, 大学院・農学研究科, 教授 (00109175)
• Project Period (FY): 2003 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥3,700,000 (Direct Cost: ¥3,700,000); Fiscal Year 2004: ¥1,200,000 (Direct Cost: ¥1,200,000); Fiscal Year 2003: ¥2,500,000 (Direct Cost: ¥2,500,000)
• Keywords: Staphylococcus aureus / gamma-hemolysin / two-component haemolysin / transmembrane pore / molecular architecture / 3-dimensional structure / electron microscopy / subunit arrangement / 二成分性溶血毒素

Research Abstract

Staphylococcal γ-hemolysin (Hlg) is a two-component cytolysin secreated by Staphylococcus aureus. We have previously shown that the two-component γ-hemolysin assembles from LukF (or Hlg1, 34 kDa) and Hlg2 (32 kDa) to form ring-shaped transmembrane pores of approximately 200 kDa on human erythrocytes. Our recent study involving electron microscopy and chemical cross-linking suggested that LukF and Hlg2 assemble in a stochastic manner to form alternate complexes with subunit stoichiometries of 3:4 and 4:3. In this research project, we attempted to construct nanogold-labeled glutathione-S-transferase (GST) fusion proteins of LukF and Hlg2 to visualize the alternate arrangements of LukF and Hlg2 under electron microscope. For nanogold labeling of the GST fusion proteins, three Cys residues (except for Cys138) of GST were successfully replaced with Ala by using primer extension procedure of polymerase chain reactions. Recombinant GST fusion proteins of LukF and Hlg2 thus obtained were hemolytically active in the presence of their counterpart. We now attempt to separate nanogold-labeled GST fusion proteins from unlabeled fusion proteins and nanogold particles. In this research project, we also analyzed fine structure of the heteroheptamers of Hlg by electron microscopy. The pore complexes isolated from human erythrocytes were negatively stained with phosphotungstic acid and observed under a transmission electron microscopy. High resolution images of the pore complexes from different angles were collected and analyzed. As a result, the pore complex of Hlg has a barrel morphology with 10-nm width and 10-nm height.

Research Products

• [Journal Article] Pleurotolysin, a novel sphingomyelin-specific two-component cytolysin from the edible mushroom Pleurotus ostreatus, assembled into a transmembrane pore complex. (2004)
  • Author(s): Tomita, T., Noguchi, K., Mimuro, H., Ukaji, F., Ito, K., Sugawara-Tomita, N., Hashimoto, Y.
  • Journal Title: J.Biol.Chem. 279 Pages: 26975-26982
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Protein sequence analysis, cloning an expression of flammutoxin a pore-forming cytolysin from Flammulina velutipes : Maturation of dimeric precursor to monomeric (2004)
  • Author(s): Tomita, T., Mizumachi, Y, Chong, K, Ogawa, Konishi, Sugawara Tomita, N., Dohmae, N, Hashimoto Y, Takio
  • Journal Title: J.Biol.Chem. 279 Pages: 54161-54172
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Cloning expression, and pore-forming properties of mature and precursor forms of pleurotolysin, a sphingomyelin-specific two-component cytolysin from the edible (2004)
  • Author(s): Sakurai, N., Kaneko, J., Kamio, Y., Tomita, T.
  • Journal Title: Biochim.Biophys.Acta 1679 Pages: 65-73
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Effect of a pore-forming protein derived from Flammulina velutipes on the Caco-2 intestinal epithelial cell monolayer. (2004)
  • Author(s): Narai, A., Watanabe, H., Iwanaga, T., Tomita, T., Shimizu, M.
  • Journal Title: Biosci.Biotechnol.Biochem. 68 Pages: 2230-2238
  • NAID: 130000030909
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Pleurotolysin, a novel sphingomyelin-specific two-component cytolysin from the edible mushroom Pleurotus ostreatus, assembles into a transmembrane pore complex. (2004)
  • Author(s): Tomita, T., Noguchi, K.Mimuro, H.Ukaji, F.Ito, K., Sugawara-Tomita, N., Hashimoto, Y.
  • Journal Title: J.Biol.Chem. 279 Pages: 26975-26982
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Protein sequence analysis, cloning and expression of flammutoxin, a pore-forming cytolysin from Flammulina velutipes : Maturation of dimeric precursor to monomeric active form by carboxyl-terminal truncation. (2004)
  • Author(s): Tomita, T., Mizumachi, Y, Chong, K, Ogawa., Konishi, Sugawara Tomita, N., Dohmae, N, Hashimoto, Y., Takio, K.
  • Journal Title: J.Biol.Chem. 279 Pages: 54161-54172
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Cloning, expression, and pore-forming properties of mature and precursor forms of pleurotolysin, a sphingomyelin-specific two-component cytolysin from the edible mushroom Pleurotus ostreatus. (2004)
  • Author(s): Sakurai, N., Kaneko, J., Kamio., Y., Tomita, T.
  • Journal Title: Biochim.Biophys.Acta 1679 Pages: 65-73
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Pleurotolysin, a novel sphingomyelin-specific two-component cytolysin from the edible mushroom Pleurotus ostreatus, assembles into a transmembrane pore complex. (2004)
  • Author(s): Tomita, T., Noguchi, K., Mimuro, H., Ukaji, F., Ito, K., Sugawara-Tomita, N., Hashimoto, Y.
  • Journal Title: J.Biol.Chem. 279 Pages: 26975-26982
• [Journal Article] Protein sequence analysis, cloning and expression of flammutoxin, a pore-forming cytolysin from Flammulina velutipes : Maturation of dimeric precursor to monomeric active form by carboxyl-terminal truncation. (2004)
  • Author(s): Tomita, T., Mizumachi, Y., Chong, K, Ogawa, Konishi, Sugawara, Tomita, N, Dohmae, N, Hashimoto Y, Takio, K.
  • Journal Title: J.Biol.Chem. 279 Pages: 54161-54172
• [Journal Article] Cloning, expression, and pore-forming properties of mature and precursor forms of pleurotolysin, a sphingomyelin-specific two-component cytolysin from the edible mushroom Pleurotus ostreatus. (2004)
  • Author(s): Sakurai, N., Kaneko, J., Kamio., Y., Tomita, T.
  • Journal Title: Biochim.Biophys.Acta, 1679 Pages: 65-73
• [Publications] Ito, Y., Tomita, T., Roy, N., Sugawara-Tomita, N., Abe, N., Kamio, Y.: "Cloning, expression, and cell-surface localization of Paenibacillus sp.W-61 xylanase 5, a multidomain xylanase"Appl.Environ.Microbiol.. 69・12. 6969-6978 (2003)
• [Publications] Sawada, H., Umedo, M., Kawase, T., Baba, M., Tomita, T.: "Sythesis and properties of fluoroalkyl end-capped sulfobetaine polymers"J.Appl.Polym.Sci.. 92. 1144-1153 (2004)
• [Publications] Sawada, H., Kawase, T., Inaba, Y., Baba, M., Tomita, T.: "Synthesis of fluoroalkyl end-capped oligomers containing pendant phosphinic acid and phosphonic acid segments"International J.Polymeric Mater.. (in press). (2004)