A soluble form of EphA7 (sEphA7) has been found to antagonize the role of full-length EphA7 (EphA7-FL) to stabilize the membrane level of the tight junction protein Claudin6 (CLDN6) during Xenopus pronephros development. However, the mechanism underlying this antagonistic effect remains unclear. In this study, we identified Nicalin, a Nicastrin-like protein, as a novel sEphA7-interacting protein using immunoprecipitation (IP)/mass spectrometry (MS). In HEK293 cells, Nicalin interacted with sEphA7 and they predominantly co-localized in the endoplasmic reticulum (ER). Interestingly, Nicalin diminished the protein level of sEphA7 in the membranous fraction but increased that in the insoluble cytoplasmic fraction with a reduced molecular weight, suggesting that Nicalin restricts the entry of sEphA7 into the ER for further modification. sEphA7 probably acted as a chaperone and enhanced the membrane level of EphA7-FL and the formation of EphA7 complex, however, this effect was reversed by Nicalin. Our work suggested that Nicalin limits sEphA7 secretion, thereby preventing the formation of EphA7 complex. These results demonstrated the potential role of Nicalin in regulating EphA7 expression and revealed a potential mechanism underlying the antagonistic effect between sEphA7 and EphA7-FL.

中文翻译：

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鉴定可溶性 EphA7 相互作用蛋白 Nicalin 作为 EphA7 表达的调节剂。

已发现 EphA7 的可溶性形式 (sEphA7) 拮抗全长 EphA7 (EphA7-FL) 在非洲爪蟾前肾发育过程中稳定紧密连接蛋白 Claudin6 (CLDN6) 的膜水平的作用。然而，这种拮抗作用的机制尚不清楚。在这项研究中，我们使用免疫沉淀 (IP)/质谱 (MS) 将 Nicalin（一种 Nicastrin 样蛋白）鉴定为一种新型 sEphA7 相互作用蛋白。在 HEK293 细胞中，尼卡林与 sEphA7 相互作用，它们主要共定位在内质网 (ER) 中。有趣的是，尼卡林降低了膜部分中 sEphA7 的蛋白质水平，但增加了不溶性细胞质部分中分子量降低的蛋白质水平，这表明尼卡林限制了 sEphA7 进入内质网进行进一步修饰。sEphA7 可能作为分子伴侣并增强 EphA7-FL 的膜水平和 EphA7 复合物的形成，然而，这种作用被 Nicalin 逆转。我们的工作表明尼卡林限制了 sEphA7 的分泌，从而阻止了 EphA7 复合物的形成。这些结果证明了尼卡林在调节 EphA7 表达中的潜在作用，并揭示了 sEphA7 和 EphA7-FL 之间拮抗作用的潜在机制。