Extended Cleavage Specificity of Human Neutrophil Elastase, Human Proteinase 3, and Their Distant Ortholog Clawed Frog PR3—Three Elastases With Similar Primary but Different Extended Specificities and Stability

Abstract

Serine proteases are major granule constituents of several of the human hematopoietic cell lineages. Four proteolytically active such proteases have been identified in human neutrophils: cathepsin G (hCG), N-elastase (hNE), proteinase 3 (hPR-3), and neutrophil serine protease 4 (hNSP-4). Here we present the extended cleavage specificity of two of the most potent and most abundant of these enzymes, hNE and hPR-3. Their extended specificities were determined by phage display and by the analysis of a panel of chromogenic and recombinant substrates. hNE is an elastase with a relatively broad specificity showing a preference for regions containing several aliphatic amino acids. The protease shows self-cleaving activity, which results in the loss of activity during storage even at +4°C. Here we also present the extended cleavage specificity of hPR-3. Compared with hNE, it shows considerably lower proteolytic activity. However, it is very stable, shows no self-cleaving activity and is actually more active in the presence of SDS, possibly by enhancing the accessibility of the target substrate. This enables specific analysis of hPR-3 activity even in the presence of all the other neutrophil enzymes with addition of 1% SDS. Neutrophils are the most abundant white blood cell in humans and one of the key players in our innate immune defense. The neutrophil serine proteases are very important for the function of the neutrophils and therefore also interesting from an evolutionary perspective. In order to study the origin and functional conservation of these neutrophil proteases we have identified and cloned an amphibian ortholog, Xenopus PR-3 (xPR-3). This enzyme was found to have a specificity very similar to hPR-3 but did not show the high stability in the presence of SDS. The presence of an elastase in Xenopus closely related to hPR-3 indicates a relatively early appearance of these enzymes during vertebrate evolution.

Evolution of multifunctionality through a pleiotropic substitution in the innate immune protein S100A9

“…Proteinase K was chosen both because of its low specificity and to mimic other serine proteases that A9 and A8/A9 encounter when released from neutrophils during an inflammatory response. [56][57][58][59]69 Proteolytic decay rates were estimated by fitting a single exponential decay function to the data (Figure 3b Human A8/A9 has been described as extremely resistant to proteases; 62 however, it has not been compared to S100 proteins besides human A8 and A9. To establish a baseline expectation for S100 protein proteolytic resistance, we characterized the proteolytic resistance of a broad set of human S100s against proteinase K. As previously shown, 62 human A9 and A8 alone were rapidly proteolytically degraded, while the human A8/A9 complex exhibited strong resistance (Figure 3c, Figure 3 supplemental figures 1-2).…”

Potent and Broad but not Unselective Cleavage of Cytokines and Chemokines by Human Neutrophil Elastase and Proteinase 3

“…In order to perform all the different functions needed at the area of inflammation, the proteases stored and secreted by the neutrophils have relatively broad specificities both concerning their primary and extended specificities. We have recently performed detailed studies of the extended specificities of three out the four active serine proteases stored in granules of human neutrophils, hCG, hNE and hPR-3 [9,15]. Phage display analysis and validation by a large panel of recombinant protein substrates have shown that hCG is a dual chymase and tryptase with a dominating chymase activity against aromatic amino acids, and with a tryptic activity primarily against lysine that is only 2-3 times lower than the major chymotryptic activity.…”

“…The three neutrophil proteases used for this study were commercial preparations, where the original source of the protein was blood neutrophils (Figure 1). The same batches for all three of these enzymes have been analyzed for purity by SDS PAGE analysis and for activity on chromogenic substrate assay, phage display and a panel of recombinant substrates in a previously published study [15] ( Figure 1). In this study we show that the enzymes are pure and that hPR3 and hNE both are elastases whereas hCG is a dual enzyme with both chymase and tryptase activity, the tryptase specificity with strong preference for Lys over Arg [9,15].…”

“…From the previous studies we have shown that hNE is much more active on a molar basis than both of the other enzymes. Therefore approximately 10 times more hPR3 enzyme was needed to obtain the same cleavage of chromogenic substrates compared to hNE [15]. Here we used 12 times more hPR3 during the cleavage reactions, which resulted in a corresponding band of approximately 28-29 kDa on the SDS-PAGE gels visualizing the reactions (Figures 2 and 3).…”

Extended Cleavage Specificities of Two Mast Cell Chymase-Related Proteases and One Granzyme B-Like Protease from the Platypus, a Monotreme

“…As neither chromogenic substrates nor phage display resulted in any information concerning the extended specificities of these three platypus enzymes, we instead tried a third alternative; to use a new type of recombinant substrate. A panel of such substrates has previously been designed and produced for the verification of the extended cleavage specificity following phage display analysis for a number of previously analysed mammalian hematopoietic and coagulation serine proteases [17,[21][22][23][24][25][26][27][28][29][30].…”