Studies on Cell Membrane Protein of Lactic Acid Bacteria Related with Adhesion

与粘附相关的乳酸菌细胞膜蛋白的研究

• 批准号: 09660083
• 负责人: YAMAMOTO Kenji
• 金额: $ 2.43万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09660083/
• 关键词: Lactic acid bacteria; Cell adhesion; Glycolipids; Membrane oligosacchariotes; Receptor protein; Lactobacillus casei; Cloning; Lactic acid bacteria therapy; Lactobacillus casel

Many viruses, pathogenic bacteria, and bacterial toxins specifically recognize and bind to sugar chains of the eukaryotic cell surface. Their binding to the cells is essential to establish infection or produce a toxic effect. For intestinal bacteria, adherence to cell surfaces for colonizing epithelial tissues is an important initial event. Lactobacillus. a representative useful bacterium, in the intestinal tract was found to bind to some specific glycosphingolipids, like the pathogenic intestinal bacteria. We found that Lactobacillus casei bound to neutral glycosphingolipids such as GAl and trihexosylceramide strongly. We also found and purified the receptor protein of L.casei from surface layer proteins, and attempted to clone the gene of the protein. The results of this investigation are as followed.We extracted the genomic DNA from L.casei and it was partially digested with Sau3AI.Then, 10-l5kbp DNA fragments was ligated with Charomid cloning vector digested with BamHI using DNA ligase. This recombinant DNA was transduced into Escherichia coli DH5 alpha using phage to construct a genomic library. Colony hybridization was performed with the probe of oligonucleotide mixture corresponding with N-terminal amino acid sequence of the protein. Finally, positive clone was obtained and named CH1, and the 3.0 kbp EcoRI fragment was sequenced and confirmed to contain an open reading frame encoding the protein. It consisted of 465 nucleotides encoding 155 amino acids. When the cell extract of recombinant E.coli was added with GAl and the mixture was subjected to electrophoresis, followed by immunoblotting using anti-GAl-monoclonal antibody, we found a staining band at the position of the protein expressed in recombinant E.coli. The protein was expressed in both the cell surface and cytosol fractions of E.coli.

许多病毒、致病细菌和细菌毒素特异性识别并结合真核细胞表面的糖链。它们与细胞的结合对于建立感染或产生毒性作用至关重要。对于肠道细菌，粘附于细胞表面以定植上皮组织是重要的初始事件。乳酸菌。肠道中的代表性有用细菌被发现与某些特定的鞘糖脂结合，如致病性肠道细菌。我们发现干酪乳杆菌与中性鞘糖脂如GA 1和三己糖神经酰胺结合强烈。我们还从干酪乳杆菌的表层蛋白中发现并纯化了干酪乳杆菌的受体蛋白，并试图克隆该蛋白的基因。本研究的主要结果如下：从干酪乳杆菌中提取基因组DNA，用Sau 3AI部分酶切，然后用DNA连接酶将10- 15 kbp的DNA片段与经BamHI酶切的Charomid克隆载体连接。利用噬菌体将该重组DNA导入大肠杆菌DH 5 α，构建基因组文库。以与该蛋白N端氨基酸序列相对应的寡核苷酸混合物为探针进行菌落杂交。最后获得阳性克隆，命名为CH 1，并对3.0 kbp的EcoRI片段进行测序，证实含有编码该蛋白的开放阅读框架。它由465个核苷酸组成，编码155个氨基酸。当向重组大肠杆菌的细胞提取物中加入GA 1并对混合物进行电泳，然后使用抗GA 1单克隆抗体进行免疫印迹时，我们在重组大肠杆菌中表达的蛋白质的位置处发现了染色带。该蛋白质在大肠杆菌的细胞表面和胞质溶胶组分中表达。

项目成果

S.Akiba et al.: "Effects of Carbohydrate Chain on Surface Net Charge and Hydrophobicity of Glycoenzymes" Biosci.Biotechnol.Biochem.62(11). 2171-2176 (1998)

S.Akiba 等人：“碳水化合物链对糖酶表面净电荷和疏水性的影响”Biosci.Biotechnol.Biochem.62(11)。

山本 憲二: "糖鎖を介した乳酸菌の細胞接着" 化学と生物. 36(1). 26-31 (1998)

Kenji Yamamoto：“乳酸菌通过糖链的细胞粘附”化学与生物学 36（1）（1998）。

K.Yamamoto: "Adhesion of Lactic Acid Bacteria to Cells Mediated by Sugar Chains" Kagaku to Seibutu. 36(1). 26-31 (1998)

K.Yamamoto：“糖链介导的乳酸菌与细胞的粘附” Kagaku 与 Seibutu。

K.Yamamoto et al.: "Chemo-enzymatic Synthesis of a Novel Glycopeptide using a Microbial Endoglycosidase" Carbohydr.Res.305. (1997)

K.Yamamoto 等人：“使用微生物糖苷内切酶化学酶法合成新型糖肽”CarboHydr.Res.305。

K.Yamamoto et al.: "Transglycosylation Activity of Endoglycosidases and Its Application" Trends in Glycoscience and Glycotechnology. 9(48). 339-354 (1997)

K.Yamamoto 等人：“糖苷内切酶的转糖基化活性及其应用”糖科学和糖技术的趋势。