Relaxin-like factor (RLF)/insulin-like peptide 3 (INSL3) is secreted from testicular Leydig cells as a monomeric protein comprising three domains B-C-A with full biological activity in boars.
Relaxin-like factor (RLF)/insulin-like peptide 3 (INSL3) is secreted from testicular Leydig cells as a monomeric protein comprising three domains B-C-A with full biological activity in boars.
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松弛素样因子（RLF）/胰岛素样肽3（INSL3）从睾丸leydig细胞中分泌为一种单体蛋白，其中包括三个结构域B-C-A，在公猪中具有完全的生物学活性。
DOI：
10.1042/bj20111107
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发表时间：
2012-01-01
期刊：
影响因子：
--
通讯作者：
Kohsaka T
中科院分区：
文献类型：
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作者：
Minagawa I;Fukuda M;Ishige H;Kohriki H;Shibata M;Park EY;Kawarasaki T;Kohsaka T
RLF (relaxin-like factor)，又称INSL3 (insulin-like peptide 3)，是在睾丸间质细胞中表达的松弛素/胰岛素基因家族的新成员。尽管RLF/INSL3在睾丸发育中起着重要作用，但其天然构象尚不清楚。本文首次证实了公猪睾丸RLF/INSL3是一个具有完整生物活性的单体结构。通过一系列的色谱步骤，在反相高效液相色谱中获得了高纯度的RLF/INSL3单峰。MS/MS（串联质谱）分析胰蛋白酶化样品提供了66%的序列覆盖率，并揭示了由先前从RLF/INSL3 cDNA推断出的B-， C-和a -结构域组成的独特单体结构。此外，n端肽比先前预测的长4个氨基酸残基。完整分子的质谱分析和100%序列覆盖率的PMF（肽质量指纹图谱）分析证实了这种结构，并表明存在三个位点特异性二硫键。RLF/INSL3在表达RXFP2（松弛素/胰岛素样家族肽受体2）（RLF/INSL3的受体）的HEK（人胚胎肾）-293细胞中保持充分的生物活性。此外，RLF/INSL3被发现从睾丸间质细胞分泌到睾丸静脉血中。总之，这些结果表明，公猪RLF/INSL3是由睾丸间质细胞分泌的一种具有充分生物活性的B-C-A单体结构。
RLF (relaxin-like factor), also known as INSL3 (insulin-like peptide 3), is a novel member of the relaxin/insulin gene family that is expressed in testicular Leydig cells. Despite the implicated role of RLF/INSL3 in testis development, its native conformation remains unknown. In the present paper we demonstrate for the first time that boar testicular RLF/INSL3 is isolated as a monomeric structure with full biological activity. Using a series of chromatography steps, the native RLF/INSL3 was highly purified as a single peak in reverse-phase HPLC. MS/MS (tandem MS) analysis of the trypsinized sample provided 66% sequence coverage and revealed a distinct monomeric structure consisting of the B-, C- and A-domains deduced previously from the RLF/INSL3 cDNA. Moreover, the N-terminal peptide was four amino acid residues longer than predicted previously. MS analysis of the intact molecule and PMF (peptide mass fingerprinting) analysis at 100% sequence coverage confirmed this structure and indicated the existence of three site-specific disulfide bonds. RLF/INSL3 retained full bioactivity in HEK (human embryonic kidney)-293 cells expressing RXFP2 (relaxin/insulin-like family peptide receptor 2), the receptor for RLF/INSL3. Furthermore, RLF/INSL3 was found to be secreted from Leydig cells into testicular venous blood. Collectively, these results indicate that boar RLF/INSL3 is secreted from testicular Leydig cells as a B–C–A monomeric structure with full biological activity.