Mobili Pablo, Serradell María de Los Angeles, Trejo Sebastián A, Avilés Puigvert Francesc X, Abraham Analía G, De Antoni Graciela L
Centro de Investigación y Desarrollo en Criotecnología de Alimentos, CCT La Plata, CONICET, 47 y 116 (s/n), La Plata, Argentina.
Antonie Van Leeuwenhoek. 2009 May;95(4):363-72. doi: 10.1007/s10482-009-9322-y. Epub 2009 Mar 22.
Since the presence of S-layer protein conditioned the autoaggregation capacity of some strains of Lactobacillus kefir, S-layer proteins from aggregating and non-aggregating L. kefir strains were characterized by immunochemical reactivity, MALDI-TOF spectrometry and glycosylation analysis. Two anti-S-layer monoclonal antibodies (Mab5F8 and Mab1F8) were produced; in an indirect enzyme-linked immunosorbent assay Mab1F8 recognized S-layer proteins from all L. kefir tested while Mab5F8 recognized only S-layer proteins from aggregating strains. Periodic Acid-Schiff staining of proteins after polyacrylamide gel electrophoresis under denaturing conditions revealed that all L. kefir S-layer proteins tested were glycosylated. Growth of bacteria in the presence of the N-glycosylation inhibitor tunicamycin suggested the presence of glycosydic chains O-linked to the protein backbone. MALDI-TOF peptide map fingerprint for S-layer proteins from 12 L. kefir strains showed very similar patterns for the aggregating strains, different from those for the non-aggregating ones. No positive match with other protein spectra in MSDB Database was found. Our results revealed a high heterogeneity among S-layer proteins from different L. kefir strains but also suggested a correlation between the structure of these S-layer glycoproteins and the aggregation properties of whole bacterial cells.
由于S层蛋白的存在决定了某些开菲尔乳杆菌菌株的自聚集能力，因此通过免疫化学反应、基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）和糖基化分析对来自聚集型和非聚集型开菲尔乳杆菌菌株的S层蛋白进行了表征。制备了两种抗S层单克隆抗体（Mab5F8和Mab1F8）；在间接酶联免疫吸附测定中，Mab1F8识别所有测试的开菲尔乳杆菌的S层蛋白，而Mab5F8仅识别来自聚集菌株的S层蛋白。在变性条件下进行聚丙烯酰胺凝胶电泳后对蛋白质进行高碘酸-希夫染色，结果显示所有测试的开菲尔乳杆菌S层蛋白都被糖基化。在N-糖基化抑制剂衣霉素存在的情况下细菌的生长表明存在与蛋白质主链O-连接的糖苷链。12株开菲尔乳杆菌菌株S层蛋白的MALDI-TOF肽图谱指纹显示，聚集菌株的图谱非常相似，与非聚集菌株的不同。在MSDB数据库中未发现与其他蛋白质谱的阳性匹配。我们的结果揭示了不同开菲尔乳杆菌菌株的S层蛋白之间存在高度异质性，但也表明这些S层糖蛋白的结构与整个细菌细胞的聚集特性之间存在相关性。
