Department of Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, China.
J Vet Med Sci. 2023 Feb 21;85(2):244-251. doi: 10.1292/jvms.22-0222. Epub 2023 Jan 2.
Locoweeds, a type of poisonous weedare, are widely distributed throughout the world and have a significant impact on the development of herbivore animal husbandry. Swainsonine (SW), the main toxin in locoweeds, can competitively inhibit lysosomes α-mannosidase (LAM) in animal cells, resulting in α-mannosidosis. However, the specifics of the interaction between SW and LAM are still unclear. Here, we used molecular docking to predicte the interaction points between SW and LAM, built mutated lysosomes α-mannosidase (LAM), and analyzed its biochemical properties changes in presumption points. The Trp at the 28th position and the Tyr at the 599th position of the LAM were interaction point candidates, and the above two amino acids in Capra hircus LAM (chLAM), were successfully mutated to glycine by constructing recombinant yeast GS115/PIC9K- LAM. The results showed that the sensitivity of Capra hircus LAM (chLAM), to SW decreased significantly compared with wild-type LAM, the enzyme activity of LAM decreased approximately threefold, the optimum temperature of LAM decreased from 55°C to 50°C, the optimum pH value increased from 4.5 to 5.0, and the effects of Mn, Fe, Al, Co, Cr, and ethylenediaminetetraacetic acid (EDTA) on LAM enzyme activity before and after point mutation changed significantly. These findings help us better understanding the molecular mechanism of the interaction mechanism between SW and chLAM, and provide new reference for solving locoweeds poisoning.
疯草，一种有毒杂草，广泛分布于世界各地，对食草动物畜牧业的发展有重大影响。疯草中的主要毒素苦马豆素（SW）可以竞争性地抑制动物细胞溶酶体α-甘露糖苷酶（LAM），导致α-甘露糖苷病。然而，SW 与 LAM 之间的相互作用的具体细节仍不清楚。在这里，我们使用分子对接预测 SW 和 LAM 之间的相互作用点，构建突变的溶酶体α-甘露糖苷酶（LAM），并在假定点分析其生化特性的变化。LAM 中的色氨酸第 28 位和酪氨酸第 599 位是相互作用点候选者，并且成功地通过构建重组酵母 GS115/PIC9K-LAM 将绵羊 LAM（chLAM）中的上述两种氨基酸突变为甘氨酸。结果表明，与野生型 LAM 相比，绵羊 LAM（chLAM）对 SW 的敏感性显著降低，LAM 的酶活性降低了约三倍，LAM 的最适温度从 55°C 降低到 50°C，最适 pH 值从 4.5 增加到 5.0，以及 Mn、Fe、Al、Co、Cr 和乙二胺四乙酸（EDTA）对突变前后 LAM 酶活性的影响发生了显著变化。这些发现有助于我们更好地理解 SW 与 chLAM 相互作用机制的分子机制，并为解决疯草中毒提供新的参考。
