Department of Physics and Astronomy, University of Exeter, Stocker Road, Exeter EX4 4QL, UK.
College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter EX4 4QD, UK.
Toxins (Basel). 2022 Nov 3;14(11):757. doi: 10.3390/toxins14110757.
epsilon toxin (Etx) is a pore forming toxin that causes enterotoxaemia in ruminants and may be a cause of multiple sclerosis in humans. To date, most in vitro studies of Etx have used the Madin-Darby canine kidney (MDCK) cell line. However, studies using Chinese hamster ovary (CHO) cells engineered to express the putative Etx receptor, myelin and lymphocyte protein (MAL), suggest that amino acids important for Etx activity differ between species. In this study, we investigated the role of amino acids Y42, Y43 and H162, previously identified as important in Etx activity towards MDCK cells, in Etx activity towards CHO-human MAL (CHO-hMAL) cells, human red blood cells (hRBCs) and synthetic bilayers using site-directed mutants of Etx. We show that in CHO-hMAL cells Y42 is critical for Etx binding and not Y43 as in MDCK cells, indicating that surface exposed tyrosine residues in the receptor binding domain of Etx impact efficiency of cell binding to MAL-expressing cells in a species-specific manner. We also show that Etx mutant H162A was unable to lyse CHO-hMAL cells, lysed hRBCs, whilst it was able to form pores in synthetic bilayers, providing evidence of the complexity of Etx pore formation in different lipid environments.
epsilon 毒素（Etx）是一种形成孔的毒素，可导致反刍动物肠毒血症，并且可能是人类多发性硬化症的病因。迄今为止，大多数 Etx 的体外研究都使用了 Madin-Darby 犬肾（MDCK）细胞系。然而，使用表达假定的 Etx 受体、髓鞘和淋巴细胞蛋白（MAL）的中国仓鼠卵巢（CHO）细胞进行的研究表明，氨基酸对于 Etx 活性的重要性在不同物种之间存在差异。在这项研究中，我们使用 Etx 的定点突变体研究了先前确定的对 MDCK 细胞 Etx 活性重要的氨基酸 Y42、Y43 和 H162 在 Etx 对 CHO-human MAL（CHO-hMAL）细胞、人红细胞（hRBC）和合成双层膜的活性中的作用。我们表明，在 CHO-hMAL 细胞中，Y42 对于 Etx 结合至关重要，而不是 MDCK 细胞中的 Y43，这表明 Etx 受体结合域中表面暴露的酪氨酸残基以物种特异性的方式影响细胞与 MAL 表达细胞结合的效率。我们还表明，Etx 突变体 H162A 无法裂解 CHO-hMAL 细胞，但可裂解 hRBC，而它能够在合成双层膜中形成孔，这为 Etx 在不同脂质环境中形成孔的复杂性提供了证据。
