Hung Chien-Wen, Koudelka Tomas, Anastasi Cyril, Becker Alexander, Moali Catherine, Tholey Andreas
Systematic Proteomics & Bioanalytics, Institut für Experimentelle Medizin, Christian-Albrechts-Universität zu Kiel, Germany.
UMR 5305, Tissue Biology and Therapeutic Engineering, CNRS/University of Lyon, 69367 Lyon, France.
J Proteomics. 2016 Apr 14;138:136-45. doi: 10.1016/j.jprot.2016.02.031. Epub 2016 Mar 2.
Bone morphogenetic protein 1 (BMP-1) is an essential metalloproteinase to trigger extracellular matrix assembly and organogenesis. Previous structural studies on the refolded catalytic domain of BMP-1 produced in E. coli have suggested the existence of a rare vicinal disulfide linkage near the active site. To confirm that this was not an artifact of the refolding procedure, the full-length human BMP-1 produced in mammalian cells was investigated via sequence-dependent enzyme cleavage under native conditions followed by high mass accuracy and high resolution LC-MS/MS analysis to interrogate the post-translational modifications. Ten disulfide linkages of BMP-1, including the vicinal disulfide linkage C185-C186 could be unambiguously identified. Further, around 50% of this vicinal disulfide bond was found to be modified by N-ethylmaleimide (NEM), a cysteine protease inhibitor supplied when the BMP-1-containing medium was collected, suggesting that this bond was highly unstable. In the absence of NEM, BMP-1 has a higher tendency to form aggregates, but after aggregate removal, C185 and C186 are almost quantitatively engaged in the vicinal disulfide bond and BMP-1 activity remains unchanged. In addition, three consensus N-glycosylation sites at N142, N363, and N599 could be identified together with a previously unknown O-glycosylation site and an Asn-hydroxylation.
An in-depth characterization of post-translational modifications of the full-length human BMP-1 produced in mammalian cells by MS was performed. A rare vicinal disulfide bond in the catalytic domain could be confirmed for the first time by mass spectrometry along with nine other proposed disulfide linkages of mature BMP-1. This vicinal disulfide bond can transiently open to form covalent adducts with the cysteine protease inhibitor (NEM) supplied in cell medium during protein harvesting. Further, we report a previously unknown O-glycosylation site and Asn-hydroxylation site, indicating a novel feature of BMP-1 in the EGF domain. The study clearly outlines the benefit of in-depth characterization of overexpressed proteins to deduce important protein modifications.
骨形态发生蛋白1（BMP-1）是触发细胞外基质组装和器官发生的必需金属蛋白酶。先前对在大肠杆菌中产生的BMP-1重折叠催化结构域的结构研究表明，在活性位点附近存在罕见的邻位二硫键。为了确认这不是重折叠过程的假象，通过在天然条件下进行序列依赖性酶切，随后进行高质量精度和高分辨率液相色谱-串联质谱分析以探究翻译后修饰，对在哺乳动物细胞中产生的全长人BMP-1进行了研究。BMP-1的十个二硫键，包括邻位二硫键C185-C1-86，可以明确鉴定。此外，发现该邻位二硫键约50%被N-乙基马来酰亚胺（NEM）修饰，NEM是在收集含BMP-1的培养基时添加的一种半胱氨酸蛋白酶抑制剂，这表明该键高度不稳定。在没有NEM的情况下，BMP-1形成聚集体的倾向更高，但去除聚集体后，C185和C186几乎定量地参与邻位二硫键的形成，且BMP-1活性保持不变。此外，在N142、N363和N599处的三个共有N-糖基化位点以及一个先前未知的O-糖基化位点和一个天冬酰胺羟基化位点得以鉴定。
通过质谱对在哺乳动物细胞中产生的全长人BMP-1的翻译后修饰进行了深入表征。首次通过质谱证实了催化结构域中罕见的邻位二硫键以及成熟BMP-1的其他九个推测的二硫键。该邻位二硫键在蛋白质收获期间可短暂打开，与细胞培养基中提供的半胱氨酸蛋白酶抑制剂（NEM）形成共价加合物。此外，我们报告了一个先前未知的O-糖基化位点和天冬酰胺羟基化位点，表明BMP-1在表皮生长因子（EGF）结构域中的一个新特征。该研究清楚地概述了对过表达蛋白质进行深入表征以推断重要蛋白质修饰的益处。
