Oxidation-resistant variants of recombinant anti-leucoprotease are better inhibitors of human-neutrophil-elastase-induced emphysema in hamsters than natural recombinant antileucoprotease

Abstract

1. Antileucoprotease, being sensitive to oxidative inactivation, can be produced by recombinant techniques. Via site-directed mutagenesis, two mutants of recombinant antileucoprotease were produced in which one or more of the oxidation-sensitive methionine residues were replaced by leucine: in rALP242, methionine-73 was replaced by leucine, and in rALP231, leucine was substituted for four methionine residues. In vitro, native antileucoprotease and the recombinant antileucoprotease preparations have similar inhibitory characteristics towards human neutrophil elastase. We hypothesized that replacement of methionine residues in the antileucoprotease molecule would result in a reduced oxidation sensitivity of the mutants. 2. After incubation of recombinant antileucoprotease and its mutants with increasing dosages of cis-platinum(II)diammine dichloride, we observed that native antileucoprotease and recombinant antileucoprotease were inactivated by this reagent to the same extent. Compared with this, rALP242 was less inactivated, whereas the inhibitory capacity of rALP231 was not influenced by cis-platinum(II)diammine dichloride at all. 3. After incubation of recombinant antileucoprotease, rALP242 and rALP231 with triggered polymorphonuclear leucocytes, which are thought to produce an excess of oxidants, we measured residual inhibitory activities towards human neutrophil elastase of 10%, 55% and 87%, respectively. 4. In vivo, the inhibitory effects of intratracheally administered rALP242 and rALP231 towards human-neutrophil-elastase-induced emphysema were significantly greater than that of recombinant antileucoprotease. There were no significant differences between the mutants.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative loss of activity of recombinant secretory leukoprotease inhibitor and alpha 1-protease inhibitor caused by different forms of oxidative stress

“…Incubating this mutant with cisplatinum (II) diaminechloride as well as triggered PMNs, resulted in reduced loss of activity compared with unmodified rSLPI. These results were confirmed in in vivo experiments: in animal emphysema models with intratracheal instillation of NE and lipopolysaccharide, the protective effect of the oxidant-resistant rSLPI mutant was superior to that of unmodified rSLPI [42,43]. These data suggest that methionine residues are critical for the anti-NE activity of both α 1 -PI and rSLPI.…”

Antibacterial activity of antileukoprotease

“…Proteins. Recombinant ALP was produced in Escherichia coli and purified as previously described (29). The separated NH 2 -and COOH-terminal domains were obtained by partial acidic hydrolysis.…”

Oxidized mucus proteinase inhibitor: a fairly potent neutrophil elastase inhibitor

“…Although there is a methionine residue at the P'u position of plasminogen activated inhibitor I, inactivation is not due to methionine oxidation, but to an oxidant-induced conformational change of the protein, as demonstrated with a mutant inhibitor in which valines replaced methionines (Strandberg et al, 1991). In this context it is worthwhile noticing that mutant MPI, in which the four methionines are replaced by leucines, no longer undergoes oxidative impairment of anti-elastase activity (Rudolphus et al, 1991a). This indicates that the effect of NCS is not an indirect one, as in the case of the plasminogen activator inhibitor, but results directly from the oxidation of the P'1 residue of the inhibitor.…”