Laboratory of Agrozoology, Department of Plants and Crops, Faculty of Bioscience Engineering, Ghent University, 9000, Ghent, Belgium.
Department of Biotechnology, Faculty of Bioscience Engineering, Ghent University, 9000, Ghent, Belgium.
Sci Rep. 2021 Sep 9;11(1):17958. doi: 10.1038/s41598-021-97161-2.
Several plant lectins, or carbohydrate-binding proteins, interact with glycan moieties on the surface of immune cells, thereby influencing the immune response of these cells. Orysata, a mannose-binding lectin from rice, has been reported to exert immunomodulatory activities on insect cells. While the natural lectin is non-glycosylated, recombinant Orysata produced in the yeast Pichia pastoris (YOry) is modified with a hyper-mannosylated N-glycan. Since it is unclear whether this glycosylation can affect the YOry activity, non-glycosylated rOrysata was produced in Escherichia coli (BOry). In a comparative analysis, both recombinant Orysata proteins were tested for their carbohydrate specificity on a glycan array, followed by the investigation of the carbohydrate-dependent agglutination of red blood cells (RBCs) and the carbohydrate-independent immune responses in Drosophila melanogaster S2 cells. Although YOry and BOry showed a similar carbohydrate-binding profiles, lower concentration of BOry were sufficient for the agglutination of RBCs and BOry induced stronger immune responses in S2 cells. The data are discussed in relation to different hypotheses explaining the weaker responses of glycosylated YOry. In conclusion, these observations contribute to the understanding how post-translational modification can affect protein function, and provide guidance in the selection of the proper expression system for the recombinant production of lectins.
几种植物凝集素，即糖结合蛋白，与免疫细胞表面的聚糖部分相互作用，从而影响这些细胞的免疫反应。ORYsata 是一种来自水稻的甘露糖结合凝集素，据报道它对昆虫细胞具有免疫调节活性。虽然天然凝集素是非糖基化的，但在巴斯德毕赤酵母（Pichia pastoris）中生产的重组 ORYsata（YOry）经过了高度甘露糖化的 N-聚糖修饰。由于尚不清楚这种糖基化是否会影响 YOry 的活性，因此在大肠杆菌（Escherichia coli）中生产了非糖基化的 rORYsata（BOry）。在对比分析中，对两种重组 ORYsata 蛋白在糖芯片上的碳水化合物特异性进行了测试，随后研究了红细胞（RBC）的碳水化合物依赖性凝集以及黑腹果蝇 S2 细胞的碳水化合物非依赖性免疫反应。尽管 YOry 和 BOry 表现出相似的碳水化合物结合谱，但 BOry 的浓度较低即可引起 RBC 的凝集，并且 BOry 在 S2 细胞中诱导更强的免疫反应。这些数据与解释糖基化 YOry 反应较弱的不同假说有关。总之，这些观察结果有助于理解翻译后修饰如何影响蛋白质功能，并为选择适当的表达系统用于重组生产凝集素提供指导。
