Cloning and functional expression of the murine homologue of proteinase 3: implications for the design of murine models of vasculitis

AbstractAnti-neutrophil cytoplasmic autoantibodies recognizing conformational epitopes (c-ANCA) of proteinase 3 (PR3) from azurophil granules are a diagnostic hallmark in Wegener's granulomatosis (WG). Because a functional PR3 homologue has not been identified in rodents, it is difficult to assess immunopathological responses in rats or mice immunized with patients' derived c-ANCA or human PR3. Here we report the full length cDNA cloning and functional expression of murine PR3 in HMC-1 cells. Recombinant murine PR3 shows highly similar substrate specificities towards synthetic peptides and is inhibited by human α1-proteinase inhibitor like human PR3. However, neither human c-ANCA, rabbit sera nor mouse monoclonal antibodies to human PR3 recognize the murine homologue. Consequently, it is unlikely that disease observed in mice after immunization with c-ANCA or human PR3 is caused by pathogenic antibodies directed against mouse PR3. Recombinant human-mouse chimaeric variants will be a valuable new tool to localize the disease-specific immunodominant epitopes in human PR3

Tailor-made inflammation: how neutrophil serine proteases modulate the inflammatory response

Neutrophil granulocytes are important mediators of innate immunity, but also participate in the pathogenesis of (auto)inflammatory diseases. Neutrophils express a specific set of proteolytic enzymes, the neutrophil serine proteases (NSPs), which are stored in cytoplasmic granules and can be secreted into the extra- and pericellular space upon cellular activation. These NSPs, namely cathepsin G (CG), neutrophil elastase (NE), and proteinase 3 (PR3), have early been implicated in bacterial defense. However, NSPs also regulate the inflammatory response by specifically altering the function of cytokines and chemokines. For instance, PR3 and NE both inactivate the anti-inflammatory mediator progranulin, which may play a role in chronic inflammation. Here, we provide a concise update on NSPs as modulators of inflammation and discuss the biological and pathological significance of this novel function of NSPs. Mounting evidence support an important proinflammatory function for PR3, which may have been underestimated in the past

Consequences of cathepsin C inactivation for membrane exposure of proteinase 3, the target antigen in autoimmune vasculitis

Membrane-bound proteinase 3 (PR3(m)) is the main target antigen of anti-neutrophil cytoplasmic autoantibodies (ANCA) in granulomatosis with polyangiitis, a systemic small-vessel vasculitis. Binding of ANCA to PR3(m) triggers neutrophil activation with the secretion of enzymatically active PR3 and related neutrophil serine proteases, thereby contributing to vascular damage. PR3 and related proteases are activated from pro-forms by the lysosomal cysteine protease cathepsin C (CatC) during neutrophil maturation. We hypothesized that pharmacological inhibition of CatC provides an effective measure to reduce PR3(m) and therefore has implications as a novel therapeutic approach in granulomatosis with polyangiitis. We first studied neutrophilic PR3 from 24 patients with Papillon-Lefevre syndrome (PLS), a genetic form of CatC deficiency. PLS neutrophil lysates showed a largely reduced but still detectable (0.5-4%) PR3 activity when compared with healthy control cells. Despite extremely low levels of cellular PR3, the amount of constitutive PR3(m) expressed on the surface of quiescent neutrophils and the typical bimodal membrane distribution pattern were similar to what was observed in healthy neutrophils. However, following cell activation, there was no significant increase in the total amount of PR3(m) on PLS neutrophils, whereas the total amount of PR3(m) on healthy neutrophils was significantly increased. We then explored the effect of pharmacological CatC inhibition on PR3 stability in normal neutrophils using a potent cell-permeable CatC inhibitor and a CD34(+) hematopoietic stem cell model. Human CD34(+) hematopoietic stem cells were treated with the inhibitor during neutrophil differentiation over 10 days. We observed strong reductions in PR3(m), cellular PR3 protein, and proteolytic PR3 activity, whereas neutrophil differentiation was not compromised

Common coding variant in SERPINA1 increases the risk for large artery stroke

Large artery atherosclerotic stroke (LAS) shows substantial heritability not explained by previous genome-wide association studies. Here, we explore the role of coding variation in LAS by analyzing variants on the HumanExome BeadChip in a total of 3,127 cases and 9,778 controls from Europe, Australia, and South Asia. We report on a nonsynonymous single-nucleotide variant in serpin family A member 1 (SERPINA1) encoding alpha-1 antitrypsin [AAT; p.V213A; P = 5.99E-9, odds ratio (OR) = 1.22] and confirm histone deacetylase 9 (HDAC9) as a major risk gene for LAS with an association in the 3?-UTR (rs2023938; P = 7.76E-7, OR = 1.28). Using quantitative microscale thermophoresis, we show that M1 (A213) exhibits an almost twofold lower dissociation constant with its primary target human neutrophil elastase (NE) in lipoprotein-containing plasma, but not in lipid-free plasma. Hydrogen/deuterium exchange combined with mass spectrometry further revealed a significant difference in the global flexibility of the two variants. The observed stronger interaction with lipoproteins in plasma and reduced global flexibility of the Val-213 variant most likely improve its local availability and reduce the extent of proteolytic inactivation by other proteases in atherosclerotic plaques. Our results indicate that the interplay between AAT, NE, and lipoprotein particles is modulated by the gate region around position 213 in AAT, far away from the unaltered reactive center loop (357-360). Collectively, our findings point to a functionally relevant balance between lipoproteins, proteases, and AAT in atherosclerosis

Therapeutic targeting of cathepsin C::from pathophysiology to treatment

Cathepsin C (CatC) is a highly conserved tetrameric lysosomal cysteine dipeptidyl aminopeptidase. The best characterized physiological function of CatC is the activation of pro-inflammatory granule-associated serine proteases. These proteases are synthesized as inactive zymogens containing an N-terminal pro-dipeptide, which maintains the zymogen in its inactive conformation and prevents premature activation, which is potentially toxic to the cell. The activation of serine protease zymogens occurs through cleavage of the N-terminal dipeptide by CatC during cell maturation in the bone marrow. In vivo data suggest that pharmacological inhibition of pro-inflammatory serine proteases would suppress or attenuate deleterious effects of inflammatory/auto-immune disorders mediated by these proteases. The pathological deficiency in CatC is associated with Papillon-Lefèvre syndrome. The patients however do not present marked immunodeficiency despite the absence of active serine proteases in immune defense cells. Hence, the transitory pharmacological blockade of CatC activity in the precursor cells of the bone marrow may represent an attractive therapeutic strategy to regulate activity of serine proteases in inflammatory and immunologic conditions. A variety of CatC inhibitors have been developed both by pharmaceutical companies and academic investigators, some of which are currently being employed and evaluated in preclinical/clinical trials

Tailor-made inflammation: how neutrophil serine proteases modulate the inflammatory response.

Neutrophil granulocytes are important mediators of innate immunity, but also participate in the pathogenesis of (auto)inflammatory diseases. Neutrophils express a specific set of proteolytic enzymes, the neutrophil serine proteases (NSPs), which are stored in cytoplasmic granules and can be secreted into the extra- and pericellular space upon cellular activation. These NSPs, namely cathepsin G (CG), neutrophil elastase (NE), and proteinase 3 (PR3), have early been implicated in bacterial defense. However, NSPs also regulate the inflammatory response by specifically altering the function of cytokines and chemokines. For instance, PR3 and NE both inactivate the anti-inflammatory mediator progranulin, which may play a role in chronic inflammation. Here, we provide a concise update on NSPs as modulators of inflammation and discuss the biological and pathological significance of this novel function of NSPs. Mounting evidence support an important proinflammatory function for PR3, which may have been underestimated in the past

NSP4, a novel elastase-related protease with unique specificity is produced by human neutrophils

Neutrophil serine proteases (NSPs) in cytoplasmic granules of neutrophils are regarded as important antimicrobial defence weapons after endocytosis and exposure of pathogens to the content of primary granules. Despite intensive studies on NSPs during the last three decades, only three active serine proteases, neutrophil elastase (NE), cathepsin G (CG) and proteinase 3 (PR3) expressed and stored by these short-lived cells were encountered. Here we report on the discovery of a fourth serine protease (NSP4) with unique elastase activity. Although amino acid residues are only 40% identical with PR3 and NE, many features like propeptide processing by cathepsin C, storage and release as an active enzyme and cleavage of Ala-Pro-Nva-thiobenzylesters are shared with them. We prepared and identified specific monoclonal antibodies to NSP4 in rats, excluded cross-reactivity to all five granzymes, NE, CG, PR3, azurocidin and stained different human tissues and blood leukocyte populations. To our surprise only granulocyte precursors and neutrophil populations from peripheral blood were positive. Upon neutrophil activation, NSP4 was released into the supernatant, but the content of NSP4 in neutrophil lysates was about 20 fold lower than that of CG using purified CG and recombinant NSP4 as protein standards in Western blotting. NSP4 was active towards some tripeptide-thiobenzylesters cleaving after aliphatic hydrophobic residues. It was moderately inhibited by the natural serine protease inhibitors a1PI and elafin indicating a distinct extended peptide specificity of this novel NSP. Functional specialization and preferred natural substrates of NSP4 remain to be determined in order to understand the biological interplay of all four NSPs during neutrophil responses