Sheares B T, Robbins P W
Proc Natl Acad Sci U S A. 1986 Apr;83(7):1993-7. doi: 10.1073/pnas.83.7.1993.
In an effort to understand the factors that influence protein glycosylation, we are studying the expression of the chicken ovalbumin gene in a heterologous cell. Ovalbumin synthesized in mouse L-353 cells is glycosylated, as judged by incorporation of [3H]mannose and susceptibility to endo-beta-N-acetylglucosaminidases. Sequential digestion of ovalbumin synthesized in L cells with trypsin and chymotrypsin yields material migrating as one peak on HPLC coincident with similarly treated material from chicken ovalbumin, suggesting that the protein synthesized in L cells is glycosylated at the correct site. As in the case of chicken ovalbumin, the protein synthesized in L cells contains large amounts of hybrid oligosaccharides. Approximately 50% of the [3H]mannose incorporated into ovalbumin secreted by L cells is found in such hybrid structures. These results suggest that it is the polypeptide chain of ovalbumin that is responsible for proper glycosylation and subsequent processing of a substantial fraction of the oligosaccharide chains to hybrid structures. However, differences do exist between ovalbumin synthesized in L cells and the chicken glycoprotein. The hybrid oligosaccharides of ovalbumin secreted by L cells are completely sialylated and do not contain a bisecting GlcNAc residue, distinguishing them from hybrid chains in chicken ovalbumin. In addition to high-mannose and hybrid oligosaccharide chains, ovalbumin synthesized in L cells contains oligosaccharides of the complex type. To date, this type of sugar chain has not been observed in chicken ovalbumin. These differences in fine structure, between the oligosaccharides derived from ovalbumin secreted by L cells and those known to be present in the chicken egg glycoprotein, suggest that the cell type also plays a role in oligosaccharide processing.
为了了解影响蛋白质糖基化的因素，我们正在研究鸡卵清蛋白基因在异源细胞中的表达。通过[3H]甘露糖的掺入以及对内切β-N-乙酰葡糖胺糖苷酶的敏感性判断，在小鼠L-353细胞中合成的卵清蛋白发生了糖基化。用胰蛋白酶和糜蛋白酶对L细胞中合成的卵清蛋白进行顺序消化，产生的物质在高效液相色谱（HPLC）上作为一个峰迁移，与来自鸡卵清蛋白的经类似处理的物质一致，这表明在L细胞中合成的蛋白质在正确的位点发生了糖基化。与鸡卵清蛋白的情况一样，在L细胞中合成的蛋白质含有大量的杂合寡糖。掺入到L细胞分泌的卵清蛋白中的[3H]甘露糖，约50%存在于这种杂合结构中。这些结果表明，是卵清蛋白的多肽链负责大部分寡糖链正确的糖基化及随后加工成杂合结构。然而，在L细胞中合成的卵清蛋白与鸡糖蛋白之间确实存在差异。L细胞分泌的卵清蛋白的杂合寡糖完全被唾液酸化，并且不含有平分型N-乙酰葡糖胺残基，这将它们与鸡卵清蛋白中的杂合链区分开来。除了高甘露糖型和杂合寡糖链外，在L细胞中合成的卵清蛋白还含有复合型寡糖。迄今为止，在鸡卵清蛋白中尚未观察到这种类型的糖链。L细胞分泌的卵清蛋白衍生的寡糖与已知存在于鸡卵糖蛋白中的寡糖在精细结构上的这些差异表明，细胞类型在寡糖加工中也起作用。
