Department of Brain Function, Division of Stress Adaptation and Protection, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Aichi, Japan.
Department of Molecular Pharmacokinetics, Nagoya University Graduate School of Medicine, Nagoya 464-8601, Aichi, Japan.
Int J Mol Sci. 2022 Mar 15;23(6):3137. doi: 10.3390/ijms23063137.
Signal peptides (SPs) not only mediate targeting to the endoplasmic reticulum (ER) but also play important roles as biomarkers and substances with physiological activity in extracellular fluids including blood. SPs are thought to be degraded intracellularly, making it unclear how they are transported from the ER to the extracellular fluid. In a recent study, we showed that a C-terminal fragment of the SP of a type I membrane protein, amyloid precursor protein (APP), was secreted into the extracellular fluid via exosomes using transformed HEK293 cells expressing APP SP flanking a reporter protein. In the present study, we demonstrate that a N-terminal fragment of the SP from a type II membrane protein, human placental secreted alkaline phosphatase (SEAP), is contained in exosomes and secreted into the extracellular fluid using HEK-Blue hTLR3 cells, which express both a human toll-like receptor 3 gene and an inducible SEAP reporter gene. When HEK-Blue hTLR3 cells were stimulated with a TLR3 ligand, a N-terminal fragment of SEAP SP in exosomes was increased in parallel with SEAP secretion in a concentration-dependent manner. These results indicated that SP fragments are exosomal components. In addition, migrating SP fragments were determined by characteristics of the signal-anchor sequence of membrane proteins. Furthermore, we found that SP fragments could bind to calmodulin (CALM), which is a cytosolic protein and also a component of exosomes, suggesting its involvement in the transportation of SP fragments from the endoplasmic reticulum to exosomes.
信号肽（SPs）不仅介导靶向内质网（ER），而且作为生物标志物和具有生理活性的物质在包括血液在内的细胞外液中发挥重要作用。SPs 被认为在细胞内降解，因此不清楚它们是如何从 ER 运输到细胞外液的。在最近的一项研究中，我们使用表达 APP SP 侧翼有报告蛋白的转化 HEK293 细胞表明，I 型膜蛋白淀粉样前体蛋白（APP）的 SP 的 C 末端片段通过外泌体被分泌到细胞外液中。在本研究中，我们证明了 II 型膜蛋白人胎盘碱性磷酸酶（SEAP）的 SP 的 N 末端片段包含在外泌体中，并使用表达人 toll 样受体 3 基因和诱导型 SEAP 报告基因的 HEK-Blue hTLR3 细胞分泌到细胞外液中。当 HEK-Blue hTLR3 细胞用 TLR3 配体刺激时，与 SEAP 分泌平行，外泌体中 SEAP SP 的 N 末端片段以浓度依赖性方式增加。这些结果表明 SP 片段是外泌体的组成部分。此外，迁移的 SP 片段通过膜蛋白的信号锚序列特征来确定。此外，我们发现 SP 片段可以与钙调蛋白（CALM）结合，CALM 是一种细胞质蛋白，也是外泌体的组成部分，这表明其参与了 SP 片段从内质网到外泌体的运输。
