Expedited Solid-Phase Synthesis of Fluorescently Labeled and Biotinylated Aminoalkane Diphenyl Phosphonate Affinity Probes for Chymotrypsin- and Elastase-Like Serine Proteases

In this study, we report on a novel, expedited solid-phase approach for the synthesis of biotinylated and fluorescently tagged irreversible affinity based probes for the chymotrypsin and elastase-like serine proteases. The novel solid-phase biotinylation or fluorescent labeling of the aminoalkane diphenyl phosphonate warhead using commercially available Biotin-PEG-NovaTag or EDANS NovaTag resin permits rapid, facile synthesis of these reagents. We demonstrate the kinetic evaluation and utilization of a number of these irreversible inactivators for chymotrypsin-like (chymotrypsin/human cathepsin G) and elastase-like serine proteases. Encouragingly, these compounds display comparable potency against their target proteases as their N-benzyloxycarbonyl (Cbz)-protected parent compounds, from which they were derived, and function as efficient active site-directed inactivators of their target proteases. We subsequently applied the biotinylated reagents for the sensitive detection of protease species via Western blot, showing that the inactivation of the protease was specifically mediated through the active site serine. Furthermore, we also demonstrate the successful detection of serine protease species with the fluorescently labeled derivatives “in-gel”, thus avoiding the need for downstream Western blotting. Finally, we also show the utility of biotinylated and pegylated affinity probes for the isolation/enrichment of serine protease species, via capture with immobilized streptavidin, and their subsequent identification via de novo sequencing. Given their selectivity of action against the serine proteases, we believe that these reagents can be exploited for the direct, rapid, and selective identification of these enzymes from biological milieu containing multiple protease subclasses.

Alpha-1-antitrypsin aerosolized augmentation abrogates neutrophil elastase-induced expression of cathepsin B and matrix metalloprotease 2 in vivo and in vitro

Background: Neutrophil elastase (NE) activity is increased in lung diseases such as a1-antitrypsin (A1AT) deficiency and pneumonia. It has recently been shown to induce expression of cathepsin B and matrix metalloprotease 2 (MMP-2) in vitro and in a mouse model. It is postulated that increased cathepsin B and MMP-2 in acute and chronic lung diseases result from high levels of extracellular NE and that expression of these proteases could be inhibited by A1AT augmentation therapy.

Methods: Cathepsin and MMP activities were assessed in bronchoalveolar lavage (BAL) fluid from patients with A1AT deficiency, pneumonia and control subjects. Macrophages were exposed to BAL fluid rich in free NE from patients with pneumonia following pretreatment with A1AT. MMP-2, cathepsin B, secretory leucoprotease inhibitor (SLPI) and lactoferrin levels were determined in BAL fluid from A1AT-deficient patients before and after aerosolisation of A1AT.

Results: BAL fluid from both patients with pneumonia and those with A1AT deficiency containing free NE had increased cathepsin B and MMP-2 activities compared with BAL fluid from healthy volunteers. The addition of A1AT to BAL fluid from patients with pneumonia greatly reduced NE-induced cathepsin B and MMP-2 expression in macrophages in vitro. A1AT augmentation therapy to A1AT-deficient individuals also reduced cathepsin B and MMP-2 activity in BAL fluid in vivo. Furthermore, A1AT-deficient patients had higher levels of SLPI and lactoferrin after A1AT augmentation therapy.

Conclusion: These findings suggest a novel role for A1AT inhibition of NE-induced upregulation of MMP and cathepsin expression both in vitro and in vivo.

Elafin, an Elastase-specific Inhibitor, Is Cleaved by Its Cognate Enzyme Neutrophil Elastase in Sputum from Individuals with Cystic Fibrosis

Elafin is a neutrophil serine protease inhibitor expressed in lung and displaying anti-inflammatory and anti-bacterial properties. Previous studies demonstrated that some innate host defense molecules of the cystic fibrosis (CF) and chronic obstructive pulmonary disease airways are impaired due to increased proteolytic degradation observed during lung inflammation. In light of these findings, we thus focused on the status of elafin in CF lung. We showed in the present study that elafin is cleaved in sputum from individuals with CF. Pseudomonas aeruginosa-positive CF sputum, which was found to contain lower elafin levels and higher neutrophil elastase (NE) activity compared with P. aeruginosa-negative samples, was particularly effective in cleaving recombinant elafin. NE plays a pivotal role in the process as only NE inhibitors are able to inhibit elafin degradation. Further in vitro studies demonstrated that incubation of recombinant elafin with excess of NE leads to the rapid cleavage of the inhibitor. Two cleavage sites were identified at the N-terminal extremity of elafin (Val-5—Lys-6 and Val-9—Ser-10). Interestingly, purified fragments of the inhibitor (Lys-6—Gln-57 and Ser-10—Gln-57) were shown to still be active for inhibiting NE. However, NE in excess was shown to strongly diminish the ability of elafin to bind lipopolysaccharide (LPS) and its capacity to be immobilized by transglutamination. In conclusion, this study provides evidence that elafin is cleaved by its cognate enzyme NE present at excessive concentration in CF sputum and that P. aeruginosa infection promotes this effect. Such cleavage may have repercussions on the innate immune function of elafin.