Komatsu S, Yamada E, Furukawa K
National Institute of Crop Science, Kannondai 2-1-18, Tsukuba, 305-8518, Japan.
Amino Acids. 2009 Jan;36(1):115-23. doi: 10.1007/s00726-008-0039-4. Epub 2008 Feb 16.
Post-translational modifications such as glycosylation are important for changing the properties and functions of proteins. To analyze the importance of glycosylation during cold stress in rice, a proteomics approach was used. Proteins extracted from the basal part of rice leaf sheaths were separated by two-dimensional polyacrylamide gel electrophoresis, and subjected to lectin blot analysis using concanavalin A. From a total of 250 detected proteins, 22 reacted with the lectin, suggesting that they were N-glycosylated proteins. To determine how N-glycosylation of these proteins is affected by cold stress, rice seedlings were incubated at 5 degrees C for 48 h, and proteins extracted from the basal parts of leaf sheaths were analyzed by the lectin blot assay. Cold stress changed the reactivity toward the lectin for 12 of the 22 glycoproteins. The identity of the 12 proteins was determined by protein sequencing and mass spectrometry with the majority of these glycoproteins being categorized as involved in energy production. Furthermore, calreticulin, one of the 12 glycoproteins, was also phosphorylated as a result of cold stress. These results indicate that cold stress of the basal parts of rice leaf sheaths changes the glycosylation and phosphorylation profiles of calreticulin, a key protein that regulates the quality control of other proteins.
糖基化等翻译后修饰对于改变蛋白质的性质和功能非常重要。为了分析糖基化在水稻冷胁迫过程中的重要性，采用了蛋白质组学方法。从水稻叶鞘基部提取的蛋白质通过二维聚丙烯酰胺凝胶电泳进行分离，并用伴刀豆球蛋白A进行凝集素印迹分析。在总共检测到的250种蛋白质中，有22种与凝集素发生反应，表明它们是N-糖基化蛋白。为了确定这些蛋白质的N-糖基化如何受到冷胁迫的影响，将水稻幼苗在5℃下培养48小时，然后通过凝集素印迹分析对从叶鞘基部提取的蛋白质进行分析。冷胁迫改变了22种糖蛋白中12种对凝集素的反应性。通过蛋白质测序和质谱确定了这12种蛋白质的身份，其中大多数糖蛋白被归类为参与能量产生。此外，这12种糖蛋白之一的钙网蛋白也因冷胁迫而发生磷酸化。这些结果表明，水稻叶鞘基部的冷胁迫改变了钙网蛋白的糖基化和磷酸化谱，钙网蛋白是一种调节其他蛋白质质量控制的关键蛋白。
