Effector activity of peanut allergens: a critical role for Ara h 2, Ara h 6, and their variants.
Effector activity of peanut allergens: a critical role for Ara h 2, Ara h 6, and their variants.
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DOI：
10.1111/j.1365-2222.2009.03273.x
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发表时间：
2009-07
期刊：
影响因子：
--
通讯作者：
Dreskin SC
中科院分区：
文献类型：
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作者：
Porterfield HS;Murray KS;Schlichting DG;Chen X;Hansen KC;Duncan MW;Dreskin SC
过敏原的一个重要特性是它们能够交联IgE并激活肥大细胞和嗜碱性粒细胞。花生过敏原的效应活性尚未得到很好的表征。新鲜花生粉的粗提物通过凝胶过滤进行分级。通过测量用来自个体血清和来自花生过敏供体血清池的IgE致敏的RBL SX-38细胞的脱粒来测定效应子功能。凝胶过滤后，在所得级分中回收了75±7%的应用蛋白和76±16%（n=3）的应用活性（用11份血清的合并液测定）。回收活性的大部分（85±2%; n=3）存在于理论平均分子量约为20 kD且范围为13-25 kD的馏分中。当所有单独的级分重新组合时，测得的活性与原始提取物的活性相似（当用血清池（n=2）测量时为140±43%，当用个体血清（n=4）测量时为66±7%）;当将除20 kD级分之外的所有单独级分重新组合时，当用血清池测定时，测得的活性仅为原始提取物的8±2%（n=2），而当用单个血清测定时，测得的活性为10±4%（n=3）。该生物活性组分的二维凝胶电泳显示>60个蛋白质点。通过基质辅助激光解吸电离飞行时间质谱法和自动串联质谱法结合在线毛细管液相色谱法对50个最突出的斑点进行分析，发现超过97%的蛋白质质量由Ara h 2.0101，Ara h 2.0201，Ara h 6同种型和这些蛋白质的变体组成。Ara h 2和Ara h 6占在粗花生提取物中发现的效应子活性的大部分。
An important property of allergens is their ability to cross-link IgE and activate mast cells and basophils. The effector activity of peanut allergens has not been well characterized. Crude extracts of fresh peanut flour were fractionated by gel filtration. Effector function was assayed by measuring degranulation of RBL SX-38 cells sensitized with IgE from individual sera and from pools of sera of peanut allergic donors. Following gel filtration, 75±7% of the applied protein and 76±16% (n=3) of the applied activity (assayed with a pool of 11 sera) were recovered in the resultant fractions. The majority (85±2%; n=3) of the recovered activity resided in a fraction with a theoretical average molecular weight of ~20 kD and a range of 13–25 kD. When all the individual fractions were recombined, the measured activity was similar to that of the original extract (140±43% when measured with a pool of serum (n=2) and 66±7% when measured with individual sera (n=4)); when all individual fractions excluding the 20 kD fraction were recombined, the measured activity was only 8±2% (n=2) of the original extract when assayed with the serum pool and 10±4% (n=3) when assayed with the individual sera. Two dimensional gel electrophoresis of this biologically active fraction by revealed >60 protein spots . Analysis of 50 of the most prominent spots by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry and of the full mixture by automated tandem mass spectrometry coupled to online capillary liquid chromatography revealed that greater than 97% of the protein mass consisted of Ara h 2.0101, Ara h 2.0201, Ara h 6 isoforms, and variants of these proteins. Ara h 2 and Ara h 6 account for the majority of the effector activity found in a crude peanut extract.