Oxidative damage to fibronectin. I. The effects of the neutrophil myeloperoxidase system and HOCl.
Exposure of purified human plasma fibronectin to the myeloperoxidase-H2O2-Cl- system of neutrophils or to reagent HOCl resulted in extensive changes to its primary and tertiary structures. When 1.14 microM fibronectin was exposed to 50-400 microM HOCl or 50-400 microM H2O2 plus myeloperoxidase and Cl-, there was progressive loss of tryptophan fluorescence and cysteines, and an increase in bityrosine fluorescence and carbonyl content. Analysis by SDS-PAGE indicated extensive crosslinking of the fibronectin, the crosslinks being stable under reducing conditions. The coincident increase of bityrosine fluorescence suggests that crosslinking may be largely due to intermolecular bityrosines rather than disulfides. All changes observed with the myeloperoxidase system were inhibited by azide or methionine, and were dependent upon the presence of chloride, indicating that they are mediated by HOCl. The reaction between HOCl and fibronectin resulted in the formation of long-lived chloramines. Exposure to increasing amounts of oxidant resulted in an increase in the susceptibility of fibronectin to proteolytic attack by purified neutrophil elastase. Analysis by SDS-PAGE showed a different fragmentation pattern for oxidant-treated fibronectin compared with the native protein. This suggests that regions of the molecule which were previously resistant to proteolysis were denatured to create susceptible sites for elastase. This demonstration that fibronectin is extensively modified by the myeloperoxidase system has implications for the mechanism of tissue injury by neutrophils in inflammation, since a loss of functional fibronectin would result in cell detachment and a distortion of normal tissue organization.
Vissers
M C
MC
Department of Pathology, Christchurch School of Medicine, Christchurch Hospital, New Zealand.
Winterbourn
C C
CC
D001386:Azides / Q000494:pharmacology; D002700:Chloramines / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism*; D005453:Fluorescence; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D006997:Hypochlorous Acid / Q000494:pharmacology*; D008715:Methionine / Q000494:pharmacology; D008970:Molecular Weight; D009504:Neutrophils / Q000201:enzymology*; D010196:Pancreatic Elastase / Q000494:pharmacology; D009195:Peroxidase / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D011489:Protein Denaturation; D014364:Tryptophan / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D001386:Azides; D002700:Chloramines; D005353:Fibronectins; D014443:Tyrosine; D006997:Hypochlorous Acid; D014364:Tryptophan; D008715:Methionine; D006861:Hydrogen Peroxide; C007543:dityrosine; D009195:Peroxidase; D010196:Pancreatic Elastase; C030816:chloramine