CARBOHYDRATE DETERMINANTS IN TRANSFERRIN RECEPTOR ACTION

哺乳动物细胞对铁的摄取涉及铁与铁的结合。 转铁蛋白，血清铁转运蛋白，转铁蛋白受体， 随后是受体-转铁蛋白复合物的内化。 铁 积累对于细胞分裂是必不可少的。 快速增殖细胞 比非分裂细胞需要更高的铁， 被鉴定为增殖特异性标记物。 转铁蛋白受体含有三个天冬酰胺连接的寡糖， 其是“高甘露糖”和“复杂”结构的混合物，并且至少 一个O-连接（丝氨酸/苏氨酸）寡糖。 既往研究使用 衣霉素处理的人类细胞已经证明， 转铁蛋白受体的形式没有功能。 因此， 这个提议是为了检验糖基化对于 转铁蛋白受体的正确折叠和转运到细胞 表面和结合或转铁蛋白中。 将使用定点诱变来测试以下各项的贡献： 天冬酰胺连接的糖基化位点的折叠和运输 转铁蛋白受体 单、双、三重组合 将制备突变并测试小鼠细胞系。 证据表明 将低聚糖加工成复合物形式， 功能和作用，这一进程将检查在糖基化 缺乏中国人卵巢细胞突变体。 这种突变体也将 研究O-连接糖基化在受体中的作用 功能 转铁蛋白受体寡糖还没有被直接 特征，并将分析它们相对于结构（高 甘露糖/复合物），其存在于所述复合物中的每个天冬酰胺连接位点上。 天然转铁蛋白受体以及在鼠和 转染后的CHO细胞。 这些分析将通过 碳水化合物和蛋白质化学技术，包括反相 凝集素上寡糖的高效液相色谱、肽段测序及分析 亲和柱和分子筛柱。

The uptake of iron into mammalian cells involves the binding of transferrin, the serum iron transport protein, to the tranferin receptor, followed by the internalization of the receptor-transferrin complex. Iron accumulation is essential for cell division. Rapidly proliferating cells have higher iron requirements than non-dividing cells and the receptor has been identified as a proliferation specific marker. The transferrin receptor contains three asparagine-linked oligosaccharides, which are a mixture of "high mannose" and "complex" structures and at least one O-linked (serine/threonine) oligosaccharide. Prior studies using tunicamycin-treated human cells have demonstrated that the unglycosylated from of the transferrin receptor is not functional. Thus, the goals of this proposal are to test the hypothesis that glycosylation is vital for the correct folding and transport of the transferrin receptor to the cell surface and in the binding or transferrin. Site-directed mutagenesis will be used to test the contribution of each of the asparagine-linked glycosylation sites to the folding and transport of the transferrin receptor. Combinations of single, double and triple mutations will be prepared and tested mouse cell lines. Evidence suggests that processing of oligosaccharides to complex forms enhances receptor function and the role of this processing will be examined in glycosylation deficient Chinese Hamster Ovary cell mutants. Such mutants will also be sued to investigate the role of O-linked glycosylation in receptor function. Transferrin receptor oligosaccharides have not been directly characterized and they will be analyzed with respect to structure (high mannose/complex) present on each of the asparagine-linked sites in the native transferrin receptor as well as the receptor expressed in murine and CHO cells following transfection. These analyses will be performed via carbohydrate and protein chemical techniques, including - reverse phase HPLC, peptides sequencing and analysis of oligosaccharides on lectin affinity and molecular sieve columns.