Graduate Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan.
PLoS One. 2012;7(4):e34627. doi: 10.1371/journal.pone.0034627. Epub 2012 Apr 10.
Pen c 13, identified as a 33-kDa alkaline serine protease, is a major allergen secreted by Penicillium citrinum. Detailed knowledge about the epitopes responsible for IgE binding would help inform the diagnosis/prognosis of fungal allergy and facilitate the rational design of hypoallergenic candidate vaccines. The goal of the present study was to characterize the IgE epitopes of Pen c 13.
METHODOLOGY/PRINCIPAL FINDINGS: Serum samples were collected from 10 patients with mold allergy and positive Pen c 13 skin test results. IgE-binding epitopes on rPen c 13 were mapped using an enzymatic digestion and chemical cleavage method, followed by dot-blotting and mass spectrometry. A B-cell epitope-predicting server and molecular modeling were used to predict the residues most likely involved in IgE binding. Theoretically predicted IgE-binding regions were further confirmed by site-directed mutagenesis assays. At least twelve different IgE-binding epitopes located throughout Pen c 13 were identified. Of these, peptides S16 (A(148)-E(166)) and S22 (A(243)-K(274)) were recognized by sera from 90% and 100% of the patients tested, and were further confirmed by inhibition assays. Peptide S22 was selected for further analysis of IgE-binding ability. The results of serum screening showed that the majority of IgE-binding ability resided in the C-terminus. One Pen c 13 mutant, G270A (T(261)-K(274)), exhibited clearly enhanced IgE reactivity, whereas another, K274A, exhibited dramatically reduced IgE reactivity.
CONCLUSIONS/SIGNIFICANCE: Experimental analyses confirmed in silico-predicted residues involved in an important antigenic region of Pen c 13. The G270A mutant of Pen c 13 has the potential to serve as an additional tool for the diagnosis/prognosis of mold allergy, and the K274A mutant, as a hypoallergenic form of the epitope, may provide a framework for the design and development of a safe and efficient therapeutic strategy for treating human allergic diseases.
Pen c 13 被鉴定为一种 33kDa 的碱性丝氨酸蛋白酶,是青霉属中主要的过敏原。详细了解引起 IgE 结合的表位有助于真菌过敏的诊断/预后,并有助于合理设计低变应原候选疫苗。本研究的目的是鉴定 Pen c 13 的 IgE 表位。
方法/主要发现:从 10 例霉菌过敏且对 Pen c 13 皮肤试验阳性的患者中采集血清样本。使用酶消化和化学裂解法、点印迹和质谱法对 rPen c 13 上的 IgE 结合表位进行定位。使用 B 细胞表位预测服务器和分子建模预测最有可能与 IgE 结合的残基。通过定点突变实验进一步验证理论预测的 IgE 结合区域。在 Pen c 13 全长鉴定出至少 12 个不同的 IgE 结合表位。其中,肽 S16(A(148)-E(166))和 S22(A(243)-K(274))分别被 90%和 100%的测试患者的血清识别,并通过抑制实验进一步确认。选择肽 S22 进一步分析其 IgE 结合能力。血清筛选结果表明,大多数 IgE 结合能力位于 C 端。一个 Pen c 13 突变体 G270A(T(261)-K(274))表现出明显增强的 IgE 反应性,而另一个突变体 K274A 则表现出明显降低的 IgE 反应性。
结论/意义:实验分析证实了 Pen c 13 中一个重要抗原区域涉及的预测残基。Pen c 13 的 G270A 突变体有望成为霉菌过敏诊断/预后的另一种工具,而 K274A 突变体作为该表位的低变应原形式,可能为设计和开发安全有效的人类过敏性疾病治疗策略提供框架。