Raftery M J, Yang Z, Valenzuela S M, Geczy C L
Cytokine Research Unit, School of Pathology, University of New South Wales, Kensington, New South Wales 2052, Australia.
J Biol Chem. 2001 Sep 7;276(36):33393-401. doi: 10.1074/jbc.M101566200. Epub 2001 Jul 9.
Hypochlorite is a major oxidant generated when neutrophils and macrophages are activated at inflammatory sites, such as in atherosclerotic lesions. Murine S100A8 (A8) is a major cytoplasmic protein in neutrophils and is secreted by macrophages in response to inflammatory stimuli. After incubation with reagent HOCl for 10 min, approximately 85% of A8 was converted to 4 oxidation products, with electrospay ionization mass spectrometry masses of m/z 10354, 10388, 10354 +/- 1, and 20707 +/- 3. All were resistant to reduction by dithiothreitol. Initial formation of a reactive Cys sulfenic acid intermediate was demonstrated by the rapid conjugation of 5,5-dimethyl-1,3-cyclohexanedione (dimedone) to HOCl-treated A8 to form stable adducts. Matrix-assisted laser desorption-reflectron time of flight peptide mass fingerprinting of isolated oxidation products confirmed the mass additions observed in the full-length proteins. Both Met(36/73) were converted to Met(36/73) sulfoxides. An additional product with an unusual mass addition of m/z 14 (+/-0.2) was identified and corresponded to the addition of oxygen to Cys(41), conjugation to various epsilon-amines of Lys(6), Lys(34/35), or Lys(87) with loss of dihydrogen and formation of stable intra- or inter-molecular sulfinamide cross-links. Specific fragmentations identified in matrix-assisted laser desorption-post source decay spectra and low energy collisional-induced dissociation tandem mass spectroscopy spectra of sulfinamide-containing digest peptides confirmed Lys(34/35) to Cys(41) sulfinamide bonds. HOCl oxidation of mutants lacking Cys(41) (Ala(41)S100A8) or specific Lys residues (e.g. Lys(34/35), Ala(34/35)S100A8) did not form sulfinamide cross-links. HOCl generated by myeloperoxidase and H(2)O(2) and by phorbol 12-myristate 13-acetate-activated neutrophils also formed these products(.) In contrast to the disulfide-linked dimer, oxidized monomer retained normal chemotactic activity for neutrophils. Sulfinamide bond formation represents a novel oxidative cross-linking process between thiols and amines and may be a general consequence of HOCl protein oxidation in inflammation not identified previously. Similar modifications in other proteins could potentially regulate normal and pathological processes during aging, atherogenesis, fibrosis, and neurogenerative diseases.
次氯酸盐是中性粒细胞和巨噬细胞在炎症部位（如动脉粥样硬化病变处）被激活时产生的主要氧化剂。小鼠S100A8（A8）是中性粒细胞中的一种主要细胞质蛋白，巨噬细胞在受到炎症刺激时会分泌该蛋白。用试剂次氯酸（HOCl）孵育10分钟后，约85%的A8转化为4种氧化产物，其电喷雾电离质谱质量分别为m/z 10354、10388、10354 +/- 1和20707 +/- 3。所有产物均对二硫苏糖醇的还原作用具有抗性。通过5,5 - 二甲基 - 1,3 - 环己二酮（达美酮）与经HOCl处理的A8快速结合形成稳定加合物，证明了反应性半胱氨酸亚磺酸中间体的初始形成。对分离出的氧化产物进行基质辅助激光解吸 - 反射电子飞行时间肽质量指纹图谱分析，证实了在全长蛋白中观察到的质量增加。甲硫氨酸（Met）（36/73）均转化为甲硫氨酸（36/73）亚砜。还鉴定出一种质量增加异常为m/z 14（+/-0.2）的额外产物，它对应于半胱氨酸（Cys）（41）加氧，与赖氨酸（Lys）（6）、赖氨酸（34/35）或赖氨酸（87）的各种ε - 氨基结合，同时失去两个氢并形成稳定的分子内或分子间亚磺酰胺交联键。在含亚磺酰胺的消化肽的基质辅助激光解吸 - 源后衰变光谱和低能碰撞诱导解离串联质谱光谱中鉴定出的特定裂解峰，证实了赖氨酸（34/35）与半胱氨酸（41）之间的亚磺酰胺键。缺乏半胱氨酸（41）的突变体（丙氨酸（Ala）（41）S100A8）或特定赖氨酸残基（如赖氨酸（34/35），丙氨酸（34/35）S100A8）经HOCl氧化后未形成亚磺酰胺交联键。髓过氧化物酶和过氧化氢（H₂O₂）以及佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯激活的中性粒细胞产生的HOCl也形成了这些产物。与二硫键连接的二聚体不同，氧化的单体对中性粒细胞仍保留正常趋化活性。亚磺酰胺键的形成代表了一种硫醇和胺之间新型的氧化交联过程，可能是炎症中HOCl蛋白氧化以前未被发现的普遍结果。其他蛋白质中的类似修饰可能在衰老、动脉粥样硬化、纤维化和神经退行性疾病过程中潜在地调节正常和病理过程。
