Cockroach allergen serine proteinases: Isolation, sequencing and signalling via proteinase‐activated receptor‐2

Abstract

SUMMARY BACKGROUND Allergy to the German cockroach (Blattella germanica) is a significant asthma risk factor for inner-city communities. Cockroach, like other allergens, contains trypsin-like enzyme activity that contributes to allergenicity and airway inflammation by activating proteinase-activated receptors (PARs). To date, the enzymes responsible for the proteolytic activity of German cockroach allergen have not been characterized. OBJECTIVES We aimed to identify, isolate and characterize the trypsin-like proteinases in German cockroach allergen extracts used for clinical skin tests. For each enzyme, we sought to determine (1) its substrate and inhibitor enzyme kinetics (Km and IC50); (2) its amino acid sequence and (3) its ability to activate calcium signaling and/or ERK1/2 phosphorylation via PAR2. METHODS Using a trypsin-specific activity-based probe, we detected three distinct enzymes that were isolated using ion-exchange chromatography. Each enzyme was sequenced by mass spectometery (deconvoluted with an expressed sequence tag library), evaluated kinetically for its substrate/inhibitor profile and assessed for its ability to activate PAR2 signaling. FINDINGS Each of the three serine proteinase-activity-based probe-labelled enzymes isolated were biochemically distinct, with different enzyme kinetic profiles and primary amino acid sequences. The three enzymes showed a 57 to 71% sequence identity with a proteinase previously cloned from the American cockroach (Per a 10). Each enzyme was found to activate both Ca++ and MAPK signaling via PAR2. CONCLUSIONS AND RELEVANCE We have identified three different serine proteinases from the German cockroach that may, via PAR2 activation, play different roles for allergen-sensitization in vivo and may represent attractive therapeutic targets for asthma.

Protease-activated receptor-2 signaling through β-arrestin-2 mediates Alternaria alkaline serine protease-induced airway inflammation

“…P Յ 0.05, *significant differences between bracketed experiments; #significant differences from saline control. mite (59,63), and cockroach (42,50,60). The protease described here differs considerably from the recently described vacuolar serine protease from Alternaria, Alt a 15 (21).…”

“…The serine protease activity-based probe (ABP) Biotin-Pro-Lys-diphenylphosphonate was synthesized and graciously provided for our studies by Dr. Brendan Gilmore, Queens University School of Pharmacy, Belfast, UK. Covalent labeling of enzymes in the Alternaria filtrates and crude cellular antigen, as well as in chromatographic column fractions isolated from these sources, was achieved essentially as described previously (28,50), with minor modifications. The Alternaria samples were diluted to a final concentration of 1.5 U/ml and incubated with 100 M of the ABP in a 10-l reaction volume consisting of: 50 mM Tris•HCl, pH 8, 0.1% NP40, and 1.5 mM CaCl2 for 2 h at room temperature to biotinylate the enzymes.…”

Functional Proteomic Profiling of Secreted Serine Proteases in Health and Inflammatory Bowel Disease

“…Obviously, detection of active proteases is dependent on their affinity towards the ABP that is used. We have previously used this approach successfully to identify active serine proteases upregulated in the setting of a murine model of infectious colitis 14 and to determine the sequences of serine proteases present in complex allergenic cockroach extracts 15 . Here, we performed a study to profile and identify active serine proteases secreted by the colonic mucosa of control and IBD patients by using ABPs.…”

Aspergillus fumigatus Increased PAR-2 Expression and Elevated Proinflammatory Cytokines Expression Through the Pathway of PAR-2/ERK1/2 in Cornea

“…11 Moreover, PAR-2 plays a crucial role in different animal models of intestinal diseases, focal segmental glomerulosclerosis, skin inflammatory diseases, myocarditis, and airway inflammation. [12][13][14][15][16][17][18][19] Recent studies have shown that human corneal epithelial cells express PAR-2. In human corneal epithelial cells, inhibition of PAR-2 by a specific antagonist prevents proinflammatory cytokine production and reduces inflammation induced by Acanthamoeba plasminogen activator, a serine protease secreted by Acanthamoeba trophozoites, that is involved in the pathogenesis of Acanthamoeba keratitis, indicating that disruption of PAR-2 activity might have a major impact on preventing inflammatory responses in Acanthamoeba keratitis.…”