Mass spectrometric characterization and activity of zinc-activated proinsulin C-peptide and C-peptide mutants
Mass spectrometric characterization and activity of zinc-activated proinsulin C-peptide and C-peptide mutants
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DOI：
10.1039/b917600d
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发表时间：
2010-01-01
期刊：
影响因子：
4.2
通讯作者：
Reid, Gavin E.
中科院分区：
文献类型：
--
作者：
Keltner, Zachary;Meyer, Jennifer A.;Reid, Gavin E.
许多报道表明，胰岛素原c肽在改善糖尿病相关的慢性并发症中具有积极作用。最近有报道称，其中一些活性依赖于某些金属离子(如Fe（II), Cr（III）或Zn(II)）对c肽的激活。为了更好地了解导致这种活性的肽-金属相互作用的结构/功能依赖性，在存在或不存在Zn（II）的情况下，对c肽进行了一系列实验，包括使用电喷雾电离（ESI）、基质辅助激光解吸/电离（MALDI）和碰撞诱导解离-串联质谱（CID-MS/MS）。此外，还分析了c肽序列中各个天冬氨酸或谷氨酸残基上存在丙氨酸取代的各种c肽突变体。锌（II）存在时，野生型c肽的CID-MS/MS显示有多个金属结合位点，定位在肽序列的酸性残基上。单个酸性残基的突变对这种断裂行为没有显著影响，这表明没有单个酸性残基对结合至关重要。然而，ESI-MS分析显示，与野生型序列相比，每个突变体的相对Zn（II）结合减少了约50%。此外，与野生型c肽相比，锌（II）激活的突变肽的活性显著下降，通过测量红细胞释放的ATP可以看出，Glu27突变体的活性下降了75%。对c肽的c端五肽EGSLQ以及突变的c端五肽序列AGSLQ的进一步研究表明，谷氨酸残基的取代导致活性完全丧失，这表明Glu27在Zn（II）介导的c肽活性中起核心作用。
Numerous reports have demonstrated an active role for proinsulin C-peptide in ameliorating chronic complications associated with diabetes mellitus. It has been recently reported that some of these activities are dependent upon activation of C-peptide with certain metal ions, such as Fe(II), Cr(III) or Zn(II). In an effort to gain a greater understanding of the structure/function dependence of the peptide-metal interactions responsible for this activity, a series of experiments involving the use of electrospray ionization (ESI), matrix assisted laser desorption/ionization (MALDI) and collision-induced dissociation-tandem mass spectrometry (CID-MS/MS) of C-peptide in the presence or absence of Zn(II) have been carried out. Additionally, various C-peptide mutants with alanine substitution at individual aspartic acid or glutamic acid residues throughout the C-peptide sequence were analyzed. CID-MS/MS of wild type C-peptide in the presence of Zn(II) indicated multiple sites for metal binding, localized at acidic residues within the peptide sequence. Mutations of individual acidic residues did not significantly affect this fragmentation behavior, suggesting that no single acidic residue is critical for binding. However, ESI-MS analysis revealed an approximately 50% decrease in relative Zn(II) binding for each of the mutants compared to the wild type sequence. Furthermore, a significant decrease in activity was observed for each of the Zn(II)-activated mutant peptides compared to the wild type C-peptide, indicated by measurement of ATP released from erythrocytes, with a 75% decrease observed for the Glu27 mutant. Additional studies on the C-terminal pentapeptide of C-peptide EGSLQ, as well as a mutant C-terminal pentapeptide sequence AGSLQ, revealed that substitution of the glutamic acid residue resulted in a complete loss of activity, implicating a central role for Glu27 in Zn(II)-mediated C-peptide activity.