Abstract
Human neutrophil elastase (HNE) is a uniquely destructive serine protease with the ability to unleash a wave of proteolytic activity by destroying the inhibitors of other proteases. Although this phenomenon forms an important part of the innate immune response to invading pathogens, it is responsible for the collateral host tissue damage observed in chronic conditions such as chronic obstructive pulmonary disease (COPD), and in more acute disorders such as the lung injuries associated with COVID-19 infection. Previously, a combinatorially selected activity-based probe revealed an unexpected substrate preference for oxidised methionine, which suggests a link to oxidative pathogen clearance by neutrophils. Here we use oxidised model substrates and inhibitors to confirm this observation and to show that neutrophil elastase is specifically selective for the di-oxygenated methionine sulfone rather than the mono-oxygenated methionine sulfoxide. We also posit a critical role for ordered solvent in the mechanism of HNE discrimination between the two oxidised forms methionine residue. Preference for the sulfone form of oxidised methionine is especially significant. While both host and pathogens have the ability to reduce methionine sulfoxide back to methionine, a biological pathway to reduce methionine sulfone is not known. Taken together, these data suggest that the oxidative activity of neutrophils may create rapidly cleaved elastase “super substrates” that directly damage tissue, while initiating a cycle of neutrophil oxidation that increases elastase tissue damage and further neutrophil recruitment.
Highlights
Neutrophils of the human innate immune system use an armamentarium of chemical and enzymatic weapons to defend against pathogen invasion
A focused sparse matrix library of tetrapeptides terminating in a para-nitroanilide reporter group was assembled to probe the substrate selectivity of Human Neutrophil Elastase (HNE)
These data confirm a strong selectivity for methionine sulfone residues at position P3, as peptide substrates that included a methionine sulfone residue at position P3 exhibited 19 of the 20 highest observed rates
Summary
Neutrophils of the human innate immune system use an armamentarium of chemical and enzymatic weapons to defend against pathogen invasion. The potency of A1AT inhibition is entirely abrogated under oxidative conditions, such as those induced by a neutrophil response to infection, due to the oxidation of a methionine residue in the P1 position (Schechter–Berger nomenclature [13]) of its protease binding surface [14,15]. Oxidation of inhibitor methionine residues may be induced by oxidative stressors, such as cigarette smoke [18] or the respiratory burst of neutrophils [19], potentiating runaway proteolysis. Since these endogenous inhibitors bind to HNE in a substrate-like manner, the effects of methionine residue oxidation within peptide substrates might be expected to be similar to the inhibitors. The oxidation of P3 methionine residues in peptide substrates appears to enhance the HNE catalysed cleavage rates of those substrates [20] and suggests a strong positional dependence
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