Inhibition of neutrophil cathepsin G by oxidized mucus proteinase inhibitor. Effect of heparin.
Oxidation of mucus proteinase inhibitor (MPI) transforms Met73, the P'1 residue of its active center into methionine sulfoxide and lowers its affinity for neutrophil elastase [Boudier, C., and Bieth, J. G. (1994) Biochem. J. 303, 61-68]. Here, we show that the oxidized inhibitor has also a decreased affinity for neutrophil cathepsin G and pancreatic chymotrypsin. The Ki of the oxidized MPI-cathepsin G complex (1.2 microM) is probably too high to be compatible with significant inhibition of cathepsin G in inflammatory lung secretions. Stopped-flow kinetics shows that, within the inhibitor concentration range used, the mechanism of inhibition of cathepsin G and chymotrypsin by oxidized MPI is consistent with a one-step reaction, [equation in text] whereas the inhibition of elastase takes place in two steps, [equation in text]. Heparin, which accelerates the inhibition of the three proteinases by native MPI, also favors their interaction with oxidized MPI. Flow calorimetry shows that heparin binds oxidized MPI with Kd, Delta H degrees, and Delta S degrees values close to those reported for native MPI. In the presence of heparin, oxidized MPI inhibits cathepsin G via a two-step reaction characterized by Ki = 0.22 microM, k2 = 0.1 s-1, k-2 = 0.023 s-1, and Ki = 42 nM. Under these conditions, in vivo inhibition of cathepsin G is again possible. Heparin also improves the inhibition of chymotrypsin and elastase by oxidized MPI by increasing their kass or k2/Ki and decreasing their Ki. Our data suggest that oxidation of MPI during chronic bronchitis may lead to cathepsin G-mediated lung tissue degradation and that heparin may be a useful adjuvant of MPI-based therapy of acute lung inflammation in cystic fibrosis.
Boudier
C
C
Laboratoire d'Enzymologie, INSERM U 392, Université Louis Pasteur de Strasbourg, France.
Cadène
M
M
Bieth
J G
JG
D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D056649:Cathepsin G; D002403:Cathepsins / Q000037:antagonists & inhibitors*; D002417:Cattle; D002918:Chymotrypsin / Q000037:antagonists & inhibitors; D006493:Heparin / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D010084:Oxidation-Reduction; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins / Q000378:metabolism / Q000494:pharmacology*; D012697:Serine Endopeptidases; D015842:Serine Proteinase Inhibitors / Q000378:metabolism / Q000494:pharmacology
D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins; D015842:Serine Proteinase Inhibitors; D006493:Heparin; D002403:Cathepsins; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C534094:CTSG protein, human; D056649:Cathepsin G; D010196:Pancreatic Elastase