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.


CONCLUSIONS
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.
Sudha
V T
VT
Institute of Genomics and Integrative Biology, Delhi University Campus, Delhi, India.
Arora
N
N
Gaur
S N
SN
Pasha
S
S
Singh
B P
BP
D000293:Adolescent; D000328:Adult; D000485:Allergens / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D016207:Cytokines / Q000032:analysis / Q000276:immunology; D005260:Female; D006636:Histamine Release; D006801:Humans; D006967:Hypersensitivity / Q000276:immunology* / Q000378:metabolism; D007073:Immunoglobulin E / Q000276:immunology; D008297:Male; D010526:Periplaneta / Q000201:enzymology / Q000276:immunology*; D012697:Serine Endopeptidases / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D012882:Skin Tests
D000485:Allergens; D016207:Cytokines; D015842:Serine Proteinase Inhibitors; D007073:Immunoglobulin E; D012697:Serine Endopeptidases