Department of Physiology and Membrane Biology, University of California Davis, Davis, CA, USA.
Department of Neurology, University of California Davis, Davis, CA, USA.
Hum Mol Genet. 2019 Aug 15;28(16):2648-2658. doi: 10.1093/hmg/ddz081.
Agrin is a large extracellular matrix protein whose isoforms differ in their tissue distribution and function. Motoneuron-derived y+z+ agrin regulates the formation of the neuromuscular junction (NMJ), while y-z- agrin is widely expressed and has diverse functions. Previously we identified a missense mutation (V1727F) in the second laminin globular (LG2) domain of agrin that causes severe congenital myasthenic syndrome. Here, we define pathogenic effects of the agrin V1727F mutation that account for the profound dysfunction of the NMJ. First, by expressing agrin variants in heterologous cells, we show that the V1727F mutation reduces the secretion of y+z+ agrin compared to wild type, whereas it has no effect on the secretion of y-z- agrin. Second, we find that the V1727F mutation significantly impairs binding of y+z+ agrin to both heparin and the low-density lipoprotein receptor-related protein 4 (LRP4) coreceptor. Third, molecular modeling of the LG2 domain suggests that the V1727F mutation primarily disrupts the y splice insert, and consistent with this we find that it partially occludes the contribution of the y splice insert to agrin binding to heparin and LRP4. Together, these findings identify several pathogenic effects of the V1727F mutation that reduce its expression and ability to bind heparan sulfate proteoglycan and LRP4 coreceptors involved in the muscle-specific kinase signaling pathway. These defects primarily impair the function of neural y+z+ agrin and combine to cause a severe CMS phenotype, whereas y-z- agrin function in other tissues appears preserved.
聚集蛋白是一种大型细胞外基质蛋白，其同工型在组织分布和功能上有所不同。运动神经元衍生的 y+z+聚集蛋白调节神经肌肉接头（NMJ）的形成，而 y-z-聚集蛋白广泛表达并有多种功能。之前我们在聚集蛋白的第二个层粘连蛋白球状（LG2）结构域中发现了一个导致严重先天性肌无力综合征的错义突变（V1727F）。在这里，我们定义了聚集蛋白 V1727F 突变的致病效应，这些效应解释了 NMJ 的严重功能障碍。首先，通过在异源细胞中表达聚集蛋白变体，我们表明 V1727F 突变与野生型相比，减少了 y+z+聚集蛋白的分泌，而对 y-z-聚集蛋白的分泌没有影响。其次，我们发现 V1727F 突变显著损害了 y+z+聚集蛋白与肝素和低密度脂蛋白受体相关蛋白 4（LRP4）核心受体的结合。第三，LG2 结构域的分子建模表明，V1727F 突变主要破坏 y 剪接插入，与这一发现一致的是，我们发现它部分阻碍了 y 剪接插入对聚集蛋白与肝素和 LRP4 核心受体结合的贡献。总之，这些发现确定了 V1727F 突变的几种致病效应，这些效应降低了其表达能力，并削弱了其结合硫酸乙酰肝素蛋白聚糖和 LRP4 核心受体的能力，而这些受体参与肌肉特异性激酶信号通路。这些缺陷主要损害了神经 y+z+聚集蛋白的功能，并共同导致严重的 CMS 表型，而其他组织中的 y-z-聚集蛋白功能似乎得到保留。
