Niu Chendi, Du Yi, Kaltashov Igor A
Chemistry Department, University of Massachusetts-Amherst, Amherst, MA.
Int J Mass Spectrom. 2021 May;463. doi: 10.1016/j.ijms.2021.116550. Epub 2021 Feb 14.
Neutrophil elastase is a serine protease released by neutrophils, and its dysregulation has been associated with a variety of debilitating pathologies, most notably cystic fibrosis. This protein is also a prominent component of the so-called neutrophil extracellular traps (NETs), whose formation is a part of the innate immunity response to invading pathogens, but also contributes to a variety of pathologies ranging from autoimmune disorders and inflammation to cancer to thrombotic complications in COVID-19. Retention of neutrophil elastase within NETs is provided by ejected DNA chains, although this protein is also capable of interacting with a range of other endogenous polyanions, such as heparin and heparan sulfate. In this work, we evaluate the feasibility of using native mass spectrometry (MS) as a means of studying interactions of neutrophil elastase with heparin oligomers ranging from structurally homogeneous synthetic pentasaccharide fondaparinux to relatively long (up to twenty saccharide units) and structurally heterogeneous chains produced by partial depolymerization of heparin. The presence of heterogeneous glycan chains on neutrophil elastase and the structural heterogeneity of heparin oligomers render the use of standard MS to study their complexes impractical. However, supplementing MS with limited charge reduction in the gas phase allows meaningful data to be extracted from MS measurements. In contrast to earlier molecular modeling studies where a single heparin-binding site was identified, our work reveals the existence of multiple binding sites, with a single protein molecule being able to accommodate up to three decasaccharides. The measurements also reveal the ability of even relatively short heparin oligomers to bridge two protein molecules, suggesting that characterization of these complexes using native MS can shed light on the structural properties of NETs. Lastly, the use of MS allows the binding preferences of heparin oligomers to neutrophil elastase to be studied with respect to specific structural properties of heparin, such as the level of sulfation ( charge density). All experimental measurements are carried out in parallel with molecular dynamics simulations of the protein/heparin oligomer systems, which are in remarkable agreement with the experimental data and highlight the role of electrostatic interactions as dominant forces governing the formation of these complexes.
中性粒细胞弹性蛋白酶是一种由中性粒细胞释放的丝氨酸蛋白酶，其失调与多种使人衰弱的病理状况有关，最显著的是囊性纤维化。这种蛋白质也是所谓中性粒细胞胞外陷阱（NETs）的一个主要成分，NETs的形成是对入侵病原体的固有免疫反应的一部分，但也会导致从自身免疫性疾病、炎症到癌症以及COVID - 19中的血栓并发症等多种病理状况。尽管这种蛋白质也能够与一系列其他内源性聚阴离子相互作用，如肝素和硫酸乙酰肝素，但NETs中中性粒细胞弹性蛋白酶的保留是由排出的DNA链提供的。在这项工作中，我们评估了使用原生质谱（MS）作为研究中性粒细胞弹性蛋白酶与肝素寡聚物相互作用的一种手段的可行性，这些肝素寡聚物范围从结构均一的合成五糖磺达肝癸钠到相对较长（多达二十个糖单元）且由肝素部分解聚产生的结构异质链。中性粒细胞弹性蛋白酶上存在异质聚糖链以及肝素寡聚物的结构异质性使得使用标准质谱研究它们的复合物不切实际。然而，可以通过在气相中进行有限的电荷减少来补充质谱，从而从质谱测量中提取有意义的数据。与早期鉴定出单个肝素结合位点的分子建模研究不同，我们的工作揭示了存在多个结合位点，单个蛋白质分子能够容纳多达三个十糖。这些测量还揭示了即使相对较短的肝素寡聚物也能够桥接两个蛋白质分子，这表明使用原生质谱对这些复合物进行表征可以揭示NETs的结构特性。最后，质谱的使用使得能够针对肝素的特定结构特性，如硫酸化水平（电荷密度），研究肝素寡聚物对中性粒细胞弹性蛋白酶的结合偏好。所有实验测量均与蛋白质/肝素寡聚物系统的分子动力学模拟并行进行，模拟结果与实验数据显著一致，并突出了静电相互作用作为支配这些复合物形成的主导力量的作用。
