The elastin-binding protein of Staphylococcus aureus (EbpS) is expressed at the cell surface as an integral membrane protein and not as a cell wall-associated protein
The elastin-binding protein of Staphylococcus aureus (EbpS) is expressed at the cell surface as an integral membrane protein and not as a cell wall-associated protein
复制标题
DOI：
10.1074/jbc.m107621200
复制
发表时间：
2002-01-04
影响因子：
4.8
通讯作者：
Foster, TJ
中科院分区：
文献类型：
--
作者：
Downer, R;Roche, F;Foster, TJ
从序列分析预测金黄色葡萄球菌菌株科万和8325-4的弹性蛋白结合蛋白EbpS包含486个残基。针对在大肠杆菌中表达为重组蛋白的N-末端结构域（残基1-267）和C-末端结构域（残基343-486）产生特异性抗体。野生型8325-4和纽曼和相应的ebpS突变体的裂解物的蛋白质印迹显示，EbpS迁移的表观分子量为83 kDa。该蛋白质被发现专门在细胞质膜组分纯化的原生质体或裂解细胞，相反，凝集因子CIFA，这是细胞壁相关的。预测EbpS具有三个疏水结构域H1-（205-224）、H2（265-280）和H3-（315-342）。在N端的EbpS与C端的碱性磷酸酶或β-半乳糖苷酶之间形成一系列杂合蛋白（EbpS-PhoA，EbpS-LacZ）。PhoA和LacZ融合到EbpS的疏水结构域H1-H2和H2-H3之间，并远离H3。在E.大肠杆菌中发现EbpS是一个完整的膜蛋白，具有两个跨膜结构域H1和H3。预测N-末端残基1-205和C-末端残基343-486暴露在细胞质膜的外表面上。从先前的研究中已知EbpS的配体结合结构域存在于残基14-34之间的N末端，并且用抗EbpS 1 -267抗体探测整个细胞表明该区域暴露在完整细胞的表面上。这也被野生型S. aureus纽曼细胞与标记的原弹性蛋白结合，而ebpS突变体的结合率低72%。相比之下，携带推定的LysM肽聚糖结合结构域的C末端并未暴露在完整细胞的表面上，并且推测仍然埋在肽聚糖内。最后，EbpS的表达与细胞在液体培养中生长至更高密度的能力相关，表明EbpS可能在调节细胞生长中起作用。
The elastin-binding proteins EbpS of Staphylococcus aureus strains Cowan and 8325-4 were predicted from sequence analysis to comprise 486 residues. Specific antibodies were raised against an N-terminal domain (residues 1-267) and a C-terminal domain (residues 343-486) expressed as recombinant proteins in Escherichia coli. Western blotting of lysates of wild-type 8325-4 and Newman and the corresponding ebpS mutants showed that EbpS migrated with an apparent molecular mass of 83 kDa. The protein was found exclusively in cytoplasmic membrane fractions purified from protoplasts or lysed cells, in contrast to the clumping factor CIfA, which was cell-wall-associated. EbpS was predicted to have three hydrophobic domains H1-(205-224), H2(265-280), and H3-(315-342). A series of hybrid proteins was formed between EbpS at the N terminus and either alkaline phosphatase or beta-galactosidase at the C terminus (EbpS-PhoA, EbpS-LacZ). PhoA and LacZ were fused to EbpS between hydrophobic domains H1-H2 and H2-H3, and distal to H3. Expression of enzymatic activity in E. coli showed that EbpS is an integral membrane protein with two membrane-spanning domains H1 and H3. N-terminal residues 1-205 and C-terminal residues 343-486 were predicted to be exposed on the outer face of the cytoplasmic membrane. The ligand-binding domain of EbpS is known from previous studies to be present in the N terminus between residues 14-34 and probing whole cells with anti-EbpS1-267 antibodies indicated that this region is exposed on the surface of intact cells. This was also confirmed by the observation that wildtype S. aureus Newman cells bound labeled tropooelastin whereas the ebpS mutant bound 72% less. In contrast, the C terminus, which carries a putative LysM peptidoglycan-binding domain, is not exposed on the surface of intact cells and presumably remains buried within the peptidoglycan. Finally, expression of EbpS was correlated with the ability of cells to grow to a higher density in liquid culture, suggesting that EbpS may have a role in regulating cell growth.