Identification of a serine protease as a major allergen (Per a 10) of Periplaneta americana

Background: Cockroach allergens are associated with the development of Asthma, but none of these has been characterized for proteolytic activity. This study was undertaken to isolate and characterize a Protease from Periplaneta americana and determine its allergenicity.

Methods: A serine Protease was isolated from P. americana Extract using benzamidine sepharose column and characterized by immunobiochemical methods. Allergenicity of the Protease was assessed by enzyme-linked immunosorbent assay, immunoblot, intradermal testing, histamine release and peripheral blood mononuclear cells (PBMCs) proliferation.

Results: Affinity purified protein of approximately 28 kDa (Per a 10) showed a single band of activity in gelatin zymogram and Agarose plate assay. N-terminal sequence (IVGGRPAQI) revealed similarity with Mite serine Protease allergens and insect trypsins. It demonstrated proteolytic activity with azocollagen > gelatin > defatted-milk > casein including serine Protease specific substrate, N-benzoyl-arginine-ethyl-ester-hydrochloride. It was inhibited by serine Protease Inhibitors, namely aprotinin > pefabloc > AEBSF > PMSF > benzamidine > antipain > leupeptin and trypsin-specific inhibitor (tosyl-lysyl-chloromethyl-ketone) suggesting it to be a trypsin-like serine Protease. Per a 10 was recognized as a major allergen, showing IgE reactivity with >80% of cockroach sensitized patients by skin tests and immunoblot. It could induce significant histamine release (P < 0.05) in blood and secretion of interleukin-4 (IL-4) (P < 0.05) and IL-5 (P < 0.05) in culture supernatant of PBMCs from cockroach hypersensitive patients, suggesting a strong allergenic potency.

Conclusion: A serine Protease isolated from P. americana was demonstrated to be a major allergen (Per a 10). It has a potential for component-based diagnosis of Allergy and will be useful in elucidating the mechanism of Allergy.