INRAE, UR 1268 Biopolymères Interactions Assemblages (BIA), 44316, Nantes, France.
UMR 1208 IATE, Univ Montpellier, INRAE, L'Institut-Agro Montpellier, 34060, Montpellier, France.
Sci Rep. 2022 Jul 18;12(1):12253. doi: 10.1038/s41598-022-15811-5.
Lipid transfer proteins (LTPs) were identified as allergens in a large variety of pollens and foods, including cereals. LTPs belong to the prolamin superfamily and display an α-helical fold, with a bundle of four α-helices held together by four disulfide bonds. Wheat LTP1 is involved in allergic reactions to food. To identify critical structural elements of antibody binding to wheat LTP1, we used site-directed mutagenesis on wheat recombinant LTP1 to target: (i) sequence conservation and/or structure flexibility or (ii) each disulfide bond. We evaluated the modifications induced by these mutations on LTP1 secondary structure by synchrotron radiation circular dichroism and on its antigenicity with patient's sera and with mouse monoclonal antibodies. Disruption of the C28-C73 disulfide bond significantly affected IgE-binding and caused protein denaturation, while removing C13-C27 bond decreased LTP1 antigenicity and slightly modified LTP1 overall folding. In addition, we showed Lys72 to be a key residue; the K72A mutation did not affect global folding but modified the local 3D structure of LTP1 and strongly reduced IgE-binding. This work revealed a cluster of residues (C13, C27, C28, C73 and K72), four of which embedded in disulfide bonds, which play a critical role in LTP1 antigenicity.
脂质转移蛋白（LTPs）在各种花粉和食物中被鉴定为过敏原，包括谷物。LTPs 属于类蛋白超家族，呈现出α-螺旋折叠结构，由四个α-螺旋组成，由四个二硫键固定在一起。小麦 LTP1 与食物过敏反应有关。为了确定抗体与小麦 LTP1 结合的关键结构元素，我们使用定点突变技术对小麦重组 LTP1 进行了靶向修饰：（i）序列保守性和/或结构灵活性，或（ii）每个二硫键。我们使用同步辐射圆二色性评估这些突变对 LTP1 二级结构的影响，以及对患者血清和小鼠单克隆抗体的抗原性的影响。破坏 C28-C73 二硫键会显著影响 IgE 结合并导致蛋白质变性，而去除 C13-C27 键会降低 LTP1 的抗原性并略微改变 LTP1 的整体折叠。此外，我们还发现 Lys72 是一个关键残基；K72A 突变不会影响整体折叠，但会改变 LTP1 的局部 3D 结构，并强烈降低 IgE 结合。这项工作揭示了一组关键残基（C13、C27、C28、C73 和 K72），其中四个残基嵌入在二硫键中，它们在 LTP1 的抗原性中起着至关重要的作用。
