Department of Biological Sciences, Sunway University, Bandar Sunway, 47500, Selangor, Malaysia.
Allergy and Molecular Immunology Laboratory, Functional Genomics Laboratories, Department of Biological Science, National University of Singapore, Block S2, Level 5, Science Drive 4, Singapore, 117543, Singapore.
Sci Rep. 2019 Feb 7;9(1):1556. doi: 10.1038/s41598-018-38313-9.
Der p 2 is a major dust mite allergen and >80% of mite allergic individuals have specific IgE to this allergen. Although it is well characterized in terms of allergenicity, there is still some ambiguity in terms of its biological function. Three-dimensional structural analysis of Der p 2 and its close homologues indicate the presence of a hydrophobic cavity which can potentially bind to lipid molecules. In this study, we aimed to identify the potential ligand of Der p 2. Using a liposome pulldown assay, we show that recombinant Der p 2 binds to liposomes prepared with exogenous cholesterol in a dose dependent fashion. Next, an ELISA based assay using immobilized lipids was used to study binding specificities of other lipid molecules. Cholesterol was the preferred ligand of Der p 2 among 11 different lipids tested. Two homologues of Der p 2, Der f 2 and Der f 22 also bound to cholesterol. Further, using liquid chromatography-mass spectrometry (LC-MS), we confirmed that cholesterol is the natural ligand of Der p 2. Three amino acid residues of Der p 2, V104, V106 and V110 are possible cholesterol binding sites, as alanine mutations of these residues showed a significant decrease in binding (p < 0.05) compared to wild-type Der p 2. These results provide the first direct experimental evidence that Der p 2 binds to cholesterol.
Der p 2 是一种主要的尘螨过敏原，超过 80%的尘螨过敏个体对这种过敏原具有特异性 IgE。尽管它在变应原性方面得到了很好的描述，但在其生物学功能方面仍存在一些模糊性。Der p 2 及其密切同源物的三维结构分析表明存在一个潜在的疏水腔，可以与脂质分子结合。在这项研究中，我们旨在鉴定 Der p 2 的潜在配体。使用脂质体下拉测定法，我们表明重组 Der p 2 以剂量依赖的方式与用外源性胆固醇制备的脂质体结合。接下来，使用固定化脂质的基于 ELISA 的测定法用于研究其他脂质分子的结合特异性。在测试的 11 种不同脂质中，胆固醇是 Der p 2 的首选配体。Der p 2 的两种同源物，Der f 2 和 Der f 22 也与胆固醇结合。此外，使用液相色谱-质谱（LC-MS），我们证实胆固醇是 Der p 2 的天然配体。Der p 2 的三个氨基酸残基 V104、V106 和 V110 可能是胆固醇结合位点，因为这些残基的丙氨酸突变与野生型 Der p 2 相比，结合显著减少（p < 0.05）。这些结果提供了 Der p 2 与胆固醇结合的第一个直接实验证据。
