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.
Staphylococcal leukocidin and gamma-hemolysin consist of LukF and LukS for leukocidin and LukF and Hlg2 for gamma-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 [gamma-32P]ATP in the presence of protein kinase A in a cell-free system. Thus, we conclude that the 5-residue segment 1242K243R244S245T246 is the pivotal segment of LukS responsible for the LukS function of staphylococcal leukocidin.
Nariya
H
H
Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, Sendai, Japan.
Nishiyama
A
A
Kamio
Y
Y
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism; D015153:Blotting, Western; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006460:Hemolysin Proteins; D006801:Humans; D007956:Leukocidins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009504:Neutrophils / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D017386:Sequence Homology, Amino Acid; D013211:Staphylococcus aureus / Q000737:chemistry*; D013912:Threonine / Q000378:metabolism
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D017931:DNA Primers; D006460:Hemolysin Proteins; D007956:Leukocidins; D010446:Peptide Fragments; C037956:gamma-hemolysin, Staphylococcus aureus; C108483:leukocidin S-component protein, Staphylococcus; D013912:Threonine; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases