Komatsuzawa H, Sugai M, Nakashima S, Yamada S, Matsumoto A, Oshida T, Suginaka H
Department of Microbiology, Hiroshima University School of Dentistry, Japan.
Microbiol Immunol. 1997;41(6):469-79. doi: 10.1111/j.1348-0421.1997.tb01880.x.
The Staphylococcus aureus autolysin gene, atl, encodes a unique 138-kDa protein (ATL) with amidase and glucosaminidase domains. ATL has been suggested to undergo proteolytic processing to generate two extracellular peptidoglycan hydrolases, 51-kDa endo-beta-N-acetylglucosaminidase (51-kDa GL) and 62-kDa N-acetylmuramyl-L-alanine amidase (62-kDa AM). To investigate cell-associated bacteriolytic enzymes for atl gene products, proteins were extracted from the cells as follows. The cells were exposed to 3 M LiCl followed by 4% SDS. Thereafter, the cells were disrupted and again extracted with 4% SDS. Whole SDS-stable cell-associated bacteriolytic proteins were extracted without disrupting the cells. Exposure to 3 M LiCl released major 138-, 115-, 85-, 62- and 51-kDa bacteriolytic proteins, and subsequent 4% SDS extraction released major 138- and 115-kDa bacteriolytic proteins. These bacteriolytic proteins were missing in extracts of atl mutant RUSAL2 (S. aureus RN450 atl::Tn551). Immunoblotting studies suggest that these are all atl gene products: the 138-kDa protein is an ATL with a cleaved signal sequence; the 115- and 85-kDa proteins are intermediates; and the 51- and 62-kDa proteins are cell-associated 51-kDa GL and 62-kDa AM, respectively. The trypsin susceptibility of these proteins suggests that they are located outside the cell membrane. Differences in extractability and immunoelectron microscopic studies suggest that atl gene products are associated with cells in two different ways, LiCl extractable and non extractable. We suggest that the 138-kDa ATL undergoes processing through intermediate proteins (115- and 85-kDa proteins) to mature as the active cell cluster-dispersing enzymes 51-kDa GL and 62-kDa AM on the cell surface.
金黄色葡萄球菌自溶素基因atl编码一种独特的138 kDa蛋白（ATL），其具有酰胺酶和氨基葡萄糖苷酶结构域。有人提出ATL会经历蛋白水解加工，产生两种细胞外肽聚糖水解酶，即51 kDa的内切-β-N-乙酰氨基葡萄糖苷酶（51 kDa GL）和62 kDa的N-乙酰胞壁酰-L-丙氨酸酰胺酶（62 kDa AM）。为了研究与细胞相关的atl基因产物的溶菌酶，按如下方法从细胞中提取蛋白质。将细胞暴露于3 M LiCl，然后用4% SDS处理。之后，将细胞破碎，再用4% SDS提取。在不破坏细胞的情况下提取了完整的SDS稳定的与细胞相关的溶菌蛋白。暴露于3 M LiCl释放出主要的138 kDa、115 kDa、85 kDa、62 kDa和51 kDa溶菌蛋白，随后用4% SDS提取释放出主要的138 kDa和115 kDa溶菌蛋白。这些溶菌蛋白在atl突变体RUSAL2（金黄色葡萄球菌RN450 atl::Tn551）的提取物中缺失。免疫印迹研究表明，这些都是atl基因产物：138 kDa蛋白是具有切割信号序列的ATL；115 kDa和85 kDa蛋白是中间体；51 kDa和62 kDa蛋白分别是与细胞相关的51 kDa GL和62 kDa AM。这些蛋白对胰蛋白酶的敏感性表明它们位于细胞膜外。提取能力的差异和免疫电子显微镜研究表明，atl基因产物以两种不同方式与细胞相关，即可被LiCl提取和不可被提取。我们认为138 kDa的ATL通过中间蛋白（115 kDa和85 kDa蛋白）进行加工，在细胞表面成熟为活性细胞簇分散酶51 kDa GL和62 kDa AM。
