Biochemical characteristics of point mutated Capra hircus lysosome α-mannosidase.
Biochemical characteristics of point mutated Capra hircus lysosome α-mannosidase.
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DOI：
10.1292/jvms.22-0222
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发表时间：
2023-02-21
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疯草是一种有毒杂草，广泛分布于世界各地，对草食畜牧业的发展具有重大影响。苦马豆素(Swainsonine，SW)是疯草中的主要毒素，能竞争性抑制动物细胞中的溶酶体甘露糖苷酶(α-mannosidase，LAM)，导致α-甘露糖苷症。然而，西南航空和LAM之间互动的细节仍不清楚。在这里，我们利用分子对接的方法预测了Sw和LAM之间的相互作用点，构建了突变的溶酶体α-甘露糖苷酶(LAMM)，并分析了其在推测点上的生化性质变化。通过构建重组酵母GS115/PIC9K-Lamm，将CAPRA hircus Lam(ChLAM)中第28位的Trp和第599位的Tyr作为候选的相互作用点，成功地将上述两个氨基酸突变为甘氨酸。结果表明，与野生型LAM相比，山羊LAMM(ChLAMM)对SW的敏感性显著降低，LAM酶活性降低近3倍，最适温度从55℃降至50℃，最适pH值从4.5升至5.0，点突变前后Mn2+、Fe3+、Al3+、Co2+、Cr3+、EDTA对LAM酶活性的影响发生显著变化。这些发现有助于我们更好地了解SW与chLAM相互作用的分子机制，为解决疯草中毒问题提供新的参考。
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 (LAMM), 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- LAMM. The results showed that the sensitivity of Capra hircus LAMM (chLAMM), to SW decreased significantly compared with wild-type LAM, the enzyme activity of LAM decreased approximately threefold, the optimum temperature of LAMM decreased from 55°C to 50°C, the optimum pH value increased from 4.5 to 5.0, and the effects of Mn2+, Fe3+, Al3+, Co2+, Cr3+, 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.