Biochemistry and Molecular Biology Unit, Biomedicine Department, Faculty of Medicine, University of Barcelona, Barcelona, Spain.
Biochemistry and Molecular Biology Unit, Biomedicine Department, Faculty of Medicine, University of Barcelona, Barcelona, Spain; Clinical and Experimental Respiratory Immunoallergy (IRCE), Institut d'Investigacions Biomediques August Pi i Sunyer, Barcelona, Spain.
J Allergy Clin Immunol. 2021 May;147(5):1855-1864.e9. doi: 10.1016/j.jaci.2020.12.637. Epub 2020 Dec 29.
Anaphylaxis is a severe allergic reaction that can be lethal if not treated adequately. The underlying molecular mechanisms responsible for the severity are mostly unknown.
This study is based on a clinical case of a patient with extremely severe anaphylaxis to paper wasp venom. This patient has a mutation in the KARS gene, which encodes lysyl-tRNA synthetase (LysRS), a moonlight protein with a canonical function in protein synthesis and a noncanonical function in antigen dependent-FcεRI activation in mast cells. In this study, the objective was to characterize the mutation at the molecular level.
Analysis of the KARS mutation was carried out using biochemical and functional approaches, cell transfection, Western blot, confocal microscopy, cell degranulation, prostaglandin D secretion, and proteases gene transcription. Structural analysis using molecular dynamics simulations and well-tempered metadynamics was also performed.
The mutation found, P542R (proline was replaced by arginine at aminoacid 542), affects the location of the protein as we show in biochemical and structural analyses. The mutation resembles active LysRS and causes a constitutive activation of the microphthalmia transcription factor, which is involved in critical mast cell functions such as synthesis of mediators and granule biogenesis. Moreover, the structural analysis provides insights into how LysRS works in mast cell activation.
A link between the aberrant LysRS-P542R function and mast cell-exacerbated activation with increase in proinflammatory mediator release after antigen-IgE-dependent response could be established.
过敏反应是一种严重的过敏反应，如果得不到充分治疗可能会致命。导致过敏反应严重程度的潜在分子机制尚不清楚。
本研究基于一名对纸黄蜂毒液发生极度严重过敏反应的患者的临床病例。该患者的 KARS 基因发生突变，该基因编码赖氨酸 tRNA 合成酶（LysRS），一种具有经典蛋白合成功能的月光蛋白，也具有非经典功能，可在肥大细胞中激活抗原依赖性-FcεRI。本研究的目的是从分子水平上对突变进行特征分析。
使用生化和功能方法、细胞转染、Western blot、共聚焦显微镜、细胞脱颗粒、前列腺素 D 分泌和蛋白酶基因转录，对 KARS 突变进行分析。还使用分子动力学模拟和温度良好的元动力学进行结构分析。
发现的突变，P542R（脯氨酸被精氨酸取代，位于第 542 位氨基酸），影响蛋白质的位置，如我们在生化和结构分析中所示。该突变类似于活性 LysRS，并导致小眼转录因子的组成性激活，小眼转录因子参与肥大细胞的关键功能，如介质的合成和颗粒生物发生。此外，结构分析提供了关于 LysRS 在肥大细胞激活中如何工作的见解。
可以建立异常 LysRS-P542R 功能与肥大细胞过度激活之间的联系，这种过度激活会导致抗原-IgE 依赖性反应后促炎介质释放增加。
