CARBOHYDRATE DETERMINANTS IN TRANSFERRIN RECEPTOR ACTION

哺乳动物细胞对铁的摄取涉及结合 转铁蛋白，血清铁转运蛋白，与转铁蛋白受体， 然后是受体-转铁蛋白复合体的内在化。铁 积累对于细胞分裂是必不可少的。快速增殖的细胞 比未分裂的细胞对铁的需求更高，受体具有 已被确定为增殖特异性标记物。 转铁蛋白受体含有三种天冬酰胺连接的寡糖， 它们是“高甘露糖”和“复杂”结构的混合体，至少 一个O-连接的(丝氨酸/苏氨酸)低聚糖。先前的研究使用 衣霉素处理的人类细胞已经证明了未糖化的 转铁蛋白受体的形式是不起作用的。因此，我们的目标是 这一提议是为了检验糖基化对 转铁蛋白受体的正确折叠和转运到细胞 表面和在结合或转铁蛋白。 定点突变将被用来测试每一种 天冬酰胺连接的糖基化位点与折叠和转运 转铁蛋白受体。单人、双人和三人的组合 将准备突变小鼠细胞系并对其进行测试。有证据表明 将低聚糖加工成复杂形式增强受体 这一过程的功能和作用将在糖基化过程中进行检验 中国仓鼠卵巢细胞缺陷型突变体。这样的突变体也将是 用来研究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直接 将根据结构(高)对它们进行分析 甘露糖/复合体)存在于天冬酰胺连接的每个位置上。 天然转铁蛋白受体及其在小鼠和小鼠体内的表达 CHO细胞转染法。这些分析将通过以下方式执行 碳水化合物和蛋白质化学技术，包括-反相 凝集素低聚糖的高效液相色谱、多肽测序及分析 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.