Molecular characterization of binding subcomponents of Clostridium botulinum type C progenitor toxin for intestinal epithelial cells and erythrocytes
Molecular characterization of binding subcomponents of Clostridium botulinum type C progenitor toxin for intestinal epithelial cells and erythrocytes
复制标题
DOI：
10.1099/mic.0.26805-0
复制
发表时间：
2004-05-01
期刊：
影响因子：
2.8
通讯作者：
Oguma, K
中科院分区：
文献类型：
--
作者：
Fujinaga, Y;Inoue, K;Oguma, K
肉毒梭菌C型16 S祖毒素由神经毒素（NTX）、无毒非HA（NTNH）和血凝素（HA）组成。HA作为粘附素，允许16 S毒素结合肠上皮细胞和红细胞。在C型中，这些结合依赖于唾液酸。HA由四个不同的亚组分组成，命名为HA 1、HA 2、HA 3a和HA 3b。为了鉴定C型16 S毒素的HA的结合亚组分，所有HA亚组分和它们的一些前体形式作为与谷胱甘肽S-转移酶（GST）融合的重组蛋白产生。评价这些蛋白质粘附于豚鼠和人红细胞的肠上皮细胞的能力。GST-HA 1、GST-HA 3b和GST-HA 3（HA 3a和HA 3b的前体形式）结合肠上皮细胞和红细胞，而单独的GST、GST-HA 2和GST-HA 3a不结合。GST-HA 3b和GST-HA 3显示神经氨酸酶敏感的结合肠上皮细胞和红细胞，而GST-HA 1显示神经氨酸酶不敏感的结合。TLC结合测定显示GST-HA 3b和GST-HA 3识别红细胞的神经节苷脂部分中的唾液酸副脑苷脂（SPG）和GM 3，类似于天然型C16 S毒素[Inoue，K.等人（1999）。Microbiology 145，2533-2542]。另一方面，除了SPG和GM 3之外，GST-HA 1还识别副裂叶糖苷（PG; SPG的脱唾液酸衍生物）。GST-HA 3b的缺失突变分析表明HA 3b的C-末端区域对其结合活性是重要的。基于这些数据，可以得出结论，HA组分包含两个不同的碳水化合物结合亚组分，HA 1和HA 3b，其以不同的特异性识别碳水化合物。
Clostridium botulinum type C 16S progenitor toxin consists of a neurotoxin (NTX), a non-toxic non-HA (NTNH), and a haemagglutinin (HA). The HA acts as an adhesin, allowing the 16S toxin to bind to intestinal epithelial cells and erythrocytes. In type C, these bindings are dependent on sialic acid. The HA consists of four distinct subcomponents designated HA1, HA2, HA3a and HA3b. To identify the binding subcomponent(s) of HA of type C 16S toxin, all of the HA-subcomponents and some of their precursor forms were produced as recombinant proteins fused to glutathione S-transferase (GST). These proteins were evaluated for their capacity to adhere to intestinal epithelial cells of guinea pig and human erythrocytes. GST-HA1, GST-HA3b and GST-HA3 (a precursor form of HA3a and HA3b) bound intestinal epithelial cells and erythrocytes, whereas GST alone, GST-HA2 and GST-HA3a did not. GST-HA3b and GST-HA3 showed neuraminidase-sensitive binding to the intestinal epithelial cells and erythrocytes, whereas GST-HA1 showed neuraminidase-insensitive binding. TLC binding assay revealed that GST-HA3b and GST-HA3 recognized sialosylparagloboside (SPG) and GM3 in the ganglioside fraction of the erythrocytes, like native type C 16S toxin [Inoue, K. et al. (1999). Microbiology 145, 2533-2542]. On the other hand, GST-HA1 recognized paragloboside (PG; an asialo- derivative of SPG) in addition to SPG and GM3. Deletion mutant analyses of GST-HA3b showed that the C-terminal region of HA3b is important for its binding activity. Based on these data, it is concluded that the HA component contains two distinct carbohydrate-binding subcomponents, HA1 and HA3b, which recognize carbohydrates in different specificities.