Nishiyama Akihito, Guerra Marie Antonette Ruth V, Sugawara Noriko, Yokota Kenji, Kaneko Jun, Kamio Yoshiyuki
Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan.
Biosci Biotechnol Biochem. 2002 Feb;66(2):328-35. doi: 10.1271/bbb.66.328.
Luk-I produced by Staphylococcus intermedius was found to be a new member of the staphylococcal bi-component pore-forming toxin family, in which staphylococcal leukocidin, Panton-Valentine leukocidin, and gamma-hemolysin are included. Luk-I consists of LukS-I and LukF-I. From the deduced amino acid sequence of LukS-I, a 4-residue sequence, K135K1361137S138, at the root of the stem region was found to be identical with that of the phosphorylated segment of a protein phosphorylated by protein kinase A. A mutant of LukS-I (MLSI-SA), in which the Ser138 residue was replaced by an alanine residue, was created, purified, and assayed for its leukocytolytic and pore-forming activities with LukF-I. Both LukS-I and MLSI-SA formed a ring-shaped complex with LukF-I on rabbit erythrocytes and human polymorphonuclear leukocytes (HPMNLs) membrane. However, MLSI-SA showed no leukocytolytic activity with LukF-I. LukS-I was phosphorylated by protein kinase A in the presence of [gamma-32P] ATP in a cell-free system, but MLSI-SA was not phosphorylated significantly. A potent and selective inhibitor of protein kinase A (N-[2(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89)) showed 50% inhibition of the Luk-I-induced cell lysis at 0.5 nM. Thus, it is concluded that the phosphorylation of the Ser138 residue in the 4-residue segment K135K1361137S138 of LukS-I is important for the leukocytolysis of HPMNLs.
中间葡萄球菌产生的Luk-I被发现是葡萄球菌双组分成孔毒素家族的一个新成员，该家族包括葡萄球菌杀白细胞素、潘顿-瓦伦丁杀白细胞素和γ-溶血素。Luk-I由LukS-I和LukF-I组成。从LukS-I推导的氨基酸序列中，发现茎区根部的一个4残基序列K135K136I137S138与蛋白激酶A磷酸化的一种蛋白质的磷酸化片段的序列相同。构建了LukS-I的一个突变体（MLSI-SA），其中Ser138残基被丙氨酸残基取代，对其进行纯化，并与LukF-I一起检测其溶白细胞和形成孔的活性。LukS-I和MLSI-SA都在兔红细胞和人多形核白细胞（HPMNLs）膜上与LukF-I形成环形复合物。然而，MLSI-SA与LukF-I一起时未显示出溶白细胞活性。在无细胞系统中，LukS-I在[γ-32P]ATP存在下被蛋白激酶A磷酸化，但MLSI-SA未被显著磷酸化。一种有效的蛋白激酶A选择性抑制剂（N-[2-(对溴肉桂酰胺基)乙基]-5-异喹啉磺酰胺（H-89））在0.5 nM时对Luk-I诱导的细胞裂解有50%的抑制作用。因此，可以得出结论，LukS-I的4残基片段K135K136I137S138中Ser138残基的磷酸化对HPMNLs的溶白细胞作用很重要。
