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


OBJECTIVE
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 signalling 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 signalling.


RESULTS
Each of the three serine proteinase activity-based probe-labelled enzymes isolated was biochemically distinct, with different enzyme kinetic profiles and primary amino acid sequences. The three enzymes showed a 57%-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 signalling via PAR2.


CONCLUSIONS
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.
Department of Physiology & Pharmacology, Inflammation Research Network-Snyder Institute for Chronic Disease, University of Calgary Cumming School of Medicine, Calgary, AB, Canada.
D J
DJ
Polley
Department of Physiology & Pharmacology, Inflammation Research Network-Snyder Institute for Chronic Disease, University of Calgary Cumming School of Medicine, Calgary, AB, Canada.
K
K
Mihara
Department of Physiology & Pharmacology, Inflammation Research Network-Snyder Institute for Chronic Disease, University of Calgary Cumming School of Medicine, Calgary, AB, Canada.
R
R
Ramachandran
Pulmonary Research Group, Department of Medicine, University of Alberta, Edmonton, AB, Canada.
H
H
Vliagoftis
Department of Physiology & Pharmacology, Inflammation Research Network-Snyder Institute for Chronic Disease, University of Calgary Cumming School of Medicine, Calgary, AB, Canada.
B
B
Renaux
Department of Physiology & Pharmacology, Inflammation Research Network-Snyder Institute for Chronic Disease, University of Calgary Cumming School of Medicine, Calgary, AB, Canada.
M
M
Saifeddine
Asthma & Airway Disease Research Center, University of Arizona, Tucson, AZ, USA.
M O
MO
Daines
Asthma & Airway Disease Research Center, University of Arizona, Tucson, AZ, USA.
S
S
Boitano
Department of Physiology & Pharmacology, Inflammation Research Network-Snyder Institute for Chronic Disease, University of Calgary Cumming School of Medicine, Calgary, AB, Canada.|Department of Medicine, University of Calgary Cumming School of Medicine, Calgary, AB, Canada.
M D
http://orcid.org/0000-0003-4070-8786
MD
Hollenberg
D000485:Allergens / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D020048:Blattellidae / Q000276:immunology; D020013:Calcium Signaling; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D003058:Cockroaches / Q000276:immunology*; D006801:Humans; D006967:Hypersensitivity / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D008024:Ligands; D044464:Receptor, PAR-2 / Q000235:genetics / Q000378:metabolism; D057057:Serine Proteases / Q000737:chemistry / Q000276:immunology*; D015398:Signal Transduction; D000071557:beta-Arrestins / Q000378:metabolism
D000485:Allergens; D008024:Ligands; D044464:Receptor, PAR-2; D000071557:beta-Arrestins; D057057:Serine Proteases
asthma; calcium signalling; cockroach; protease; proteinase-activated receptor-2 (PAR2)