Steindl Christian, Schäffer Christina, Wugeditsch Thomas, Graninger Michael, Matecko Irena, Müller Norbert, Messner Paul
Institut für Chemie, Johannes-Kepler-Universität Linz, A-4040 Linz, Austria.
Biochem J. 2002 Dec 1;368(Pt 2):483-94. doi: 10.1042/BJ20020988.
The cell surface of Aneurinibacillus thermoaerophilus DSM 10155 is covered with a square surface (S)-layer glycoprotein lattice. This S-layer glycoprotein, which was extracted with aqueous buffers after a freeze-thaw cycle of the bacterial cells, is the only completely water-soluble S-layer glycoprotein to be reported to date. The purified S-layer glycoprotein preparation had an overall carbohydrate content of 19%. Detailed chemical investigations indicated that the S-layer O-glycans of previously established structure accounted for 13% of total glycosylation. The remainder could be attributed to a peptidoglycan-associated secondary cell wall polymer. Structure analysis was performed using purified secondary cell wall polymer-peptidoglycan complexes. NMR spectroscopy revealed the first biantennary secondary cell wall polymer from the domain Bacteria, with the structure alpha-L-Glc p NAc-(1-->3)-beta-L-Man p NAc-(1-->4)-beta-L-Gal p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->3)-beta-L-Man p NAc-(1-->4)-beta-L-Gal p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->4)-[alpha-L-Glc p NAc-(1-->3)-beta-L-Man p NAc-(1-->4)-beta-L-Gal p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->3)-beta-L-Man p NAc-(1-->4)-beta-L-Gal p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->3)]-beta-L-Man p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->3)-beta-L-Man p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->O)-PO(2)(-)-O-PO(2)(-)-(O-->6)-MurNAc- (where MurNAc is N -acetylmuramic acid). The neutral polysaccharide is linked via a pyrophosphate bond to the C-6 atom of every fourth N -acetylmuramic acid residue, in average, of the A1gamma-type peptidoglycan. In vivo, the biantennary polymer anchored the S-layer glycoprotein very effectively to the cell wall, probably due to the doubling of motifs for a proposed lectin-like binding between the polymer and the N-terminus of the S-layer protein. When the cellular support was removed during S-layer glycoprotein isolation, the co-purified polymer mediated the solubility of the S-layer glycoprotein in vitro. Initial crystallization experiments performed with the soluble S-layer glycoprotein revealed that the assembly property could be restored upon dissociation of the polymer by the addition of poly(ethylene glycols). The formed two-dimensional crystalline S-layer self-assembly products exhibited the same lattice symmetry as observed on intact bacterial cells.
嗜热栖热放线菌DSM 10155的细胞表面覆盖着一个方形表面（S）-层糖蛋白晶格。这种S-层糖蛋白在细菌细胞经过冻融循环后用水性缓冲液提取，是迄今为止报道的唯一一种完全水溶性的S-层糖蛋白。纯化后的S-层糖蛋白制剂的总碳水化合物含量为19%。详细的化学研究表明，先前确定结构的S-层O-聚糖占总糖基化的13%。其余部分可归因于与肽聚糖相关的次生细胞壁聚合物。使用纯化的次生细胞壁聚合物-肽聚糖复合物进行结构分析。核磁共振光谱揭示了来自细菌域的首个双天线次生细胞壁聚合物，其结构为α-L-Glc p NAc-(1→3)-β-L-Man p NAc-(1→4)-β-L-Gal p NAc-(1→3)-α-L-Glc p NAc-(1→3)-β-L-Man p NAc-(1→4)-β-L-Gal p NAc-(1→3)-α-L-Glc p NAc-(1→4)-[α-L-Glc p NAc-(1→3)-β-L-Man p NAc-(1→4)-β-L-Gal p NAc-(1→3)-α-L-Glc p NAc-(1→3)-β-L-Man p NAc-(1→4)-β-L-Gal p NAc-(1→3)-α-L-Glc p NAc-(1→3)]-β-L-Man p NAc-(1→3)-α-L-Glc p NAc-(1→3)-β-L-Man p NAc-(1→3)-α-L-Glc p NAc-(1→3)-α-L-Glc p NAc-(1→O)-PO₂⁻-O-PO₂⁻-(O→6)-MurNAc-（其中MurNAc是N-乙酰胞壁酸）。中性多糖通过焦磷酸键平均连接到A1γ型肽聚糖每隔四个N-乙酰胞壁酸残基的C-6原子上。在体内，双天线聚合物非常有效地将S-层糖蛋白锚定在细胞壁上，这可能是由于聚合物与S-层蛋白N端之间拟议的凝集素样结合基序加倍。当在S-层糖蛋白分离过程中去除细胞支持物时，共纯化的聚合物在体外介导了S-层糖蛋白的溶解性。对可溶性S-层糖蛋白进行的初步结晶实验表明，通过添加聚乙二醇使聚合物解离后，组装特性可以恢复。形成的二维结晶S-层自组装产物表现出与完整细菌细胞上观察到的相同晶格对称性。
