Smiljanic K, Apostolovic D, Trifunovic S, Ognjenovic J, Perusko M, Mihajlovic L, Burazer L, van Hage M, Cirkovic Velickovic T
Faculty of Chemistry, Centre of Excellence for Molecular Food Sciences, University of Belgrade, Belgrade, Serbia.
Immunology and Allergy Unit, Department of Medicine Solna, Karolinska Institute and University Hospital, Stockholm, Sweden.
Clin Exp Allergy. 2017 Jun;47(6):815-828. doi: 10.1111/cea.12874. Epub 2017 Jan 19.
Short ragweed (Ambrosia artemisiifolia) allergies affect more than 36 million people annually. Ragweed pollen grains release subpollen particles (SPP) of respirable size upon hydration or a change in air electrical conditions. The aim of this study was to characterize the proteomes and allergomes of short ragweed SPP and total pollen protein extract (TOT), and compare their effects with those of standard aqueous pollen protein extract (APE) using sera from short ragweed pollen-sensitized patients.
Quantitative 2D gel-based and shotgun proteomics, 1D and 2D immunoblotting, and quantitative ELISA were applied. Novel SPP extraction and preparation protocols enabled appropriate sample preparation and further downstream analysis by quantitative proteomics.
The SPP fraction contained the highest proportion (94%) of the allergome, with the largest quantities of the minor Amb a 4 and major Amb a 1 allergens, and as unique, NADH dehydrogenases. APE was the richest in Amb a 6, Amb a 5 and Amb a 3, and TOT fraction was the richest in the Amb a 8 allergens (89% and 83% of allergome, respectively). Allergenic potency correlated well among the three fractions tested, with 1D immunoblots demonstrating a slight predominance of IgE reactivity to SPP compared to TOT and APE. However, the strongest IgE binding in ELISA was noted against APE. New allergenic candidates, phosphoglycerate mutase and phosphoglucomutase, were identified in all the three pollen fractions. Enolase, UTP-glucose-1-phosphate uridylyltransferase and polygalacturonase were observed in SPP and TOT fractions as novel allergens of the short ragweed pollen, as previously described.
We demonstrated that the complete major (Amb a 1 and 11) and almost all minor (Amb a 3, 4, 5, 6, 8 and 9) short ragweed pollen allergen repertoire as well as NADH oxidases are present in SPP, highlighting an important role for SPP in allergic sensitization to short ragweed.
短豚草（Ambrosia artemisiifolia）过敏每年影响超过3600万人。豚草花粉粒在水合作用或空气电学条件变化时会释放出可吸入大小的亚花粉颗粒（SPP）。本研究的目的是对短豚草SPP和总花粉蛋白提取物（TOT）的蛋白质组和过敏原组进行表征，并使用短豚草花粉致敏患者的血清，将它们与标准水性花粉蛋白提取物（APE）的作用进行比较。
应用基于二维凝胶的定量蛋白质组学和鸟枪法蛋白质组学、一维和二维免疫印迹以及定量酶联免疫吸附测定。新颖的SPP提取和制备方案实现了适当的样品制备，并通过定量蛋白质组学进行进一步的下游分析。
SPP组分含有过敏原组中最高比例（94%）的成分，含有大量次要的Amb a 4和主要的Amb a 1过敏原，以及独特的NADH脱氢酶。APE富含Amb a 6、Amb a 5和Amb a 3，TOT组分富含Amb a 8过敏原（分别占过敏原组的89%和83%）。在测试的三个组分中，致敏效力相关性良好，一维免疫印迹显示与TOT和APE相比，IgE对SPP的反应性略占优势。然而，酶联免疫吸附测定中最强的IgE结合是针对APE的。在所有三个花粉组分中都鉴定出了新的致敏候选物，磷酸甘油酸变位酶和磷酸葡萄糖变位酶。如前所述，烯醇化酶、UTP - 葡萄糖 - 1 - 磷酸尿苷酰转移酶和多聚半乳糖醛酸酶在SPP和TOT组分中被观察到是短豚草花粉的新过敏原。
我们证明了完整的主要（Amb a 1和11）和几乎所有次要（Amb a 3、4、5、6、8和9）短豚草花粉过敏原库以及NADH氧化酶都存在于SPP中，突出了SPP在短豚草过敏致敏中的重要作用。
