Chou H, Lin W L, Tam M F, Wang S R, Han S H, Shen H D
Department of Medical Research, Veterans General Hospital Taipei, Taiwan, Republic of China.
Int Arch Allergy Immunol. 1999 Aug;119(4):282-90. doi: 10.1159/000024205.
Aspergillus species are prevalent indoor airborne fungi and have been identified to be a causative agent of human allergic disorders. In the present study, we identified, purified and characterized the allergen(s) from Aspergillus flavus, a predominant airborne Aspergillus species in the Taipei area.
The IgE-binding components of A. flavus were identified by SDS-PAGE immunoblotting with sera from asthmatic patients. The N-terminal amino acid sequences of the major allergens were determined by Edman degradation. The allergenic cross-reactivity among allergens from different fungi was analyzed by immunoblot inhibition using sera from asthmatic patients. The detected major allergen was purified from the culture medium. It was further characterized in terms of its N-terminal amino acid sequence, its IgE-binding activity and its enzymatic activity.
The results of the immunoblot analysis indicate that a 34-kD component that has high IgE-binding (63%) frequency is a major allergen of A. flavus. The N-terminus of this 34-kD major allergen (GLTTQKSAP) has high sequence identity with that of the 34-kD alkaline serine proteinase major allergen of A. oryzae. Results from immunoblot inhibition studies indicate that IgE cross-reactivity occurs among the 34-kD major allergens of A. flavus, A. fumigatus and Penicillium citrinum. The 34-kD major allergen of A. flavus was purified from the culture medium by ammonium sulfate precipitation and DEAE ion exchange chromatography. The N-terminal amino acid sequence of the purified allergen (Asp fl 13) is identical to that determined previously for the 34-kD major allergen in the crude extract of A. flavus. The IgE immunoblot reactivity to the 34-kD major allergen in the crude extract can be dose-dependently inhibited by the purified Asp fl 13. The degree of IgE binding to the 34-kD major allergen in the crude extract correlates with that of the purified Asp fl 13 in sera of 8 asthmatic patients. The purified Asp fl 13 has proteolytic activity with casein as substrate at pH 8.0. This enzymatic activity can be inhibited by either phenylmethylsulfonyl fluoride or diethylpyrocarbonate.
Our results suggest that the 34-kD alkaline serine proteinase is a major allergen of A. flavus. There was IgE cross-reactivity among allergens of A. flavus, A. fumigatus and P. citrinum.
曲霉菌是常见的室内空气传播真菌，已被确认为人类过敏性疾病的致病因子。在本研究中，我们从黄曲霉中鉴定、纯化并表征了过敏原，黄曲霉是台北地区空气中主要的曲霉菌种。
用哮喘患者的血清通过SDS-PAGE免疫印迹法鉴定黄曲霉的IgE结合成分。通过埃德曼降解法测定主要过敏原的N端氨基酸序列。使用哮喘患者的血清通过免疫印迹抑制法分析来自不同真菌的过敏原之间的过敏交叉反应性。从培养基中纯化检测到的主要过敏原。进一步根据其N端氨基酸序列、IgE结合活性和酶活性对其进行表征。
免疫印迹分析结果表明，一种具有高IgE结合（63%）频率的34-kD成分是黄曲霉的主要过敏原。这种34-kD主要过敏原（GLTTQKSAP）的N端与米曲霉的34-kD碱性丝氨酸蛋白酶主要过敏原的N端具有高度序列同一性。免疫印迹抑制研究结果表明，黄曲霉、烟曲霉和桔青霉的34-kD主要过敏原之间存在IgE交叉反应性。通过硫酸铵沉淀和DEAE离子交换色谱从培养基中纯化了黄曲霉的34-kD主要过敏原。纯化过敏原（Asp fl 13）的N端氨基酸序列与先前在黄曲霉粗提物中测定的34-kD主要过敏原的序列相同。纯化的Asp fl 13可以剂量依赖性地抑制粗提物中对34-kD主要过敏原的IgE免疫印迹反应性。在8名哮喘患者的血清中，粗提物中与34-kD主要过敏原的IgE结合程度与纯化的Asp fl 13的结合程度相关。纯化的Asp fl 13在pH 8.0时以酪蛋白为底物具有蛋白水解活性。这种酶活性可以被苯甲基磺酰氟或焦碳酸二乙酯抑制。
我们的结果表明，34-kD碱性丝氨酸蛋白酶是黄曲霉的主要过敏原。黄曲霉、烟曲霉和桔青霉的过敏原之间存在IgE交叉反应性。
