Vertel B M, Hitti Y
Coll Relat Res. 1987 Apr;7(1):57-75. doi: 10.1016/s0174-173x(87)80021-3.
Early steps in the biosynthesis of chondroitin sulfate proteoglycan (CSPG) and collagenous cartilage matrix molecules were examined by the comparison of products translated in mRNA-directed cell-free reactions and those synthesized by intact cartilage cells. RNA isolated from embryonic chicken sterna was used to direct cell-free translation reactions. Chicken sternal chondrocytes in culture were pulse-labeled with [35S]-methionine. The CSPG core protein was identified by immunoprecipitation. The Mr of the cartilage cell-synthetized core protein was determined to be 370K, approximately 10-15K greater than that of the comparable cell-free translation product. Experimental results strongly support the view that the observed difference in Mr reflects the cotranslational addition of mannose-rich, N-asparagine-linked oligosaccharides to the cell-synthesized core protein: 1) the cell-synthesized product was labeled with [3H]-mannose and precipitated by concanavalin A-sepharose beads; 2) the incorporated [3H]-mannose could be subsequently removed by digestion with endoglycosidase H (Endo H); 3) the Mr of the cell-synthesized core protein was reduced by Endo H digestion to that of the comparable cell-free translation product; 4) the core protein synthesized by tunicamycin-treated chondrocytes (inhibited in their ability to add N-asparagine-linked mannose-rich oligosaccharides to proteins) was comparable in electrophoretic mobility to that of the core protein cell-free translation product; and 5) the core protein translated in microsome-coupled cell-free reactions had an Mr 8-10K greater than that of the core protein translated in the absence of microsomes. For the purpose of examining biosynthetic intermediates, chondrocytes were labeled continuously or pulse-chase labeled for varying times. No biosynthetic CSPG intermediates migrating between the core protein and the CSPG monomer were detected. However, a band of 355Kdal appeared to share certain characteristics with the 307Kdal core protein (including its immunoprecipitability with CSPG antibodies), and a 340Kdal band was noted. Type II procollagen and other collagenase-sensitive products of 205Kdal and 110Kdal were observed among translation and chondrocyte-synthesized products. In chondrocytes, all three products exhibited labeling or chase time-dependent increases in Mr which were accelerated by ascorbate supplements and inhibited by the addition of alpha, alpha'-dipyridyl. These results suggest that the observed time-dependent increases in Mr are a consequence of collagen hydroxylation. The 110Kdal and 205Kdal collagenous proteins may be related to the minor collagens recently described in cartilage.
通过比较在mRNA指导的无细胞反应中翻译的产物与完整软骨细胞合成的产物，研究了硫酸软骨素蛋白聚糖（CSPG）和胶原软骨基质分子生物合成的早期步骤。从胚胎鸡胸骨中分离的RNA用于指导无细胞翻译反应。培养的鸡胸骨软骨细胞用[35S]-甲硫氨酸进行脉冲标记。通过免疫沉淀鉴定CSPG核心蛋白。软骨细胞合成的核心蛋白的Mr测定为370K，比相应的无细胞翻译产物大约大10 - 15K。实验结果有力地支持了这样一种观点，即观察到的Mr差异反映了富含甘露糖的N-天冬酰胺连接的寡糖在共翻译过程中添加到细胞合成的核心蛋白上：1）细胞合成的产物用[3H]-甘露糖标记，并用伴刀豆球蛋白A-琼脂糖珠沉淀；2）随后可以通过用内切糖苷酶H（Endo H）消化去除掺入的[3H]-甘露糖；3）Endo H消化后，细胞合成的核心蛋白的Mr降低到与相应的无细胞翻译产物相同；4）用衣霉素处理的软骨细胞合成的核心蛋白（其向蛋白质添加N-天冬酰胺连接的富含甘露糖的寡糖的能力受到抑制）在电泳迁移率上与核心蛋白无细胞翻译产物相当；5）在微粒体偶联的无细胞反应中翻译的核心蛋白的Mr比在没有微粒体的情况下翻译的核心蛋白大8 - 10K。为了研究生物合成中间体，对软骨细胞进行连续标记或脉冲追踪标记不同时间。未检测到在核心蛋白和CSPG单体之间迁移的生物合成CSPG中间体。然而，一条355Kdal的条带似乎与307Kdal的核心蛋白具有某些共同特征（包括其与CSPG抗体的免疫沉淀性），并且注意到一条340Kdal的条带。在翻译产物和软骨细胞合成产物中观察到II型前胶原以及205Kdal和110Kdal的其他胶原酶敏感产物。在软骨细胞中，所有这三种产物在Mr上均表现出标记或追踪时间依赖性增加，抗坏血酸补充剂可加速这种增加，而添加α，α'-联吡啶则可抑制这种增加。这些结果表明，观察到的Mr时间依赖性增加是胶原羟基化的结果。110Kdal和205Kdal的胶原蛋白可能与最近在软骨中描述的次要胶原有关。
