Identification of the minimum segment in which the threonine246 residue is a potential phosphorylated site by protein kinase A for the LukS‐specific function of staphylococcal leukocidin

FEBS Letters

Nariya, Hirofumi¹  Kamio, Yoshiyuki¹  Nishiyama, Akihito¹ 

[1] Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, 1-1 Amamiya-machi, Tsutsumi-dori, Aoba-ku, Sendai 981, Japan

关键词: Staphylococcal leukocidin;    γ-Hemolysin;    Bi-component cytolysin;    LukS;    Hlg2;    Toxin phosphorylated by protein kinase A;    LukS;    LukS component of leukocidin;    LukF;    LukF component of leukocidin/γ-hemolysin;    Hlg2;    Hlg2 component of γ-hemolysin;    PMSF;    phenylmethylsulfonyl fluoride;   

DOI  :  10.1016/S0014-5793(97)01100-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Staphylococcal leukocidin and γ-hemolysin consist of LukF and LukS for leukocidin and LukF and Hlg2 for γ-hemolysin. In this report, we identify the minimum segment responsible for the LukS-specific function of leukocidin. After chemical analysis and homology study of the amino acid sequence of the C-terminal region between LukS and Hlg2, we found a unique 5-residue sequence I²⁴²K²⁴³R²⁴⁴S²⁴⁵T²⁴⁶ in LukS in which the 4-residue KRST is identical with that of the phosphorylated segment of a protein phosphorylated by protein kinase A. To elucidate whether the 5-residue segment is essential for the LukS function, we created plasmids containing a series of mutant genes corresponding to the 5-residue sequence and expressed them in Escherichia coli. The mutant proteins were purified and assayed for their leukocytolytic activity with LukF. The mutant MLS-TS, in which the T²⁴⁶ in the 5-residue sequence was replaced by S, showed leukocidin activity 10 times higher than that of the intact LukS. However, neither mutant MLS-TY nor MLS-TA, in which T²⁴⁶ was replaced by Y or A, respectively, showed leukocidin activity. The 5-residue segment was found to be deleted in Hlg2. The mutant of Hlg2, in which the 5-residue segment was inserted at the position that the segment is deleted, showed leukocidin activity. The boiled LukS, MLS-TS, and MHS-Z were strongly phosphorylated with [γ-³²P]ATP in the presence of protein kinase A in a cell-free system. Thus, we conclude that the 5-residue segment I²⁴²K²⁴³R²⁴⁴S²⁴⁵T²⁴⁶ is the pivotal segment of LukS responsible for the LukS function of staphylococcal leukocidin.

【 授权许可】

Unknown