Domains in the S-layer protein CbsA of Lactobacillus crispatus involved in adherence to collagens, laminin and lipoteichoic acids and in self-assembly
Domains in the S-layer protein CbsA of Lactobacillus crispatus involved in adherence to collagens, laminin and lipoteichoic acids and in self-assembly
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DOI：
10.1046/j.1365-2958.2002.03180.x
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发表时间：
2002-10-01
影响因子：
3.6
通讯作者：
Korhonen, TK
中科院分区：
文献类型：
--
作者：
Antikainen, J;Anton, L;Korhonen, TK
卷曲乳杆菌JCM 5810的S-层蛋白CbsA中的蛋白质区域，需要结合到胶原蛋白和层粘连蛋白，锚定到细菌细胞壁，以及自组装，通过从大肠杆菌中分离的His-标记的肽的缺失分析和干酪乳杆菌上的异源表达进行映射。成熟的CbsA是410个氨基酸长，并且在两端的逐步遗传截短揭示了区域32-271携带CbsA自组装成周期性结构的信息。乳酸杆菌S层蛋白在其组装结构域中表现出序列变异，但CbsA中的边界区域30-34和269-274在富含缬氨酸的短序列中是保守的。在这些区域的短缺失或取代影响His-CbsA聚合物的形态，其从片状到圆柱形管状聚合物变化，并且超过编码残基32和271的DNA的进一步截短导致非周期性聚集。截短肽的自组装，如通过电子显微镜观察到的，与它们在交联研究中的行为相关。通过交联研究和质谱法观察到不形成规则聚合物的较短肽形成二聚体、三聚体和四聚体，而其他肽仅交联成大的多聚体。用His-CbsA肽1-274和31-287观察到溶解的I型和IV型胶原的结合，但不与较小的肽结合，不管它们形成规则聚合物的能力如何。菌株JCM 5810也粘附于固定化层粘连蛋白，并且为了分析CbsA与层粘连蛋白的可能结合，将CbsA及其片段表达于L. casei。CbsA肽1-274、1287、28-287和31-287在L.酪蛋白赋予固定在玻璃上的层粘连蛋白和胶原蛋白以及鸡结肠和回肠中的含层粘连蛋白和胶原蛋白的区域染色。C端肽251-410和288-410与L.通过化学提取去除S层的卷曲肌JCM 5810细胞，而没有观察到与His-CbsA肽1-250或1-269或与具有完整S层的细胞的结合。His-CbsA肽251-410和288-410与几种细菌物种的磷壁酸结合。结果表明，CbsA是一种粘附复合物，具有对细胞周围组织组分表现出亲和力的N-末端组装结构域和与带负电荷的细胞壁组分结合的阳离子C-末端结构域。
The protein regions in the S-layer protein CbsA of Lactobacillus crispatus JCM 5810, needed for binding to collagens and laminin, anchoring to bacterial cell wall, as well as self-assembly, were mapped by deletion analysis of His-tagged peptides isolated from Escherichia coli and by heterologous expression on Lactobacillus casei. Mature CbsA is 410 amino acids long, and stepwise genetic truncation at both termini revealed that the region 32-271 carries the information for self-assembly of CbsA into a periodic structure. The lactobacillar S-layer proteins exhibit sequence variation in their assembly domain, but the border regions 30-34 and 269-274 in CbsA are conserved in valine-rich short sequences. Short deletions or substitutions at these regions affected the morphology of His-CbsA polymers, which varied from sheet-like to cylindrical tubular polymers, and further truncation beyond the DNA encoding residues 32 and 271 leads to a non-periodic aggregation. The self-assembly of the truncated peptides, as seen by electron microscopy, was correlated with their behaviour in a cross-linking study. The shorter peptides not forming a regular polymer were observed by the cross-linking study and mass spectrometry to form dimers, trimers and tetramers, whereas the other peptides were cross-linked to large multimers only. Binding of solubilized type I and IV collagens was observed with the His-CbsA peptides 1-274 and 31-287, but not with the smaller peptides regardless of their ability to form regular polymers. Strain JCM 5810 also adheres to immobilized laminin and, in order to analyse the possible laminin binding by CbsA, cbsA and its fragments were expressed on the surface of L. casei. Expression of the CbsA peptides 1-274, 1287, 28-287 and 31-287 on L. casei conferred adhesiveness to both laminin and collagen immobilized on glass as well as to laminin-and collagen-containing regions in chicken colon and ileum. The C-terminal peptides 251-410 and 288-410 bound to L. crispatus JCM 5810 cells from which the S-layer had been depleted by chemical extraction, whereas no binding was seen with the His-CbsA peptides 1-250 or 1-269 or to cells with an intact S-layer. The His-CbsA peptides 251-410 and 288-410 bound to teichoic acids of several bacterial species. The results show that CbsA is an adhesive complex with an N-terminal assembly domain exhibiting affinity for pericellular tissue components and a cationic C-terminal domain binding to negatively charged cell wall components.