Laboratory of Animal Reproduction and Physiology, Faculty of Agriculture, Department of Applied Biological Chemistry, Shizuoka University, Suruga-ku, Shizuoka 422-8529, Japan.
Biol Chem. 2013 Sep;394(9):1181-94. doi: 10.1515/hsz-2012-0357.
Relaxin-like factor (RLF), also called insulin-like peptide 3 (INSL3), is a member of the insulin/relaxin gene family and is produced by testicular Leydig cells. While the understanding of its effects is growing, very little is known about the structural and functional properties of native INSL3. Here, we demonstrate that native INSL3 isolated from goat testes is a single-chain structure with full biological activity, and is constitutively expressed and secreted by Leydig cells. Using a series of chromatography steps, native INSL3 was highly purified as a single 12-kDa peak as revealed by SDS-PAGE. MS/MS analysis provided 81% sequence coverage and revealed a distinct single-chain structure consisting of the B-, C-, and A-domains deduced previously from the INSL3 cDNA sequence. Moreover, the N-terminal peptide was six amino acid residues longer than predicted. Native INSL3 exhibited full bioactivity in HEK-293 cells expressing the receptor for INSL3. Immunoelectron microscopy and Western blot analysis revealed that INSL3 was secreted by Leydig cells through the constitutive pathway into blood and body fluids. We conclude, therefore, that goat INSL3 is constitutively secreted from Leydig cells as a B-C-A single-chain structure with full biological activity.
松弛素样因子（RLF），也称为胰岛素样肽 3（INSL3），是胰岛素/松弛素基因家族的成员，由睾丸间质细胞产生。尽管对其作用的理解正在不断深入，但对天然 INSL3 的结构和功能特性知之甚少。在这里，我们证明从山羊睾丸中分离出的天然 INSL3 是具有完整生物活性的单链结构，并且由间质细胞组成性表达和分泌。通过一系列色谱步骤，天然 INSL3 被高度纯化，在 SDS-PAGE 上显示为单一的 12 kDa 峰。MS/MS 分析提供了 81%的序列覆盖度，并揭示了先前从 INSL3 cDNA 序列推断出的独特的单链结构，由 B、C 和 A 结构域组成。此外，N 端肽比预测的长六个氨基酸残基。天然 INSL3 在表达 INSL3 受体的 HEK-293 细胞中表现出完整的生物活性。免疫电子显微镜和 Western blot 分析显示，INSL3 通过组成性途径由间质细胞分泌到血液和体液中。因此，我们得出结论，山羊 INSL3 作为具有完整生物活性的 B-C-A 单链结构组成性分泌自间质细胞。
