Department of Nutrition, Food and Exercise Sciences, The Florida State University, Tallahassee, Florida 32306, United States.
J Agric Food Chem. 2011 Sep 14;59(17):9542-52. doi: 10.1021/jf2017447. Epub 2011 Aug 4.
Among tree nut allergens, pecan allergens remain to be identified and characterized. The objective was to demonstrate the IgE-binding ability of pecan 11S legumin and characterize its sequential IgE-binding epitopes. The 11S legumin gene was amplified from a pecan cDNA library and expressed as a fusion protein in Escherichia coli. The native 11S legumin in pecan extract was identified by mass spectrometry/mass spectrometry (MS/MS). Sequential epitopes were determined by probing the overlapping peptides with three serum pools prepared from different patients' sera. A three-dimensional model was generated using almond legumin as a template and compared with known sequential epitopes on other allergenic tree nut homologues. Of 28 patients tested by dot blot, 16 (57%) bound to 11S legumin, designated Car i 4. MS/MS sequencing of native 11S legumin identified 33 kDa acidic and 20-22 kDa basic subunits. Both pecan and walnut seed protein extracts inhibited IgE binding to recombinant Car i 4, suggesting cross-reactivity with Jug r 4. Sequential epitope mapping results of Car i 4 revealed weak, moderate, and strong reactivity of serum pools against 10, 5, and 4 peptides, respectively. Seven peptides were recognized by all three serum pools, of which two were strongly reactive. The strongly reactive peptides were located in three discrete regions of the Car i 4 acidic subunit sequence (residues 118-132, 208-219, and 238-249). Homology modeling of Car i 4 revealed significant overlapping regions shared in common with other tree nut legumins.
在坚果过敏原中，山核桃过敏原仍有待鉴定和表征。本研究旨在证明山核桃 11S 豆球蛋白的 IgE 结合能力，并对其顺序 IgE 结合表位进行表征。从山核桃 cDNA 文库中扩增 11S 豆球蛋白基因，并在大肠杆菌中表达为融合蛋白。通过质谱/质谱（MS/MS）鉴定山核桃提取物中原位 11S 豆球蛋白。通过用来自不同患者血清的三个血清池探测重叠肽来确定顺序表位。使用杏仁豆球蛋白作为模板生成三维模型，并与其他过敏原性树坚果同源物上已知的顺序表位进行比较。在 28 名经斑点印迹测试的患者中，有 16 名（57%）与 11S 豆球蛋白结合，命名为 Car i 4。对天然 11S 豆球蛋白的 MS/MS 测序鉴定出 33 kDa 酸性亚基和 20-22 kDa 碱性亚基。山核桃和胡桃种子蛋白提取物均抑制 IgE 与重组 Car i 4 结合，表明与 Jug r 4 发生交叉反应。Car i 4 的顺序表位映射结果显示，血清池对 10、5 和 4 个肽的反应性分别为弱、中、强。三个血清池均识别 7 个肽，其中 2 个反应强烈。强反应肽位于 Car i 4 酸性亚基序列的三个离散区域（残基 118-132、208-219 和 238-249）。Car i 4 的同源建模显示，与其他树坚果豆球蛋白共有显著重叠区域。
