Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, United States.
Peptides. 2010 Dec;31(12):2159-64. doi: 10.1016/j.peptides.2010.09.004. Epub 2010 Sep 15.
GmSubPep, a 12-amino acid peptide isolated from soybean leaves, induces the expression of genes in soybean suspension-cultured cells that encode proteins involved in defense against pathogens. The peptide is derived from an extracellular subtilisin-like protease (subtilase) and binds a putative cell-surface receptor that initiates a defense signaling cascade. Interaction of the peptide with its receptor results in alkalinization of soybean suspension cell media which can be utilized to analyze the kinetics of receptor binding. Substitutions of alanine at each of the 12 amino acid positions revealed that the amino acids at positions 10 (arginine) and 12 (histidine) were essential for activity. Both analogs were able to reduce the physiological effects of GmSubPep associated with receptor binding. Deletion of the C-terminal histidine [GmSubPep(1-11)] abolished the alkalinizing activity and this peptide was also a strong competitor for receptor binding. Deletion of N-terminal amino acids from GmSubPep caused a sequential loss of activity with no alkalinizing activity for GmSubPep(4-12). However, the N-terminal deleted peptides did not compete with GmSubPep for receptor binding. Further modifications at the arginine-10 position indicated that an ionizable proton was not essential for activity as an attenuated response was found for a citrulline substitution. Substitution of the histidine-12 with methylated histidine at position N-1 of the imidazole group abolished activity, whereas substitution at N-3 was completely active, indicating that the N-3 analog retains important receptor binding properties. This study indicates that the extreme C-terminal of GmSubPep has important signal transduction properties while the C-terminal is essential for receptor interaction.
GmSubPep 是一种从大豆叶片中分离出的 12 个氨基酸肽，它能诱导大豆悬浮细胞培养物中编码抗病原体蛋白的基因表达。该肽来自于一种细胞外的枯草杆菌蛋白酶（subtilase）类似物，并与一种假定的细胞表面受体结合，从而启动防御信号级联反应。肽与受体的相互作用导致大豆悬浮细胞培养基的碱化，这可用于分析受体结合的动力学。在 12 个氨基酸位置的每个位置用丙氨酸取代发现，位置 10（精氨酸）和 12（组氨酸）的氨基酸对于活性是必需的。这两种类似物都能够减轻与受体结合相关的 GmSubPep 的生理效应。C 末端组氨酸的缺失[GmSubPep(1-11)]使碱化活性丧失，并且该肽也是受体结合的强竞争物。从 GmSubPep 中删除 N 端氨基酸导致活性的顺序丧失，而 GmSubPep(4-12)则没有碱化活性。然而，N 端缺失的肽不能与 GmSubPep 竞争受体结合。在精氨酸 10 位置的进一步修饰表明，可电离质子对于活性不是必需的，因为发现瓜氨酸取代有减弱的反应。用组氨酸 N-1 位的甲基组氨酸取代组氨酸 12 位完全丧失了活性，而 N-3 位的取代则完全具有活性，这表明 N-3 类似物保留了重要的受体结合特性。这项研究表明，GmSubPep 的极端 C 末端具有重要的信号转导特性，而 C 末端对于受体相互作用是必需的。
