Are the Surface Layer Homology Domains Essential for Cell Surface Display and Glycosylation of the S-Layer Protein from Paenibacillus alvei CCM 2051T?
Are the Surface Layer Homology Domains Essential for Cell Surface Display and Glycosylation of the S-Layer Protein from Paenibacillus alvei CCM 2051T?
复制标题
DOI：
10.1128/jb.01487-12
复制
发表时间：
2013-02-01
影响因子：
3.2
通讯作者：
Schaeffer, Christina
中科院分区：
文献类型：
--
作者：
Janesch, Bettina;Messner, Paul;Schaeffer, Christina
用糖基化的s层蛋白SpaA组成的二维（2D）晶体阵列修饰泡状芽孢杆菌CCM 2051(T)细胞。在其N端，SpaA具有三个连续的表面层（s层）同源（SLH）结构域，其中包含氨基酸基序TRAE，已知在细胞壁结合及其TVEE和TRAQ变异中起关键作用。SpaA通过SLH结构域与肽聚糖（PG）相关的非经典丙酮酰化次级细胞壁聚合物（SCWP）的相互作用被锚定在细胞壁上。在这项研究中，我们分析了三个预测的结合基序在SLH结构域内的作用，将它们突变为TAAA基序，无论是单独的，成对的，还是全部的。通过由突变的s层蛋白和增强的绿色荧光蛋白组成的嵌合体的同源表达，以及使用his标记的SpaA变体和天然含pg的细胞壁sacsaculi（含有SCWP或不含SCWP）的体外结合实验，可以在体内观察到效果。实验数据表明：(i) TRAE、TVEE和TRAQ基序对SLH结构域的结合功能至关重要；（ii）无论结构域的位置如何，两个功能基序都足以实现细胞壁结合；（iii） SLH结构域对SCWP和PG具有双重识别功能；（iv） SpaA糖基化不需要细胞壁锚定。此外，我们发现SpaA的SLH结构域足以在肺泡假单胞菌的细胞表面显示外源蛋白。
Paenibacillus alvei CCM 2051(T) cells are decorated with a two-dimensional (2D) crystalline array comprised of the glycosylated S-layer protein SpaA. At its N terminus, SpaA possesses three consecutive surface layer (S-layer) homology (SLH) domains containing the amino acid motif TRAE, known to play a key role in cell wall binding, as well as the TVEE and TRAQ variations thereof. SpaA is predicted to be anchored to the cell wall by interaction of the SLH domains with a peptidoglycan (PG)-associated, nonclassical, pyruvylated secondary cell wall polymer (SCWP). In this study, we have analyzed the role of the three predicted binding motifs within the SLH domains by mutating them into TAAA motifs, either individually, pairwise, or all of them. Effects were visualized in vivo by homologous expression of chimeras made of the mutated S-layer proteins and enhanced green fluorescent protein and in an in vitro binding assay using His-tagged SpaA variants and native PG-containing cell wall sacculi that either contained SCWP or were deprived of it. Experimental data indicated that (i) the TRAE, TVEE, and TRAQ motifs are critical for the binding function of SLH domains, (ii) two functional motifs are sufficient for cell wall binding, regardless of the domain location, (iii) SLH domains have a dual-recognition function for the SCWP and the PG, and (iv) cell wall anchoring is not necessary for SpaA glycosylation. Additionally, we showed that the SLH domains of SpaA are sufficient for in vivo cell surface display of foreign proteins at the cell surface of P. alvei.