University of Strasbourg, CNRS, Institute of Chemistry UMR 7177, F-67000 Strasbourg, France.
Plateforme Protéomique Strasbourg-Esplanade, Institut de Biologie Moléculaire et Cellulaire, CNRS FRC1589, Université de Strasbourg, F-67000 Strasbourg, France.
Chem Res Toxicol. 2021 Sep 20;34(9):2087-2099. doi: 10.1021/acs.chemrestox.1c00172. Epub 2021 Aug 9.
Chemical skin and respiratory allergies are becoming a major health problem. To date our knowledge on the process of protein haptenation is still limited and mainly derived from studies performed in solution using model nucleophiles. In order to better understand chemical interactions between chemical allergens and the skin, we have investigated the reactivity of phthalic anhydride (PA), a chemical respiratory sensitizer, toward reconstructed human epidermis (RHE). This study was performed using a new approach combining HRMAS NMR to investigate the in situ chemical reactivity and LC-MS/MS to identify modified epidermal proteins. In RHE, the reaction of PA appeared to be quite fast and the major product formed was phthalic acid. Two amide type adducts on lysine residues were observed and after 8h of incubation, we also observed the formation of an imide type cyclized adducts with lysine. In parallel, RHE samples topically exposed to phthalic anhydride (C)-1 were analyzed using the shotgun proteomics method. Thus, 948 different proteins were extracted and identified, 135 of which being modified by PA, i.e., 14.2% of the extracted proteome. A total of 211 amino acids were modified by PA and validated by fragmentation spectra. We thus identified 154 modified lysines, 22 modified histidines, 30 modified tyrosines, and 5 modified arginines. The rate of modified residues, as a proportion of the total number of modifiable nucleophilic residues in RHE, was rather low (1%). At the protein level, modified proteins were mainly type I and type II keratins and other proteins which are abundant in the epidermis such as protein S100A, Caspase 14, annexin A2, serpin B3, fatty-acid binding protein 5, histone H2, H3, H4, etc. However, the most modified protein, mainly on histidine residues, was filaggrin, a protein that is of low abundance (0.0266 mol %) and rich in histidine.
化学性皮肤和呼吸道过敏正在成为一个主要的健康问题。迄今为止，我们对蛋白质半抗原化过程的了解仍然有限，并且主要来源于使用模型亲核试剂在溶液中进行的研究。为了更好地理解化学性过敏原与皮肤之间的化学相互作用，我们研究了邻苯二甲酸酐（PA）的反应性，邻苯二甲酸酐是一种化学性呼吸致敏原，作用于重建的人表皮（RHE）。这项研究使用了一种新方法，结合高分辨率魔角旋转核磁共振（HRMAS NMR）来研究原位化学反应性，并结合液相色谱-串联质谱（LC-MS/MS）来鉴定修饰的表皮蛋白。在 RHE 中，PA 的反应似乎很快，形成的主要产物是邻苯二甲酸。观察到赖氨酸残基上的两种酰胺型加合物，孵育 8 小时后，还观察到赖氨酸的环状酰亚胺型加合物的形成。同时，用邻苯二甲酸酐（C）-1处理的 RHE 样品使用鸟枪法蛋白质组学方法进行分析。因此，提取并鉴定了 948 种不同的蛋白质，其中 135 种被 PA 修饰，即提取的蛋白质组的 14.2%。PA 修饰了总共 211 个氨基酸，并通过碎片谱进行了验证。因此，我们鉴定了 154 个修饰的赖氨酸、22 个修饰的组氨酸、30 个修饰的酪氨酸和 5 个修饰的精氨酸。修饰残基的比例，相对于 RHE 中可修饰亲核残基的总数，相当低（1%）。在蛋白质水平上，修饰的蛋白质主要是 I 型和 II 型角蛋白和其他在表皮中丰富的蛋白质，如 S100A 蛋白、半胱天冬酶 14、膜联蛋白 A2、丝氨酸蛋白酶抑制剂 B3、脂肪酸结合蛋白 5、组蛋白 H2、H3、H4 等。然而，被修饰最多的蛋白质，主要是组氨酸残基上的丝聚蛋白，一种丰度较低（0.0266mol%）但富含组氨酸的蛋白质。
