Shao Baohai, Belaaouaj Abderrazzaq, Verlinde Christophe L M J, Fu Xiaoyun, Heinecke Jay W
Department of Medicine, University of Washington, Seattle, WA 98195, USA.
J Biol Chem. 2005 Aug 12;280(32):29311-21. doi: 10.1074/jbc.M504040200. Epub 2005 Jun 20.
Using myeloperoxidase and hydrogen peroxide, activated neutrophils produce high local concentrations of hypochlorous acid (HOCl). They also secrete cathepsin G, a serine protease implicated in cytokine release, receptor activation, and degradation of tissue proteins. Isolated cathepsin G was inactivated by HOCl but not by hydrogen peroxide in vitro. We found that activated neutrophils lost cathepsin G activity by a pathway requiring myeloperoxidase, suggesting that oxidants generated by myeloperoxidase might regulate cathepsin G activity in vivo. Tandem mass spectrometric analysis of oxidized cathepsin G revealed that loss of a peptide containing Asp108, which lies in the active site, associated quantitatively with loss of enzymatic activity. Catalytic domain peptides containing Asp108 were lost from the oxidized protein in concert with the conversion of Met110 to the sulfoxide. Release of this peptide was blocked by pretreating cathepsin G with phenylmethylsulfonyl fluoride, strongly implying that oxidation introduced proteolytic cleavage sites into cathepsin G. Model system studies demonstrated that methionine oxidation can direct the regiospecific proteolysis of peptides by cathepsin G. Thus, oxidation of Met110 may contribute to cathepsin G inactivation by at least two distinct mechanisms. One involves direct oxidation of the thioether residue adjacent to the aspartic acid in the catalytic domain. The other involves the generation of new sites that are susceptible to proteolysis by cathepsin G. These observations raise the possibility that oxidants derived from neutrophils restrain pericellular proteolysis by inactivating cathepsin G. They also suggest that methionine oxidation could render cathepsin G susceptible to autolytic cleavage. Myeloperoxidase may thus play a previously unsuspected role in regulating tissue injury by serine proteases during inflammation.
活化的中性粒细胞利用髓过氧化物酶和过氧化氢产生高浓度的局部次氯酸（HOCl）。它们还分泌组织蛋白酶G，这是一种丝氨酸蛋白酶，与细胞因子释放、受体激活及组织蛋白降解有关。体外实验中，分离出的组织蛋白酶G被HOCl灭活，但未被过氧化氢灭活。我们发现活化的中性粒细胞通过一种需要髓过氧化物酶的途径丧失组织蛋白酶G活性，这表明髓过氧化物酶产生的氧化剂可能在体内调节组织蛋白酶G的活性。对氧化后的组织蛋白酶G进行串联质谱分析发现，位于活性位点的含天冬氨酸108的肽段丢失与酶活性丧失在数量上相关。含天冬氨酸108的催化结构域肽段随着甲硫氨酸110向亚砜的转化从氧化蛋白中丢失。用苯甲基磺酰氟预处理组织蛋白酶G可阻断该肽段的释放，这强烈暗示氧化作用在组织蛋白酶G中引入了蛋白水解切割位点。模型系统研究表明，甲硫氨酸氧化可指导组织蛋白酶G对肽段进行区域特异性蛋白水解。因此，甲硫氨酸110的氧化可能通过至少两种不同机制导致组织蛋白酶G失活。一种机制涉及催化结构域中天冬氨酸相邻的硫醚残基的直接氧化。另一种机制涉及产生易被组织蛋白酶G进行蛋白水解的新位点。这些观察结果增加了中性粒细胞衍生的氧化剂通过使组织蛋白酶G失活来抑制细胞周围蛋白水解的可能性。它们还表明甲硫氨酸氧化可能使组织蛋白酶G易于发生自溶切割。因此，髓过氧化物酶可能在炎症过程中通过丝氨酸蛋白酶调节组织损伤方面发挥了之前未被怀疑的作用。
