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: 7394514
• 负责人: MICHAEL J BETENBAUGH
• 金额: $ 20.54万
• 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 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7394514
• 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（申请人提供）：生物技术行业中许多有价值的生物治疗药物都是哺乳动物细胞分泌的糖蛋白产物。这些分泌的糖蛋白通常经历翻译后修饰，包括N-糖基化（N-糖基化），其涉及通过称为寡糖转移酶（OST）的多亚基酶将寡糖从长链类异戊二烯脂质（多萜醇）整体转移到含有共有序列Asn-XSer/Thr的新生多肽上。这些寡糖连接（N-聚糖）可能对蛋白质性质至关重要，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处产生不完全N-糖基化的蛋白产物表明在将寡糖转移到靶多肽上的多醇连接的寡糖（DLO）底物或OST酶的水平上的缺陷。不能获得适当的N-糖基化降低了产品质量和生物活性，增加了生产成本，并抑制了产品商业化。未能获得适当的N-糖基化也与一系列被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。目前拟议的项目的目标是应用代谢工程策略，以克服瓶颈的N-糖基化途径，导致生物技术和生物医学利益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，下一个目标将是工程化相关的哺乳动物细胞系，以过表达限制酶或酶，并增加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为控制N-糖基化的代谢步骤提供见解，这对改善生物过程，治疗CDGs疾病以及可能表征酒精中毒的影响至关重要。更好地了解N-连接糖基化将最终降低拯救生命疗法的医疗保健生产成本，并为治疗患有N-糖基化疾病的患者提供新的见解。

项目成果

Structure and synthesis of polyisoprenoids used in N-glycosylation across the three domains of life.

• DOI: 10.1016/j.bbagen.2009.03.030
• 发表时间: 2009-06
• 期刊: BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS
• 影响因子: 3
• 作者: Jones, Meredith B.;Rosenberg, Julian N.;Betenbaugh, Michael J.;Krag, Sharon S.
• 通讯作者: Krag, Sharon S.

Analysis and metabolic engineering of lipid-linked oligosaccharides in glycosylation-deficient CHO cells.

• DOI: 10.1016/j.bbrc.2010.03.117
• 发表时间: 2010-04-23
• 期刊: BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
• 影响因子: 3.1
• 作者: Jones, Meredith B.;Tomiya, Noboru;Betenbaugh, Michael J.;Krag, Sharon S.
• 通讯作者: Krag, Sharon S.

Characterization of intact glycopeptides reveals the impact of culture media on site-specific glycosylation of EPO-Fc fusion protein generated by CHO-GS cells.

完整糖肽的表征揭示了培养基对 CHO-GS 细胞产生的 EPO-Fc 融合蛋白位点特异性糖基化的影响。

• DOI: 10.1002/bit.27009
• 发表时间: 2019
• 期刊: Biotechnology and bioengineering
• 影响因子: 3.8
• 作者: Wang,Qiong;Yang,Ganglong;Wang,Tiexin;Yang,Weiming;Betenbaugh,MichaelJ;Zhang,Hui
• 通讯作者: Zhang,Hui