Plant-produced Asialo-erythropoietin and its Antiapoptotic Functions

描述（由申请人提供）：在这个项目中，我们正在利用植物表达系统缺乏添加唾液酸的能力的独特优势，对植物进行基因修饰，以获得不含唾液酸的特殊糖蛋白asialo-rhuEPO。尽管几乎所有的人类糖蛋白都需要唾液酸残基来维持其体内功能，但我们发现的一个例外是大黄epo，它具有造血和组织保护功能。当大黄epo用作抗凋亡药物时，其造血活性会导致红细胞质量的有害增加，从而引起不良的副作用。这种活性可以通过去除唾液酸而被破坏。由大黄EPO的总酶脱盐作用产生的亚洲黄-大黄EPO已被证明保留了EPO的细胞和组织保护特性，但没有造血活性。这类EPO衍生物可作为抗凋亡药物进行探索。然而，目前还没有能直接产生亚细亚-大黄epo的表达系统。由于供应有限，也不太可能使用仅在哺乳动物细胞中产生的市售大黄生成素来制造亚洲紫草-大黄生成素。基于现有的知识和技术，很可能对植物进行基因改造，以生产不含唾液酸的人类糖蛋白。我们在烟草植物中共表达了人GalT基因和融合er信号肽的EPO基因，并尝试低成本生产具有大规模生产潜力的亚细亚-大黄EPO。我们期望植物合成的亚细亚黄-大黄epo与酶合成的亚细亚黄-大黄epo具有相似的细胞保护功能。本项目的研究结果也将为揭示植物糖基化机制和植物产生的亚细亚-大黄epo的抗凋亡机制提供新的思路。此外，所建立的蛋白质纯化方法和糖蛋白分析方法将对未来的植物糖工程项目具有重要的应用价值。

DESCRIPTION (provided by applicant): In this project, we are taking the distinct advantage of plant expression systems, which is the lack of the capacity to add sialic acids, to genetically modify plants for asialo-rhuEPO - a special glycoprotein without sialic acids. Although nearly all human glycoproteins require sialic acid residues for their in vivo functions, the one exception we could find is rhuEPO, which has both hematopoietic and tissue protective functions. When rhuEPO is used as an antiapoptotic agent, its hematopoietic activity can cause bad side effects by leading to harmful increases in red blood cell mass. This activity can be disrupted by removing sialic acids. Asialo-rhuEPO generated by total enzymatic desialylation of rhuEPO has been demonstrated to retain the cell- and tissue-protective properties of EPO without hematopoietic activity. This type of EPO derivative can be explored as the antiapoptotic agent. However, there is no expression system that can produce asialo-rhuEPO directly. It is also unlikely to use commercially available rhuEPO, which is produced only in mammalian cells, to make asialo-rhuEPO because of its limited supply. Based on available knowledge and techniques, it is likely to genetically modify plants to produce human glycoproteins without sialic acids. We have co- expressed human GalT gene and EPO gene fusing with ER-signal peptides in tobacco plants, and try to inexpensively produce asialo-rhuEPO with the potential of large scale production. We expect plant-produced asialo-rhuEPO will have similar cytoprotective function as the enzymatically produced asialo-rhuEPO. The results of this project will also provide insights into the plant glycosylation mechanism and the antiapoptotic mechanism of the plant- produced asialo-rhuEPO. In addition, the protein purification methodologies that will be established and the glycoprotein analysis methods will be very valuable for future plant glycoengineering projects. PUBLIC HEALTH RELEVANCE: We aim to genetically modify plants for production of asialo-rhuEPO - a special human protein. This type of biopharmaceutical protein can be explored as the neurotherapeutic and cardioprotective agent. Using a plant expression system, we expect to produce an inexpensive and functional asialo-rhuEPO to prevent cell death with the potential of large scale production.