Methionine sulfoxide and proteolytic cleavage contribute to the inactivation of cathepsin G by hypochlorous acid: an oxidative mechanism for regulation of serine proteinases by myeloperoxidase.
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.
Shao
Baohai
B
Department of Medicine, University of Washington, Seattle, WA 98195, USA.
Belaaouaj
Abderrazzaq
A
Verlinde
Christophe L M J
CL
Fu
Xiaoyun
X
Heinecke
Jay W
JW
D000818:Animals; D020134:Catalytic Domain; D056649:Cathepsin G; D002403:Cathepsins / Q000737:chemistry* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006997:Hypochlorous Acid / Q000737:chemistry*; D007249:Inflammation; D013058:Mass Spectrometry; D008715:Methionine / Q000031:analogs & derivatives* / Q000737:chemistry; D051379:Mice; D008956:Models, Chemical; D008958:Models, Molecular; D009504:Neutrophils / Q000378:metabolism; D016877:Oxidants / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D009195:Peroxidase / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013997:Time Factors
D016877:Oxidants; D010455:Peptides; D006997:Hypochlorous Acid; D008715:Methionine; D006861:Hydrogen Peroxide; D009195:Peroxidase; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; C534096:Ctsg protein, mouse; D010100:Oxygen; C013111:methionine sulfoxide