Sudha V T, Arora N, Gaur S N, Pasha S, Singh B P
Institute of Genomics and Integrative Biology, Delhi University Campus, Delhi, India.
Allergy. 2008 Jun;63(6):768-76. doi: 10.1111/j.1398-9995.2007.01602.x.
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.
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.
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.
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.
蟑螂过敏原与哮喘的发生有关，但这些过敏原均未被鉴定出具有蛋白水解活性。本研究旨在从美洲大蠊中分离并鉴定一种蛋白酶，并确定其致敏性。
使用苯甲脒琼脂糖柱从美洲大蠊提取物中分离出一种丝氨酸蛋白酶，并通过免疫生化方法进行鉴定。通过酶联免疫吸附测定、免疫印迹、皮内试验、组胺释放和外周血单核细胞（PBMC）增殖评估该蛋白酶的致敏性。
亲和纯化的约28 kDa蛋白（Per a 10）在明胶酶谱和琼脂糖平板试验中显示出单一活性条带。N端序列（IVGGRPAQI）显示与螨类丝氨酸蛋白酶过敏原和昆虫胰蛋白酶相似。它对偶氮胶原蛋白>明胶>脱脂牛奶>酪蛋白表现出蛋白水解活性，包括丝氨酸蛋白酶特异性底物N-苯甲酰-L-精氨酸乙酯盐酸盐。它被丝氨酸蛋白酶抑制剂抑制，即抑肽酶>苯甲磺酰氟>4-(2-氨基乙基)苯磺酰氟盐酸盐>苯甲基磺酰氟>苯甲脒>抗蛋白酶>亮抑肽酶和胰蛋白酶特异性抑制剂（甲苯磺酰-L-赖氨酰氯甲基酮），表明它是一种类胰蛋白酶丝氨酸蛋白酶。Per a 10被认为是主要过敏原，通过皮肤试验和免疫印迹显示，>80%的蟑螂致敏患者对其有IgE反应。它可在血液中诱导显著的组胺释放（P < 0.05），并在蟑螂过敏患者的PBMC培养上清液中诱导白细胞介素-4（IL-4）（P < 0.05）和IL-5（P < 0.05）的分泌，表明其具有很强的致敏能力。
从美洲大蠊中分离出的一种丝氨酸蛋白酶被证明是主要过敏原（Per a 10）。它具有基于成分的过敏诊断潜力，将有助于阐明过敏机制。
