Compared Action of Neutrophil Proteinase 3 and Elastase on Model Substrates

Abstract

Neutrophil proteinase 3 (Pr3) and elastase (NE) may cause lung tissue destruction in emphysema and cystic fibrosis. These serine proteinases have similar P 1 specificities. We have compared their catalytic activity using acyl-tetrapeptide-p-nitroanilides, which occupy the S 5 -S 1 subsites of their substrate binding site, and intramolecularly quenched fluorogenic heptapeptides, which bind at S 5 -S 4 . Most p-nitroanilide substrates are turned over slowly by Pr3 as compared with NE. These differences disappear with the fluorogenic heptapeptides, some of which are hydrolyzed even faster by Pr3 than by NE. Elongation of substrates strongly increases the catalytic efficiency of Pr3, whereas it has little effect on NE catalysis. These different sensitivities to S-P interactions show that Pr3 and NE are not interchangeable enzymes despite their similar P 1 specificity.The azurophilic granules of polymorphonuclear neutrophils contain three serine proteinases: elastase (NE), 1 cathepsin G, and proteinase 3 (Pr3), which participate in lysosomal bacterial digestion and neutrophil migration through the extracellular matrix at sites of inflammation. These enzymes are ϳ30-kDa glycoproteins, which belong to the chymotrypsin family of serine proteinases. Pr3 is the most recently discovered, the most difficult to isolate, and hence the less well studied proteinase of the three. It is identical to three independently discovered proteins: (i) myeloblastin, which regulates the growth and differentiation of leukemic cells; (ii) p29b, which has microbicidal activity; and (iii) the target antigen of antineutrophil cytoplasmic autoantibodies detected in patients with Wegner's granulomatosis (2). Pr3 cleaves extracellular matrix proteins including elastin, type IV collagen, fibronectin, laminin, and vitronectin (3, 4). It is able to produce lung emphysema in hamsters (3), and thus, in concert with NE and cathepsin G, it may be responsible for lung tissue destruction occurring in emphysema and cystic fibrosis.Although many model substrates have been used to map the active site of NE (5-8), literature on the substrate specificity and the catalytic activity of Pr3 is poorly documented. Fuginaga et al. (9) have shown that the two enzymes have similar substrate binding sites. This explains why both proteinases cleave the oxidized insulin A and B chains at peptide bonds involving small aliphatic amino acid residues (4). The specificity of Pr3 for non-bulky residues has been confirmed with a limited number of model substrates (4, 10 -12). As a rule, these substrates were turned over at a much lower rate by Pr3 than by NE, an unexplained observation. We have undertaken the present work to understand this puzzling finding. EXPERIMENTAL PROCEDURESMaterials-NE was isolated, and active site was titrated as described previously (13). Pr3 came from Athens Research Technology (Athens, GA) and was titrated with ␣ 1 -proteinase inhibitor (4). This enzyme preparation was electrophoretically pure. The presence of NE was tested by reacting 0.14 M Pr3 wi...

Influence of Charge Distribution at the Active Site Surface on the Substrate Specificity of Human Neutrophil Protease 3 and Elastase

“…Experiments must therefore be carried out in the presence of reducing agents that may interfere or modulate the activity of other components in the reaction mixture. We have avoided this problem by using the unnatural Cys homologue norvaline at P1, which gives similar or even better results than a P1-Cys, but Val residues can also be used (9,25). Nevertheless, some of the protein substrates of Pr3 are cleaved at Cys residues (19).…”

“…Developing molecular tools that specifically interact with Pr3 may therefore help elucidate its biological function. However, no specific Pr3 substrate was available until recently due to the importance of the SЈ subsites for substrate binding (8,9,29). SЈ subsites cannot be investigated using conventional chromogenic or fluorogenic substrates but require FRET peptides whose sequences extend on both sides of the cleavage site.…”

“…SЈ subsites cannot be investigated using conventional chromogenic or fluorogenic substrates but require FRET peptides whose sequences extend on both sides of the cleavage site. We and others prepared such substrates for neutrophil serine proteases and demonstrated the importance of the S2Ј subsite for discriminating between the activities of Pr3 and HNE (8,9,29). Inspection of the cell surface potential of Pr3 and HNE active sites based on the Pr3 three-dimensional structure and molecular modeling studies revealed that they differ not only at S2Ј but also at S4, S2, S1Ј, and S3 subsites (25), but it is surprising that Pr3 and HNE, whose primary sequences are very similar, differ mainly by their charged surface residues.…”

“…This is why there was no substrate that discriminated between the two proteases until fluorescence resonance energy transfer (FRET) peptides became available. These can be used to study the specificity on both sides of the cleavage site (8,9). However, kinetic and structural studies have shown that the substrate binding site in Pr3 is more polar, and some subsites are more restrictive than those in HNE (5,10).…”

Inhibition of the Activation of Multiple Serine Proteases with a Cathepsin C Inhibitor Requires Sustained Exposure to Prevent Pro-enzyme Processing

“…Debris was removed by centrifugation at 15,000 ϫ g for 10 min, and supernatants were retained. The extracts were mixed with assay buffer (100 mM Tris-HCl, pH 7.5, and 1 M NaCl) supplemented with a peptide substrate specific for the serine protease assessed: for NE, 400 M methoxysuccinyl-Ala-Ala-Pro-Val-AMC; for CG, 1 mM Suc-Ala-Ala-Pro-Phe-p-nitroanilide; and for Pr-3, 10 M L2p-Tyr-Asp-Ala-Lys-Gly-Asp-Dpa-NH 2 (47). Cleavage of the substrate was monitored spectrophotometrically using Spectramax plate readers (Molecular Devices), and kinetic rates were calculated from the linear portion of the reaction.…”