Determination of Histidine pKa Values in the Propeptides of Furin and Proprotein Convertase 1/3 Using Histidine Hydrogen-Deuterium Exchange Mass Spectrometry.
Determination of Histidine pKa Values in the Propeptides of Furin and Proprotein Convertase 1/3 Using Histidine Hydrogen-Deuterium Exchange Mass Spectrometry.
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DOI：
10.1021/acs.analchem.5b01721
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发表时间：
2015-08-04
影响因子：
7.4
通讯作者：
Shinde U
中科院分区：
文献类型：
--
作者：
Elferich J;Williamson DM;David LL;Shinde U
前蛋白转化酶的前肽通过感应分泌途径内的细胞器pH来调节其蛋白酶结构域的活化。早期的实验工作强调了一个保守的组氨酸残基内的广泛研究的成员，弗林蛋白酶的前肽的重要性。随后的进化分析发现，与原核同源物相比，分泌的真核蛋白酶的前肽中组氨酸含量增加。然而，弗林蛋白酶激活的反式高尔基体网络在pH值为6.5，而paradox，前蛋白转化酶1/3，激活的分泌囊泡在pH值为5.5。目前还不清楚保守的组氨酸如何在两个不同的pH值下介导活化。在这份手稿中，我们测量了组氨酸的pKa值内的弗林蛋白酶和前蛋白转化酶1/3的前肽使用组氨酸氢-氘交换质谱法。高密度的组氨酸残基与丰富的碱性残基的组合为产生具有独特组氨酸残基的肽离子提供了挑战，这通过采用ETD片段化来克服。在此分析过程中，我们发现缓慢的氢-氘交换的残基，而不是组氨酸在碱性pH值。最后，我们证明了保守的组氨酸在前蛋白转化酶1/3的pKa是酸转移与弗林蛋白酶相比，是一致的，其较低的pH值的激活。
Propeptides of proprotein convertases regulate activation of their protease domains by sensing the organellar pH within the secretory pathway. Earlier experimental work highlighted the importance of a conserved histidine residue within the propeptide of a widely studied member, furin. A subsequent evolutionary analysis found an increase in histidine content within propeptides of secreted eukaryotic proteases compared with their prokaryotic orthologs. However, furin activates in the trans-golgi network at a pH of 6.5 while a paralog, proprotein convertase 1/3, activates in secretory vesicles at a pH of 5.5. It is unclear how a conserved histidine can mediate activation at two different pH values. In this manuscript, we measured the pKa values of histidines within the propeptides of furin and proprotein convertase 1/3 using a histidine hydrogen–deuterium exchange mass spectrometry approach. The high density of histidine residues combined with an abundance of basic residues provided challenges for generation of peptide ions with unique histidine residues, which were overcome by employing ETD fragmentation. During this analysis, we found slow hydrogen–deuterium exchange in residues other than histidine at basic pH. Finally, we demonstrate that the pKa of the conserved histidine in proprotein convertase 1/3 is acid-shifted compared with furin and is consistent with its lower pH of activation.