Ronsein Graziella E, Winterbourn Christine C, Di Mascio Paolo, Kettle Anthony J
Departamento de Bioquí;mica, Instituto de Química, Universidade de São Paulo, 05508-900 São Paulo, SP, Brazil.
The Centre for Free Radical Research, Department of Pathology, University of Otago at Christchurch, Christchurch, New Zealand.
Free Radic Biol Med. 2014 May;70:278-87. doi: 10.1016/j.freeradbiomed.2014.01.023. Epub 2014 Jan 28.
Irreversible cross-links are increasingly being recognized as important posttranslational oxidative protein modifications that contribute to tissue injury during oxidative stress and inflammation. They also have a structural function in extracellular matrix proteins such as collagen IV. Likely contenders for forming such cross-links are the reactive halogen species that are generated by neutrophils and eosinophils, including hypochlorous acid, hypobromous acid, and their related haloamines. Methionine residues are kinetically preferred targets for these oxidants and oxidation can potentially result in sulfilimine (>S=N-) bonds with amines. Therefore, we investigated whether oxidation of methionine in the model peptide formyl-Met-Leu-Phe-Lys (fMLFK) produces cross-links with lysine residues, using mass spectrometry to characterize the products. As expected, the sulfoxide was the major product with each reactive halogen species. However, intra- and intermolecular cross-linked products were also formed. Isomers of an intramolecular sulfilimine were readily produced by hypobromous acid and bromamines, with hypochlorous acid forming lesser amounts. The predominant cross-link with chloramines was an intermolecular bond between the sulfur of fMLFK and the amine derived from the chloramine. Reactive halogen species also formed these sulfilimine cross-links in other peptides that contain methionine. We propose that protein cross-links involving methionine and amine residues will form via this mechanism when granulocytes are activated at sites of inflammation. Our results also support the proposal that reactive halogen species generated by the peroxidase peroxidasin could be responsible for the sulfilimine bonds that are integral to the structure of collagen IV.
不可逆交联越来越被认为是重要的翻译后氧化蛋白修饰，在氧化应激和炎症过程中导致组织损伤。它们在细胞外基质蛋白如IV型胶原蛋白中也具有结构功能。形成这种交联的可能候选物是由中性粒细胞和嗜酸性粒细胞产生的活性卤素物种，包括次氯酸、次溴酸及其相关的卤胺。甲硫氨酸残基在动力学上是这些氧化剂的首选靶点，氧化可能会导致与胺形成亚磺酰亚胺（>S=N-）键。因此，我们使用质谱对产物进行表征，研究了模型肽甲酰 - 甲硫氨酸 - 亮氨酸 - 苯丙氨酸 - 赖氨酸（fMLFK）中甲硫氨酸的氧化是否会与赖氨酸残基形成交联。正如预期的那样，亚砜是每种活性卤素物种的主要产物。然而，分子内和分子间的交联产物也会形成。次溴酸和溴胺很容易产生分子内亚磺酰亚胺的异构体，次氯酸产生的量较少。与氯胺的主要交联是fMLFK的硫与氯胺衍生的胺之间的分子间键。活性卤素物种在其他含甲硫氨酸的肽中也形成了这些亚磺酰亚胺交联。我们提出，当粒细胞在炎症部位被激活时，涉及甲硫氨酸和胺残基的蛋白质交联将通过这种机制形成。我们的结果还支持这样的提议，即过氧化物酶过氧联蛋白产生的活性卤素物种可能是IV型胶原蛋白结构中不可或缺的亚磺酰亚胺键的原因。
