Shankar Jay, Singh Bhanu P, Gaur Shailendra N, Arora Naveen
Institute of Genomics and Integrative Biology, Mall Road, Delhi, 110007, India.
J Clin Immunol. 2009 Jan;29(1):63-70. doi: 10.1007/s10875-008-9224-1. Epub 2008 Aug 6.
Epitopes were delineated for allergenic proteins, but studies are required to identify residues mediating IgE binding. In the present study, the in silico approach was used to identify IgE-binding residues of Alt a 13(1-50) fragment and confirmed by experimental approach.
IgE-binding epitopes of Alt a 13 mapped computationally were cloned, expressed, purified, and characterized using various immunochemical and biophysical methods. Among four fragments of Alt a 13, Alt a 13(1-50) demonstrated maximum IgE binding with two immunodominant regions and was mutated at these regions. The mutation in first region, Alt a 13(1-50)-K4A_S6F, did not show any change in immunological and biophysical properties of protein. However, mutations in the second region, Alt a 13(1-50)-T21F_N27I, caused reduced IgE binding, histamine release, and low IL-4 release on stimulation of Alternaria alternata positive patients peripheral blood mononuclear cells in vitro.
This suggests that residues T21 and N27 are important for the secondary structure. In conclusion, Alt a 13(1-50)-T21F_N27I with reduced Th 2 response and intact T-cell proliferation capacity has potential for clinical use.
已确定了变应原蛋白的表位，但需要开展研究来鉴定介导IgE结合的残基。在本研究中，采用计算机模拟方法鉴定了Alt a 13(1-50)片段的IgE结合残基，并通过实验方法进行了验证。
对通过计算机模拟定位的Alt a 13的IgE结合表位进行克隆、表达、纯化，并使用各种免疫化学和生物物理方法进行表征。在Alt a 13的四个片段中，Alt a 13(1-50)表现出与两个免疫显性区域的最大IgE结合，并在这些区域发生了突变。第一个区域的突变体Alt a 13(1-50)-K4A_S6F在蛋白质的免疫和生物物理特性方面未显示任何变化。然而，第二个区域的突变体Alt a 13(1-50)-T21F_N27I在体外刺激链格孢阳性患者外周血单个核细胞时，导致IgE结合减少、组胺释放减少以及IL-4释放降低。
这表明残基T21和N27对二级结构很重要。总之，具有降低的Th 2反应和完整的T细胞增殖能力的Alt a 13(1-50)-T21F_N27I具有临床应用潜力。
