Engineering Sialylation Pathways in Insect Cells

昆虫细胞中的唾液酸化途径工程

• 批准号: 6602823
• 负责人: MICHAEL J BETENBAUGH
• 金额: $ 28.6万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-01-08 至 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6602823
• 关键词: Insecta; N acylation; Sf9 cell line; arthropod genetics; biotechnology; cell line; fluorimetry; galactose; glycoprotein biosynthesis; glycoproteins; oligosaccharides; polymerase chain reaction; protein engineering; sialate; sialyltransferases

DESCRIPTION (provided by applicant): Insect cells are widely utilized for the production of heterologous glycoproteins, which are proteins that include oligosaccharide attachments. Many of the most valuable therapeutics are glycoproteins and the composition of the attached oligosaccharide can significantly impact the properties and value of these therapeutics. In particular, the presence or absence of sialic acid on an oligosaccharide can alter the glycoprotein's structure, stability, biological activity, and in vivo circulatory half-life. Glycoproteins containing oligosaccharides missing sialic acid are removed from human circulation and this rapid clearance will diminish the therapeutic effectiveness of a glycoprotein biopharmaceutical. Unfortunately, insect cells do not generate significant levels, if any, of sialylated glycoproteins, and this inability to sialylate severely limits the further application of the insect cell expression system. The objective of this project is to manipulate the metabolic pathways in insect cells so that these cells will produce high levels of complex, fully sialylated glycoproteins. A critical sialylation pathway involves the post-translational addition of the donor sialic acid substrate, cytidine monophosphate-N-acetylneuraminic acid (CMP-Neu5Ac or CMP-sialic acid) onto a specific acceptor carbohydrate substrate ending in galactose (Gal) via a reaction catalyzed by the enzyme sialyltransferase. Each of these three critical components (donor substrate, acceptor substrate, and sialyltransferase enzyme) is limiting or absent in insect cell lines so metabolic engineering strategies will be implemented to eliminate the bottlenecks. Limitations in CMP-Neu5Ac will be overcome by expressing essential enzymes in order to complete the intracellular synthesis of the donor substrate. Expression of heterologous glycosyltransferase enzymes, suppression of unfavorable processing reactions, and examination of alternative insect cell hosts will overcome limitations in Gal acceptors. Combining these pathway modifications with sialyltransferase expression should ensure full sialylation in insect cells. Completion of this project will lead to an increase in the number of expression systems that can produce the high-value sialylated therapeutic glycoproteins desired by the health care community. In this way, sialylation pathway engineering may increase glycoprotein quality and lower health care costs for patients receiving these therapeutics.

描述(申请人提供)：昆虫细胞被广泛用于生产异源糖蛋白，这是一种含有寡糖附着体的蛋白质。许多最有价值的治疗药物是糖蛋白，结合的寡糖的组成可以显著影响这些治疗药物的性质和价值。特别是，寡糖上唾液酸的存在或不存在会改变糖蛋白的结构、稳定性、生物活性和体内循环半衰期。含有缺失唾液酸的寡糖的糖蛋白被从人体循环中清除，这种快速清除将降低糖蛋白生物制药的治疗效果。不幸的是，昆虫细胞不会产生显著水平的唾液酸化糖蛋白，这种不能唾液酸化的能力严重限制了昆虫细胞表达系统的进一步应用。这个项目的目标是操纵昆虫细胞中的代谢途径，使这些细胞产生高水平的复杂的、完全唾液酸化的糖蛋白。一个关键的唾液酸化途径涉及到供体唾液酸底物胞苷一磷酸-N-乙酰神经氨酸(cMP-Neu5Ac或cMP-sialic酸)通过唾液酸转移酶催化的反应在特定的受体碳水化合物底物上加成，最终形成半乳糖(Gal)。在昆虫细胞系中，这三个关键成分(供体底物、受体底物和唾液酸转移酶)中的每一个都是有限的或不存在的，因此将实施代谢工程策略来消除瓶颈。通过表达必要的酶来完成供体底物的细胞内合成，将克服CMP-Neu5Ac的局限性。异源糖基转移酶的表达，不利加工反应的抑制，以及昆虫细胞替代宿主的检测，将克服Gal受体的局限性。结合这些途径的修饰和唾液酸基转移酶的表达，应该可以确保在昆虫细胞中完全唾液酸化。该项目的完成将导致能够产生医疗界所需的高价值唾液酸化治疗性糖蛋白的表达系统的数量增加。通过这种方式，唾液酸化途径工程可以提高糖蛋白质量，降低接受这些疗法的患者的医疗成本。