Purification and identification of native forms of goldfish neuromedin U from brain and gut
Purification and identification of native forms of goldfish neuromedin U from brain and gut
复制标题
从脑和肠道中纯化和鉴定金鱼神经调节素 U 的天然形式
DOI：
10.1016/j.bbrc.2019.07.108
复制
发表时间：
2019
影响因子：
3.1
通讯作者：
Matsuda Kouhei
中科院分区：
文献类型：
--
作者：
Maruyama Keisuke;Kaiya Hiroyuki;Miyazato Mikiya;Murakami Noboru;Nakahara Keiko;Matsuda Kouhei
神经介肽U（NMU）在啮齿动物和鸟类的能量平衡中起重要作用。以前，我们的小组已经从金鱼的大脑和肠道中分离出四个编码NMU前体蛋白的cDNA，并假设这些转录本是通过选择性剪接产生的。我们还表明，脑室内（ICV）注射假定金鱼NMU抑制食物摄入。然而，由于原生金鱼NMU尚未被鉴定，我们试图从金鱼脑和肠道提取物中纯化它。为了评估每个纯化步骤中组分中的NMU活性，我们使用表达金鱼NMU-R1或-R2的HEK 293细胞测量了细胞内Ca 2+浓度的变化。我们从脑和肠道中分离到一个25个氨基酸的肽（NMU-25），发现它的一级结构与哺乳动物的NMU相似。另一种21-氨基酸肽（NMU-21）是从大脑中纯化的，但不是从肠道中纯化的。此外，还从脑和肠中分离出与NMU-21和NMU-25的C-末端相同的9-氨基酸肽（NMU-9）。用合成的NMU-9、NMU-21和NMU-25处理可剂量依赖性地增加表达金鱼NMU-R1和NMU-R2的哺乳动物细胞中的细胞内Ca 2+浓度。我们还研究了ICV施用的合成金鱼NMU对金鱼食物摄入的影响。NMU-25抑制食物摄入的程度与NMU-21相同。然而，NMU-9的抑制作用略弱于NMU-21和NMU-25。这些结果表明，几种分子形式的NMU存在于金鱼的大脑和肠道中，并且它们都通过NMU-R1和NMU-R2发挥生理作用。
Neuromedin U (NMU) plays important roles in energy homeostasis in rodents and birds. Previously, our group has isolated four cDNAs encoding precursor proteins of NMU from the goldfish brain and gut, and it was assumed that these transcripts are produced by alternative splicing. We have also demonstrated that intracerebroventricular (ICV) injection of putative goldfish NMU inhibits food intake. However, as native goldfish NMU has not yet been identified, we attempted to purify it from goldfish brain and gut extracts. To assess NMU activity in fractions at each purification step, we measured changes in the intracellular concentrations of Ca2+using HEK293 cells expressing goldfish NMU-R1 or -R2. We isolated a 25-amino-acid peptide (NMU-25) from the brain and gut and found that its primary structure is similar to that of mammalian NMU. Another 21-amino-acid peptide (NMU-21) was purified from the brain, but not from the gut. Furthermore, a 9-amino-acid peptide (NMU-9) identical to the C-terminus of NMU-21 and -25 was also isolated from the brain and gut. Treatment with synthetic NMU-9, -21 and -25 dose-dependently increased the intracellular Ca2+concentration in mammalian cells expressing goldfish NMU-R1 and -R2. We also examined the effect of ICV-administered synthetic goldfish NMUs on goldfish food intake. NMU-25 inhibited food intake to the same degree as NMU-21. However, the inhibitory effect of NMU-9 was slightly weaker than those of NMU-21 and -25. These results indicate that several molecular forms of NMU exist in the goldfish brain and gut, and that all of them play physiological roles via NMU-R1 and NMU-R2.