OLIGOSACCHARIDE ASSEMBLY ON RECOMBINANT PROTEINS

重组蛋白上的寡糖组装

• 批准号: 2225273
• 负责人: FRANCIS J CASTELLINO
• 金额: $ 19.86万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2225273
• 关键词: Baculoviridae; Lepidoptera; N acetylglucosamine; SDS polyacrylamide gel electrophoresis; complementary DNA; dolichol; enzyme activity; enzyme mechanism; fucose; galactosyltransferases; glycosylation; mannosidase; molecular cloning; oligosaccharides; plasminogen; proteoglycan; recombinant proteins; sialyltransferases; tissue /cell culture; urokinase; western blottings

The long-range goal of this research is to identify some of the factors that govern the nature of glycan assembly on glycoproteins, with emphasis on specificity controls dictated by different cells and by the protein substrate. Since our previous investigations have suggested that recombinant (r) human plasminogen (HPg), a key zymogen of the fibrinolytic system, may contain structural determinants for the types of glycans assembled on its sole N-linked glycosylation site, emphasis has been placed on studies with this protein. Five specific aims are proposed: (1) to investigate the temporal nature of the appearance of different types of N- and O-linked glycans on r-HPg, as well as on r- urokinase (UK), that are expressed in several r-baculovirus-infected lepidopteran insect cell lines; (2) to evaluate whether temporal processing of oligosaccharides on r-HPg occurs in a virally-infected mammalian cell line; (3) to determine whether the stimulations of Man6- mannosidase and Nacetylglucosaminyl-transferase-1 (GlcNAc-TI) activities in baculovirus-infected insect cells are general properties of their glycosylation machinery; (4) to assay the enzymatic activities of GlcNAc- TII, GlcNAc-TIII, mannosidase II, alpha-fucosyltransferase, beta- galactosyltransferase, and alpha-sialyltransferases, as well as transferases important for O-linked glycosylation of r-HPg, in subcellular fractions from noninfected, r-virus infected, and control- virus infected insect cells; and (5) to purify the Man6-mannosidase for further characterization, and to purify GlcNAc-TI for characterization, cloning and evaluation of the molecular basis of its stimulation in baculovirus-infected cells. The design of this project involves determination of glycan structures on r-proteins that have been expressed in different virally-infected cell systems. The activities of key glycoprocessing enzymes will be also be studied in these same infected cell lines. The results obtained will allow development of strategies to control the nature of the glycosylation of rglycoproteins so that they will closely resemble their natural states, a factor particularly important when therapeutic use of such proteins is contemplated. Since the glycans present on proteins may affect their structures, functions, immunogenicities, turnover characteristics, and/or binding to cellular determinants, among other properties, the ability to understand how to control the nature of the glycans present will be a most valuable scientific asset.

这项研究的远程目标是确定一些因素 在糖蛋白上管理聚糖大会的性质，重点 关于不同细胞和蛋白质决定的特异性控制 基材。 自从我们以前的调查表明 重组（R）人纤溶酶原（HPG），这是一种关键的Zymogen 纤维蛋白水解系统可能包含类型的结构决定因素 聚集在其唯一的N连接糖基化位点上的聚糖的重点 已被放置在该蛋白质的研究上。 五个具体目标是 提议：（1）研究外观的时间性质 R-HPG以及R-上的不同类型的N-和O连接的聚糖 尿激酶（英国），在几种R-baculovirus感染者中表达 鳞翅目昆虫细胞系； （2）评估是否暂时 R-HPG上寡糖的加工发生在病毒感染中 哺乳动物细胞系； （3）确定MAN6-的刺激是否 甘露糖苷酶和nacetyl-葡萄氨酰转移酶-1（GlcNAC-TI）活性 在杆状病毒感染的昆虫细胞中是其一般特性 糖基化机制； （4）分析GlcNAC-的酶促活性 TII，GlcNAC-TIII，甘露糖苷酶II，α-凝血基转移酶，β- 半乳糖基转移酶和α-溶解酶以及 转移酶对R-HPG的O连接糖基化重要的转移酶，在 来自未感染，R-病毒感染和控制的亚细胞级分 病毒感染的昆虫细胞； （5）净化Man6-甘露糖苷酶 进一步表征，并纯化glcnac-ti进行表征， 克隆和评估其刺激的分子基础 杆状病毒感染的细胞。 该项目的设计涉及确定聚糖结构 关于在不同病毒感染细胞中表达的R蛋白 系统。 关键糖化酶的活动也将是 在这些相同的感染细胞系中进行了研究。 获得的结果将 允许制定策略来控制 rglycoprotins的糖基化，以使它们非常类似于它们 自然状态，当治疗使用时尤其重要 考虑了这种蛋白质。 由于蛋白质上存在的聚糖可能 影响其结构，功能，免疫原性，周转率 特征和/或与细胞决定因素的结合，以及其他 属性，理解如何控制本质的能力 在场的聚糖将是最有价值的科学资产。