A novel form of neurotensin post-translationally modified by arginylation
A novel form of neurotensin post-translationally modified by arginylation
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DOI：
10.1074/jbc.m502567200
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发表时间：
2005-10-21
影响因子：
4.8
通讯作者：
Sillard, R
中科院分区：
文献类型：
--
作者：
Eriste, E;Norberg, Å;Sillard, R
从猪肠中分离到一种新的生物活性形式的神经降压素，其在Glu残基处进行后修饰。通过检测SK-N-SH神经母细胞瘤细胞中的细胞内Ca 2+释放来指导肽的纯化。在离子阱傅立叶变换质谱仪上进行高分辨精确质量分析，确定其翻译后修饰为精氨酸通过异肽键连接到Glu的γ-羧基上，并将新鉴定的肽命名为“N-（neurotensin-C5-4-yl）arginine”（R-NT，N-（neurotensin-C5-4-yl）arginine）。虽然N-末端氨基修饰是蛋白质中已知的修饰，但其在Glu侧链上的存在是独特的。这一发现将神经降压素置于少数生理活性肽之列，这些肽既以后修饰形式出现，也以未修饰形式出现。在药理学上，我们表征了R-NT对三种已知神经降压素受体NTR 1、NTR-2和NTR-3的配体活性，并发现R-NT具有与神经降压素相似的药理学性质，然而，对所有三种受体的亲和力略高。我们将细胞内受体NTR 3表达为分泌到细胞培养基中的可溶性蛋白，这允许表征其R-NT和神经降压素结合特性。可溶性NTR 3的产生也提供了一种在体内和体外中和神经降压素作用的潜在工具。我们已经表明，SK-N-SH神经母细胞瘤细胞表达NTR 1和NTR 3，但不表达NTR 2，这表明R-NT引起的Ca 2+动员是通过NTR 1。
A novel bioactive form of neurotensin post-translationally modified at a Glu residue was isolated from porcine intestine. Purification of the peptide was guided by detection of intracellular Ca2+ release in SK-N-SH neuroblastoma cells. Using high resolution accurate mass analysis on an ion trap Fourier transform mass spectrometer, the post-translational modification was identified as arginine linked to the gamma-carboxyl of Glu via an isopeptide bond, and we named the newly identified peptide "arginylated neurotensin" (R-NT, N-(neurotensin-C5-4-yl)arginine). Although arginylation is a known modification of N- terminal amino groups in proteins, its presence at a Glu side chain is unique. The finding places neurotensin among the few physiologically active peptides that occur both in post-translationally modified and unmodified forms. Pharmacologically, we characterized R-NT for its ligand activity on three known neurotensin receptors, NTR1, -2, and -3, and found that R-NT has similar pharmacological properties to those of neurotensin, however, with a slightly higher affinity to all three receptors. We expressed the intracellular receptor NTR3 as a soluble protein secreted into the cell culture medium, which allowed characterization of its R-NT and neurotensin binding properties. The creation of soluble NTR3 also provides a potential tool for neutralizing neurotensin action in vivo and in vitro. We have shown that SK-N-SH neuroblastoma cells express NTR1 and NTR3 but not NTR2, suggesting that the Ca2+ mobilization elicited by R-NT is via NTR1.