Metabolic Engineering to Overcome N-Glycosylation Deficiency in Mammalian Cells

克服哺乳动物细胞 N-糖基化缺陷的代谢工程

• 批准号: 7215240
• 负责人: MICHAEL J BETENBAUGH
• 金额: $ 20.59万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7215240
• 关键词: Alcoholism; Biological Response Modifier Therapy; Biotechnology; Cell Line; Collection; Congenital Disorders; Consensus Sequence; Disease; Dolichol; Endopeptidases; Engineering; Enzymes; Failure; Functional disorder; Generations; Glycoproteins; Goals; Growth; Healthcare; Industry; Lead; Link; Lipids; Living Costs; Mammalian Cell; Metabolic; Metabolic Clearance Rate; Metabolic Pathway; Oligosaccharides; Organ failure; Pathway interactions; Patients; Peptide Hydrolases; Polysaccharides; Positioning Attribute; Post-Translational Protein Processing; Production; Property; Protein Glycosylation; Protein Overexpression; Proteins; Resistance; Therapeutic; commercialization; cost; dolichyl-diphosphooligosaccharide - protein glycotransferase; glycosylation; improved; in vivo; insight; interest; isoprenoid; mutant; polypeptide; relating to nervous system

DESCRIPTION (provided by applicant): Many valuable biotherapeutics in the biotechnology industry are glycoprotein products secreted from mammalian cells. These secreted glycoproteins often undergo post-translational modifications including Nlinked glycosylation (N-glycosylation), which involves the en bloc transfer of an oligosaccharide from a longchain isoprenoid lipid (dolichol) onto a nascent polypeptide containing the consensus sequence Asn-XSer/Thr via a multi-subunit enzyme called oligosaccharide transferase (OST). These oligosaccharide attachments (N-glycans) can be critical to protein properties including folding, stability, resistance to proteases, bioactivity, and in vivo clearance rates. The generation of incompletely N-glycosylated proteins products at positions normally glycosylated indicates a deficiency in either the levels of the dolichol-linked oligosaccharide (DLO) substrate or the OST enzyme that transfers the oligosaccharide onto the target polypeptide. The inability to attain proper N-glycosylation lowers product quality and bioactivity, increases production costs, and inhibits product commercialization. The failure to obtain proper N-glycosylation is also linked to a collection of diseases known as Congenital Disorders of Glycosylation (CDGs) with patients suffering from neural dysfunction, organ failure, and growth retardation. The objective of the current proposed project is to apply metabolic engineering strategies to overcome bottlenecks in the N-glycosylation pathway that lead to the formation of underglycosylated proteins in mammalian cells of biotechnology and biomedical interest. The initial objective will be to examine the intermediates in the DLO metabolic pathway in order to determine which step(s) are causing N-glycosylation deficiency. Once the bottleneck enzyme is elucidated, the next goal will be to engineer relevant mammalian cell lines to overexpress the limiting enzyme or enzymes and increase the efficiency of N-glycosylation of proteins in wild type and mutant mammalian cells. Applying metabolic engineering strategies will provide insights into the metabolic steps controlling N-glycosylation that will be important to improving bioprocesses, treating CDGs disease, and perhaps characterizing the effects of alcoholism. A better understanding of N-linked glycosylation will ultimately lower health care production costs for life-saving therapeutics and provide new insights into ways to treat patients suffering from N-glycosylation disorders.

描述（由申请人提供）：生物技术行业中许多有价值的生物治疗药物是哺乳动物细胞分泌的糖蛋白产品。这些分泌的糖蛋白经常经历翻译后修饰，包括 N 连接糖基化（N-糖基化），其中涉及通过称为 寡糖转移酶（OST）。这些寡糖附着物（N-聚糖）对于蛋白质特性至关重要，包括折叠、稳定性、蛋白酶抗性、生物活性和体内清除率。在正常糖基化的位置产生不完全的 N-糖基化蛋白质产物表明多醇连接寡糖 (DLO) 底物或将寡糖转移到目标多肽上的 OST 酶水平存在缺陷。无法获得适当的 N-糖基化会降低产品质量和生物活性，增加生产成本并抑制产品商业化。未能获得适当的 N-糖基化还与一系列称为先天性糖基化障碍 (CDG) 的疾病有关，患者患有神经功能障碍、器官衰竭和生长迟缓。当前拟议项目的目标是应用代谢工程策略来克服 N-糖基化途径中的瓶颈，这些瓶颈导致生物技术和生物医学领域的哺乳动物细胞中糖基化不足的蛋白质的形成。最初的目标是检查 DLO 代谢途径中的中间体，以确定哪些步骤导致 N-糖基化缺陷。一旦阐明了瓶颈酶，下一个目标将是设计相关的哺乳动物细胞系，以过度表达限制酶或酶，并提高野生型和突变哺乳动物细胞中蛋白质的 N-糖基化效率。应用代谢工程策略将深入了解控制 N-糖基化的代谢步骤，这对于改善生物过程、治疗 CDGs 疾病以及可能表征酒精中毒的影响非常重要。对 N-连接糖基化的更好理解将最终降低救生疗法的医疗保健生产成本，并为治疗 N-糖基化疾病患者的方法提供新的见解。