The neutrophil serine proteases (NSPs) elastase, proteinase 3 and cathepsin G are multifunctional proteases involved in pathogen destruction and the modulation of inflammatory processes. A fraction of secreted NSPs remains bound to the external plasma membrane, where they remain enzymatically active. This protocol describes the spectrofluorometric measurement of NSP activities on neutrophil surfaces using highly sensitive Abz-peptidyl-EDDnp fluorescence resonance energy transfer (FRET) substrates that fully discriminate between the three human NSPs. We describe FRET substrate synthesis, neutrophil purification and handling, and kinetic experiments on quiescent and activated cells. These are used to measure subnanomolar concentrations of membrane-bound or free NSPs in low-binding microplates and to quantify the activities of individual proteases in biological fluids like expectorations and bronchoalveolar lavages. The whole procedure, including neutrophil purification and kinetic measurements, can be done in 4-5 h and should not be longer because of the lifetime of neutrophils. Using this protocol will help identify the contributions of individual NSPs to the development of inflammatory diseases and may reveal these proteases to be targets for therapeutic inhibitors.

The biological functions of human neutrophil protease 3 (Pr3) differ from those of neutrophil elastase despite their close structural and functional resemblance. Although both proteases are strongly cationic, their sequences differ mainly in the distribution of charged residues. We have used these differences in electrostatic surface potential in the vicinity of their active site to produce fluorescence resonance energy transfer (FRET) peptide substrates for investigating individual Pr3 subsites. The specificities of subsites S5 to S3 were investigated both kinetically and by molecular dynamic simulations. Subsites S2, S1, and S2 were the main definers of Pr3 specificity. Combinations of results for each subsite were used to deduce a consensus sequence that was complementary to the extended Pr3 active site and was not recognized by elastase. Similar sequences were identified in natural protein substrates such as NFB and p21 that are specifically cleaved by Pr3. FRET peptides derived from these natural sequences were specifically hydrolyzed by Pr3 with specificity constants k cat /K m in the 10 6 M ؊1 s ؊1 range. The consensus Pr3 sequence may also be used to predict cleavage sites within putative protein targets like the proform of interleukin-18, or to develop specific Pr3 peptide-derived inhibitors, because none is available for further studies on the physiopathological function of this protease.Protease 3 (Pr3) 3 was initially described as an elastin-degrading protease whose structural and functional properties are similar to those of human neutrophil elastase (HNE) (1, 2). Pr3 is stored as an active enzyme within the primary granules of human neutrophils, together with HNE and cathepsin G, and is released from activated cells as a free or membrane-bound protease (3, 4). Its three-dimensional structure is very similar to that of HNE, with which its sequence is 57% identical (5). Pr3 and HNE also have extended interaction sites that greatly influence substrate binding and specificity (5). The active sites of the two proteases are also very similar, and both preferentially accommodate small aliphatic residues in their S1 subsite 4 (6, 7). 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). This partly explains why Pr3, but not HNE, is not inhibited by the low molecular weight inhibitor SLPI present in the upper airways, even though both proteases are inhibited by the ␣1-protease inhibitor (␣1-Pi) in lung secretions (6,11).Although the primary function of Pr3 and HNE is commonly thought to be the intralysosomal degradation of phagocytized microorganisms, both also act extracellularly to break down matrix proteins (6, 12, 13), release cytokines from their ...

It is widely accepted that neutrophil serine proteases (NSPs) play a critical role in neutrophil-associated lung inflammatory and tissue-destructive diseases. To investigate NSP pathogenic role(s), various mouse experimental models have been developed that mimic acutely or chronically injured human lungs. We and others are using mouse exposure to cigarette smoke as a model for chronic obstructive pulmonary disease with or without exacerbation. However, the relative contribution of NSPs to lung disease processes as well as their underlying mechanisms remains still poorly understood. And the lack of purified mouse NSPs and their specific substrates have hampered advances in these studies. In this work, we compared mouse and human NSPs and generated three-dimensional models of murine NSPs based on three-dimensional structures of their human homologs. Analyses of these models provided compelling evidence that peptide substrate specificities of human and mouse NSPs are different despite their conserved cleft and close structural resemblance. These studies allowed us to synthesize for the first time novel sensitive fluorescence resonance energy transfer substrates for individual mouse NSPs. Our findings and the newly identified substrates should better our understanding about the role of NSPs in the pathogenesis of cigarette-associated chronic obstructive pulmonary disease as well as other neutrophils-associated inflammatory diseases.Neutrophil serine proteases (NSPs), 3 neutrophil elastase (NE), cathepsin G (CG), and proteinase 3 (Pr3), are mainly stored in neutrophil primary granules in readily active forms. NSPs are structurally related and share the conserved chargerelay triad, His 57 -Asp , where Ser is the active residue (chymotrypsinogen numbering) (1). NSPs contribute to neutrophil oxygen-independent system-mediated protection of the host against invading pathogens (2). Indeed, NSPs serve a physiological role for killing of microbes (3). Activated neutrophils are also known to release NSPs in the setting of inflammation. In vitro, NSPs are capable of cleaving a panoply of substrates. These include extracellular matrix proteins, pro-inflammatory mediators, coagulation factors, and immunoglobulins (4). NE degrades pro-inflammatory mediators such as tumor necrosis factor-␣ and interleukin-1␤, hence could alter the inflammatory response. The enzyme is capable of inducing the secretion of granulocyte macrophage-colony stimulating factor and interleukin-8, which could amplify the inflammation. Consequently, the release of these potent proteinases in diseased situations could create a proteolytic environment where degradation of different host molecules might occur and result in inappropriate inflammatory response. Because of their large substrate repertoire, NSPs have been implicated in the pathogenesis of various inflammatory and tissue-destructive diseases, including acute lung injury, cystic fibrosis, and COPD (5-7).COPD is recognized as a major health problem whose worldwide incidence is increasing dramatically...