Gehlhar Kirsten, Rajashankar Kanagalaghata R, Hofmann Eckhard, Betzel Christian, Weber Wolfgang, Werner Sandra, Bufe Albrecht
Department of Experimental Pneumology, Ruhr University Bochum, Bochum, Germany.
Int Arch Allergy Immunol. 2006;140(4):285-94. doi: 10.1159/000093706. Epub 2006 May 21.
Allergens induce the formation of specific immunoglobulin (Ig)E and harbor at least two IgE-binding regions (epitopes) to facilitate crosslinking of basophilic or mast-cell-bound specific IgE antibodies. Studies mapping linear epitopes have shown that these regions often contain charged or hydrophobic amino acids. Nevertheless, these studies are hampered by limited significance due to the often conformational nature of IgE epitopes. This prompted us to study the role of lysines in the context of an intact 3-dimensional model.
Major allergen Phl p 5b from timothy grass bears 12 lysines in its C-terminal half. Using site-directed mutagenesis, we substituted all 10 surface-exposed lysines by alanines.
Although structural integrity of the lysine-deficient mutant was not altered, IgE-binding capacity measured by ELISA inhibition tests and crosslinking activity in ex vivo basophil stimulation and in vivo skin prick tests were significantly diminished. Interestingly, binding of specific IgG antibodies was considerably less reduced by loss of lysines.
Lysine is an important amino acid for IgE binding in more than one epitope of major grass pollen allergen Phl p 5b C terminus. Allergenicity, but not IgG binding of the molecule, is substantially diminished by single amino acid substitutions without structural integrity being hampered.
变应原诱导特异性免疫球蛋白（Ig）E的形成，并具有至少两个IgE结合区域（表位），以促进嗜碱性粒细胞或肥大细胞结合的特异性IgE抗体的交联。绘制线性表位的研究表明，这些区域通常含有带电荷或疏水的氨基酸。然而，由于IgE表位通常具有构象性质，这些研究的意义有限。这促使我们在完整的三维模型背景下研究赖氨酸的作用。
来自梯牧草的主要变应原Phl p 5b在其C端一半含有12个赖氨酸。使用定点诱变，我们将所有10个表面暴露的赖氨酸用丙氨酸替代。
尽管赖氨酸缺陷型突变体的结构完整性未改变，但通过ELISA抑制试验测量的IgE结合能力以及体外嗜碱性粒细胞刺激和体内皮肤点刺试验中的交联活性均显著降低。有趣的是，赖氨酸缺失对特异性IgG抗体结合的影响要小得多。
赖氨酸是主要草花粉变应原Phl p 5b C端多个表位中IgE结合的重要氨基酸。单个氨基酸替代可显著降低分子的变应原性，但不影响IgG结合，且不损害结构完整性。
