成谷 宏文 東北大学, 農学部, 学術振興会特別研究員
KANEKO Jun Tohoku University, Faculty of Agriculture, Research Associate, 農学部, 助手 (30221188)
TOMITA Toshio Tohoku University, Faculty of Agriculture, Associate Professor, 農学部, 助教授 (00126129)
|Budget Amount *help
¥13,400,000 (Direct Cost : ¥13,400,000)
Fiscal Year 1998 : ¥4,800,000 (Direct Cost : ¥4,800,000)
Fiscal Year 1997 : ¥8,600,000 (Direct Cost : ¥8,600,000)
It is well known that the proteins which show gross similarities in their biological function(s) have primary structures which are similar to each other. However, recently even proteins which show dissimilarity in primary structures have been reported to have identical biological functions and the three-dimensional structures. The Staphylococcal channel-forming toxins are taken as an example. Staphylococcus aureus secretes leukocidin and gamma-hemolysin gamma -HL) into a culture medium as water soluble monomers. Leukocidin and r -HL contain two separate and synergistic protein components, LukF and LukS, and LukF and Hlg2 (or H gamma H), respectively. LukF and LukS of leukocidin, and LukF and Hlg2 of gamma -HL assemble into a ring-shaped complexes, in which the molar ratios of LukF to LukS or Hlg2, respectively are both 1 : 1, within human polymorphonuclear leukocyte membranes and erythrocyte membranes. In each case a transmembrane pore results with a functional diameter of 2.1-2.5 nm ;
this leads to lyse the respective target cell. The cell specificities of leukocidin and gamma -HL are determined by LukS and Hlg2, respectively. Despite the 72% overall identity between the LukS and Hlg2 aminoacid sequences, the respective cell specificities are strictly defined, Within the year suported by Grant-in-Aid for Scientific Research (B), we clarified the following evidences.
[I] The staphylococcal Panton-Valentine leukocidin (PVL) genes, [lukS-PV-lukF-PV] exist on the genome of a temperate bacteriophage phiPVL isolated from mitomycin C-induced Staphylococcus aureus V8 (ATCC 49775) [Kaneko et al., 1997. Biosci. Biotechnol. Biochem. 61, 1960-l962]. In this study, the complete nucleotide sequence of the phiPVL genome was analyzed, and the att sites (attL, attR, and attB) required for site-specific integration of phiPVL into the host chromosome were also determined.
[II] Identification of the minimum segment essential for the Hlg2-specific function of staphylococcal gamma Hemolysin : The 5-residue K^<23>RLA^<127> of Hlg2 is the minimum segment essential for the Hlg2-specificfunction of staphylococcal gamma-hemolysin.
[III] Identification of the minimum segment in which threonine 246 residue is a phosphorylation site by protein kinase A for the LukS-specific function of staphylococcal leukocidin : The minimum segment responsible for the LukS specific funcion of leukocidin was identified. We conclude that the 5-residue segment I^<242>K^<243>R^<244>S^<245>T^<246> is pivotal segment of LukS responsible for the LukS-function of the staphylococcal leukocidin.
[IV] The Phosphorylation of LukS by Protein Kinase A is Crucial for the LukS-Specific Function of the Staphylococcal Leukocidin on Human Polymorphonuclear Leukocytes
[V] Crystal structure of staphylococcal LukF delineates conformational changes accompaning formation of a transmembrane channel. Less