Ayyar B Vijayalakshmi, Atassi M Zouhair
Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA; Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030, USA.
Biochim Biophys Acta. 2016 Dec;1864(12):1678-1685. doi: 10.1016/j.bbapap.2016.09.001. Epub 2016 Sep 3.
Binding behaviors of the H and the H domains of BoNT/A were investigated individually to identify if there exist any differences in their interaction with the cell membrane. Recombinant fragments corresponding to both BoNT/A H and H regions were prepared (H519-845 and H967-1296) and their binding to synaptic proteins was verified. The binding behaviors of these heavy-chain domains were analyzed by treating the Neuro 2a, a murine neuroblastoma cell line, with compounds known to alter membrane properties. Cholesterol depletion and lipid raft inhibition increased the binding of H519-845 to Neuro 2a cells without affecting H967-1296-cell interaction. Sphingolipid depletion decreased the binding of cells to both H967-1296 and H519-845 whereas, loading exogenous GD1a, on to the Neuro 2a cells, increased the binding of both the peptides to cells. Microtubule disruption of the Neuro 2a cells by nocodazole decreased the binding of both H967-1296 and H519-845 to the treated cells. Inhibition of the clathrin-mediated endocytosis using dynasore, chlorpromazine or potassium (K) depletion buffer lowered the binding of both H967-1296 and H519-845 to the cells, but seemed to exert a more pronounced effect on the binding of H967-1296 than on the binding of H519-845. Results indicate that while both the H and H domains are involved in the binding of the toxin to neuronal cells there are differences in their behavior which probably stem from their respective amino acid composition and structural location in the toxin three-dimensional structure along with their intended role in translocation and internalization into the cells.
对肉毒杆菌神经毒素A（BoNT/A）的H和H'结构域的结合行为进行了单独研究，以确定它们与细胞膜相互作用是否存在差异。制备了与BoNT/A H和H'区域相对应的重组片段（H519 - 845和H967 - 1296），并验证了它们与突触蛋白的结合。通过用已知可改变膜特性的化合物处理小鼠神经母细胞瘤细胞系Neuro 2a，分析了这些重链结构域的结合行为。胆固醇耗竭和脂筏抑制增加了H519 - 845与Neuro 2a细胞的结合，而不影响H967 - 1296与细胞的相互作用。鞘脂耗竭降低了细胞与H967 - 1296和H519 - 845的结合，而向Neuro 2a细胞加载外源性GD1a则增加了两种肽与细胞的结合。用诺考达唑破坏Neuro 2a细胞的微管降低了H967 - 1296和H519 - 845与处理后细胞的结合。使用dynasore、氯丙嗪或钾（K）耗竭缓冲液抑制网格蛋白介导的内吞作用降低了H967 - 1296和H519 - 845与细胞的结合，但似乎对H967 - 1296结合的影响比对H519 - 845结合的影响更明显。结果表明，虽然H和H'结构域都参与毒素与神经元细胞的结合，但它们的行为存在差异，这可能源于它们各自的氨基酸组成、在毒素三维结构中的结构位置以及它们在转运和内化进入细胞中的预期作用。
