Bailey D S, Burke J, Sinclair R, Mukherjee B B
Biochem J. 1981 Apr 1;195(1):139-51. doi: 10.1042/bj1950139.
Glycoprotein biosynthesis was studied with mouse L-cells grown in suspension culture. Glucose-deprived cells incorporated [3H]mannose into 'high-mannose' protein-bound oligosaccharides and a few relatively high-molecular-weight lipid-linked oligosaccharides. The latter were retained by DEAE-cellulose and turned over quite slowly during pulse--chase experiments. Increased heterogeneity in size of lipid-linked oligosaccharides developed during prolonged glucose deprivation. Sequential elongation of lipid-linked oligosaccharides was also observed, and conditions that prevented the assembly of the higher lipid-linked oligosaccharides also prevented the formation of the larger protein-bound 'high-mannose' oligosaccharides. In parallel experiments, [3H]mannose was incorporated into a total polyribosome fraction, suggesting that mannose residues were transferred co-translationally to nascent protein. Membrane preparations from these cells catalysed the assembly from UDP-N-acetyl-D-[6-3H]glucosamine and GDP-D-[U-14C]mannose of polyisoprenyl diphosphate derivatives whose oligosaccharide moieties were heterogeneous in size. Elongation of the N-acetyl-D-[6-3H]glucosamine-initiated glycolipids with mannose residues produced several higher lipid-linked oligosaccharides similar to those seen during glucose deprivation in vivo. Glucosylation of these mannose-containing oligosaccharides from UDP-D-[6-3H]glucose was restricted to those of a relatively high molecular weight. Protein-bound saccharides formed in vitro were mainly smaller in size than those assembled on the lipid acceptors. These results support the involvement of lipid-linked saccharides in the synthesis of asparagine-linked glycoproteins, but show both in vivo and in vitro that protein-bound 'high-mannose' oligosaccharide formation can occur independently of higher lipid-linked oligosaccharide synthesis.
利用悬浮培养的小鼠L细胞研究了糖蛋白的生物合成。缺乏葡萄糖的细胞将[3H]甘露糖掺入“高甘露糖型”蛋白结合寡糖和一些相对高分子量的脂连接寡糖中。后者能被二乙氨基乙基纤维素保留，并且在脉冲追踪实验中周转相当缓慢。在长时间葡萄糖缺乏期间，脂连接寡糖的大小异质性增加。还观察到脂连接寡糖的顺序延伸，并且阻止较高脂连接寡糖组装的条件也阻止了较大的蛋白结合“高甘露糖型”寡糖的形成。在平行实验中，[3H]甘露糖掺入了总多核糖体组分中，这表明甘露糖残基是共翻译转移到新生蛋白质上的。这些细胞的膜制剂催化了由UDP-N-乙酰-D-[6-3H]葡萄糖胺和GDP-D-[U-14C]甘露糖组装聚异戊二烯二磷酸衍生物，其寡糖部分大小各异。用甘露糖残基延伸以N-乙酰-D-[6-3H]葡萄糖胺起始的糖脂产生了几种较高的脂连接寡糖，类似于在体内葡萄糖缺乏期间所见的那些。来自UDP-D-[6-3H]葡萄糖的这些含甘露糖寡糖的糖基化仅限于相对高分子量的那些。体外形成的蛋白结合糖类在大小上主要比在脂受体上组装的糖类小。这些结果支持脂连接糖类参与天冬酰胺连接糖蛋白的合成，但在体内和体外均表明，蛋白结合“高甘露糖型”寡糖的形成可以独立于较高脂连接寡糖的合成而发生。
