Sundberg-Kövamees M, Holme T, Sjögren A
Microbiology and Tumorbiology Center, Karolinska Institute, Stockholm, Sweden.
Microb Pathog. 1996 Oct;21(4):223-34. doi: 10.1006/mpat.1996.0057.
C-polysaccharide (PnC) is the major surface component of pneumococci containing phosphoryl choline residues. In order to investigate the possibility that PnC can bind to glycolipid receptors present on epithelial cells we extracted carbohydrate material from a nonencapsulated strain of pneumococci. The components of the extract were separated by gel permeation chromatography. An ELISA was used for detection of fractions binding to the pneumococcal glycolipid receptor asialo GM1. These fractions were pooled and analysed by nuclear magnetic resonance spectroscopy (NMR). The 1H NMR spectrum showed good agreement with a reference spectrum of pure PnC showing that this substance was the major component. Binding of the purified PnC to asialo-GM1 was unaffected by protease K treatment. Immunoblots of the purified PnC after separation by SDS-PAGE resulted in a characteristic banding pattern. PnC could be released from pneumococci by heat treatment of whole bacteria in buffer as shown by reaction with a monoclonal antibody specific for the phosphoryl choline determinant. After separation by SDS-PAGE of the components of the heat extract, immunoblots showed the presence of bands characteristic for PnC. Eluates from the characteristic bands in the gel were shown to contain material binding to asialo-GM1. This binding was not reduced upon treatment with protease K. Pneumococci deprived of choline by cultivation in a medium containing ethanolamine as the only amino alcohol source did not bind to asialo-GM1, indicating that the phosphoryl choline residue of PnC is essential for the interaction between PnC and the glycolipid receptor. These data provide evidence that PnC containing an intact phosphoryl choline residue is a ligand responsible for binding of pneumococci to the receptor asialo-GM1.
C多糖（PnC）是肺炎球菌的主要表面成分，含有磷酰胆碱残基。为了研究PnC能否与上皮细胞上存在的糖脂受体结合，我们从一株非包膜肺炎球菌菌株中提取了碳水化合物物质。提取物的成分通过凝胶渗透色谱法进行分离。采用酶联免疫吸附测定法（ELISA）检测与肺炎球菌糖脂受体脱唾液酸GM1结合的组分。将这些组分合并并用核磁共振光谱法（NMR）进行分析。1H NMR光谱与纯PnC的参考光谱显示出良好的一致性，表明该物质是主要成分。纯化的PnC与脱唾液酸-GM1的结合不受蛋白酶K处理的影响。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离纯化的PnC后进行免疫印迹，得到了特征性的条带模式。如与针对磷酰胆碱决定簇的单克隆抗体反应所示，通过在缓冲液中对全菌进行热处理，可从肺炎球菌中释放出PnC。对热提取物的成分进行SDS-PAGE分离后，免疫印迹显示存在PnC特征性的条带。凝胶中特征条带的洗脱液显示含有与脱唾液酸-GM1结合的物质。用蛋白酶K处理后，这种结合并未减少。在以乙醇胺作为唯一氨基醇源的培养基中培养而缺乏胆碱的肺炎球菌不与脱唾液酸-GM1结合，这表明PnC的磷酰胆碱残基对于PnC与糖脂受体之间的相互作用至关重要。这些数据提供了证据，表明含有完整磷酰胆碱残基的PnC是负责肺炎球菌与受体脱唾液酸-GM1结合的配体。
