Site-specific DOTA/europium-labeling of recombinant human relaxin-3 for receptor-ligand interaction studies
Site-specific DOTA/europium-labeling of recombinant human relaxin-3 for receptor-ligand interaction studies
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重组人松弛素 3 的位点特异性 DOTA/铕标记用于受体-配体相互作用研究
DOI：
10.1007/s00726-011-1164-z
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发表时间：
2011-12
期刊：
影响因子：
3.5
通讯作者：
Guo, Zhan-Yun
中科院分区：
文献类型：
--
作者：
Zhang, Wei-Jie;Luo, Xiao;Liu, Ya-Li;Shao, Xiao-Xia;Wade, John D.;Bathgate, Ross A. D.;Guo, Zhan-Yun
松弛素-3（也称为INSL7）是最近发现的一种属于胰岛素/松弛素超家族的神经肽。据推测，它通过激活同源的g蛋白偶联受体RXFP3，在应激反应、食物摄入和繁殖的调节中起作用。它还能在体外结合并激活松弛素家族肽受体RXFP1和RXFP4。为了获得铕标记的松弛素-3作为研究这些受体与各种配体相互作用的示踪剂，在本工作中，我们提出了一种新的位点特异性标记策略，用于先前在我们实验室制备的重组人松弛素-3。首先，空气单胺肽酶去除单链松弛素-3前体的n端6×His-tag，合成肽的所有初级胺被柠檬酸酸酐可逆阻断。其次，阻断肽的a链n端通过内源性蛋白酶Asp-N切割释放，去除B链和a链之间的连接肽。第三，通过与高活性伯胺特异性n -羟基琥珀酰亚胺酯反应，将炔段引入新释放的a链n端。第四，在温和酸性条件下去除可逆堵塞后，通过点击化学将带有叠氮基团的载铕DOTA引入到带有炔基基团的双链松弛素-3中。使用这种位点特异性标记策略，可以获得均匀的单铕标记的人松弛素-3，总体收率较高。相比之下，传统的随机标记导致了一个复杂的混合物，很难解决，因为人类松弛素-3有四个伯胺部分，都与修饰试剂反应。饱和和竞争结合实验均表明，DOTA/Eu3+标记的松弛素-3对人RXFP3、RXFP4和RXFP1具有很高的结合亲和力，因此是一种合适的非放射性和稳定的示踪剂，用于研究各种天然或设计的配体与这些受体的相互作用。利用这种位点特异性标记策略，其他功能探针，如荧光染料、生物素或纳米颗粒也可以引入重组人松弛素-3的a链n端。此外，我们改进了DOTA结合的铕离子的时间分辨荧光分析，这为在未来的研究中使用DOTA作为镧系元素螯合剂用于蛋白质和肽的标记铺平了道路。
Relaxin-3 (also known as INSL7) is a recently identified neuropeptide belonging to the insulin/relaxin superfamily. It has putative roles in the regulation of stress responses, food intake, and reproduction by activation of its cognate G-protein-coupled receptor RXFP3. It also binds and activates the relaxin family peptide receptors RXFP1 and RXFP4 in vitro. To obtain a europium-labeled relaxin-3 as tracer for studying the interaction of these receptors with various ligands, in the present work we propose a novel site-specific labeling strategy for the recombinant human relaxin-3 that has been previously prepared in our laboratory. First, the N-terminal 6×His-tag of the single-chain relaxin-3 precursor was removed byAeromonasaminopeptidase and all of the primary amines of the resultant peptide were reversibly blocked by citroconic anhydride. Second, the A-chain N-terminus of the blocked peptide was released by endoproteinase Asp-N cleavage that removed the linker peptide between the B- and A-chains. Third, an alkyne moiety was introduced to the newly released A-chain N-terminus by reaction with the highly active primary amine-specificN-hydroxysuccinimide ester. Fourth, after removal of the reversible blockage under mild acidic condition, europium-loaded DOTA with an azide moiety was introduced to the two-chain relaxin-3 carrying the alkyne moiety through click chemistry. Using this site-specific labeling strategy, homogeneous monoeuropium-labeled human relaxin-3 could be obtained with good overall yield. In contrast, conventional random labeling resulted in a complex mixture that was poorly resolved because human relaxin-3 has four primary amine moieties that all react with the modification reagent. Both saturation and competition binding assays demonstrated that the DOTA/Eu3+-labeled relaxin-3 retained high binding affinity for human RXFP3, RXFP4, and RXFP1 and was therefore a suitable non-radioactive and stable tracer to study the interaction of various natural or designed ligands with these receptors. Using this site-specific labeling strategy, other functional probes, such as fluorescent dyes, biotin, or nanoparticles could also be introduced to the A-chain N-terminal of the recombinant human relaxin-3. Additionally, we improved the time-resolved fluorescence assay for the DOTA-bound europium ion which paves the way for the use of DOTA as a lanthanide chelator for protein and peptide labeling in future studies.
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影响因子：
3
作者：
Watanabe Y;Tsujimura A;Takao K;Nishi K;Ito Y;Yasuhara Y;Nakatomi Y;Yokoyama C;Fukui K;Miyakawa T;Tanaka M
通讯作者：
Tanaka M
影响因子：
3.5
作者：
Craig M. Smith;I. T. Hosken;Steve W. Sutton;Andrew J. Lawrence;A. L. Gundlach
通讯作者：
Craig M. Smith;I. T. Hosken;Steve W. Sutton;Andrew J. Lawrence;A. L. Gundlach
影响因子：
4.8
作者：
Liu, CL;Chen, JC;Lovenberg, TW
通讯作者：
Lovenberg, TW
影响因子：
--
作者：
McGowan, B. M.;Stanley, S. A.;Bloom, S. R.
通讯作者：
Bloom, S. R.
影响因子：
2.8
作者：
Craig M. Smith;P. Ryan;I. T. Hosken;Sherie Ma;A. Gundlach
通讯作者：
Craig M. Smith;P. Ryan;I. T. Hosken;Sherie Ma;A. Gundlach