OLIGOSACCHARIDE ASSEMBLY ON RECOMBINANT PROTEINS

这项研究的长期目标是确定一些因素， 控制糖蛋白上聚糖组装的性质，重点是 由不同细胞和蛋白质决定的特异性控制 衬底 因为我们之前的调查显示 重组（r）人纤溶酶原（HPg）， 纤维蛋白溶解系统，可能包含结构决定因素的类型 在其唯一的N-连接糖基化位点上组装的聚糖，强调 已经开始研究这种蛋白质。 五个具体目标是 建议：（1）调查外观的时间性质 不同类型的N-和O-连接的聚糖在r-HPg上，以及在r- 尿激酶（UK），在几种r-杆状病毒感染的 鳞翅目昆虫细胞系;（2）评估是否时间 r-HPg上的寡糖加工发生在病毒感染的 哺乳动物细胞系;（3）确定Man 6- 甘露糖苷酶和N乙酰葡糖胺转移酶-1（GlcNAc-TI）活性 在杆状病毒感染的昆虫细胞中， （4）测定GlcNAc-1的酶活性。 TII、GlcNAc-TIII、甘露糖苷酶II、α-岩藻糖基转移酶、β- 半乳糖基转移酶和α-唾液酸转移酶，以及 对于r-HPg的O-连接糖基化重要的转移酶， 来自未感染、R病毒感染和对照的亚细胞组分- 病毒感染的昆虫细胞;和（5）纯化Man 6-甘露糖苷酶， 进一步表征，并纯化GlcNAc-TI用于表征， 克隆和评估其刺激的分子基础， 杆状病毒感染的细胞。 本项目的设计涉及聚糖结构的测定 在不同病毒感染细胞中表达的R蛋白上 系统. 关键糖加工酶的活性也将被 在这些相同的感染细胞系中进行了研究。 获得的结果将 允许制定战略，以控制 糖蛋白的糖基化，使它们与它们的 自然状态，一个特别重要的因素，当治疗使用 考虑了这样的蛋白质。 由于蛋白质上的聚糖 影响其结构、功能、免疫原性、周转 特征和/或与细胞决定簇的结合， 属性，理解如何控制 存在的聚糖将是最有价值的科学资产。

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.