Plant-produced Asialo-erythropoietin and its Antiapoptotic Functions

描述(申请人提供)：在这个项目中，我们利用植物表达系统的独特优势，即缺乏添加唾液酸的能力，对植物进行基因改造，以获得asialo-rHuEPO--一种不含唾液酸的特殊糖蛋白。虽然几乎所有的人类糖蛋白在体内的功能都需要唾液酸残基，但我们可以发现的一个例外是rHuEPO，它同时具有造血和组织保护功能。当rHuEPO被用作抗凋亡剂时，其造血活性可能会导致有害的红细胞质量增加，从而导致不良副作用。这种活性可以通过去除唾液酸而被破坏。由rHuEPO的全酶解制得的Asialo-rHuEPO已被证明保留了EPO的细胞和组织保护特性，而不具有造血活性。这种类型的EPO衍生物可以作为抗细胞凋亡剂进行开发。然而，目前还没有能直接生产asialo-rHuEPO的表达系统。由于供应有限，它也不太可能使用商业上可获得的rHuEPO来制造asialo-rHuEPO，rHuEPO只在哺乳动物细胞中产生。根据现有的知识和技术，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。我们在烟草中实现了人GalT基因和EPO基因与ER信号肽的融合表达，并试图以较低的成本生产具有规模化生产潜力的asialo-rHuEPO。我们预计植物生产的asialo-rHuEPO将具有与酶法生产的asialo-rHuEPO类似的细胞保护功能。该项目的结果还将为深入了解植物糖基化机制和植物生产的asialo-rHuEPO的抗凋亡机制提供依据。此外，将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非常重要的价值。 公共卫生相关性：我们的目标是对植物进行基因改造，以生产asialo-rHuEPO-一种特殊的人类蛋白质。这种类型的生物药物蛋白可以作为神经治疗和心脏保护剂进行开发。利用植物表达系统，我们有望生产出价格低廉、功能齐全的asialo-rHuEPO，以防止细胞死亡，具有大规模生产的潜力。

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.