Glycosaminoglycans regulate elastase inhibition by oxidized secretory leukoprotease inhibitor.

Abstract

Secretory leukoprotease inhibitor (SLPI) is one of the major physiological inhibitors protecting respiratory epithelium from attack by excess human leukocyte elastase (HLE), a serine protease released by neutrophils upon activation in response to inflammatory stimuli. Reaction with N-chlorotaurine, a major long-lived oxidant generated by activated neutrophils, oxidized all four methionine residues, but no other amino acids, in SLPI, resulting in substantial diminution of its elastase inhibitory activity. Oxidation of the P1' residue, Met73, accounted for most of the diminution in activity since a site-directed mutant of SLPI with leucine at the P1' position retained much higher residual activity after reaction with N-chlorotaurine. The diminished activity of oxidized SLPI could be almost completely restored when an iduronate-containing glycosaminoglycan, such as heparin, heparan sulfate, or dermatan sulfate, was added to the reaction medium. Addition of a sulfated glucuronate-containing glycosaminoglycan, chondroitin 4- or 6-sulfate, to the medium resulted in smaller but significant restoration of the lost activity, whereas the effects of hyaluronic acid and keratan sulfate were negligible. Kinetic analysis revealed that glycosaminoglycans greatly accelerated the association of oxidized SLPI and HLE, whereas iduronate-containing glycosaminoglycans also stabilized the enzyme-inhibitor complex formed. Based on these findings, we suggest that oxidized SLPI is a functionally active form of the inhibitor but that expression of its elastase inhibitory activity is regulated by sulfated uronate-containing glycosaminoglycans. Because its methionine residues have already been oxidized, this form of SLPI is resistant to the oxidant species that selectively attacks methionine residues in proteins. These findings indicate that SLPI may play a previously unexpected role in elastase inhibitory function in the lungs when significant inflammation is present.