Olden K, Hunter V A, Yamada K M
Biochim Biophys Acta. 1980 Oct 15;632(3):408-16. doi: 10.1016/0304-4165(80)90236-6.
We have examined the maturation or processing of the oligosaccharides of cellular fibronectin in cultured chick embryo fibroblasts. Fibronectin was pulse-labeled with [2-3H]mannose of [35S]methionine, and the turnover rates of carbohydrate and polypeptide portions of immunoprecipitated fibronectin were compared. The oligosaccharides on fibronectin were analyzed by gel electrophoresis for alterations in sensitivity to the enzyme endo-beta-N-acetylgluosaminidase H, which specifically cleaves the 'high-mannose' class of asparagine-linked oligosaccharide. Incorporated mannose was removed only at early time points, suggesting that the structure of fibronectin oligosaccharides was altered due to processing. This possibility was confirmed by the analysis of glycopeptides generated by exhaustive pronase digestion. Two major glycopeptide structures were detected; their properties correspond to a 'high-mannose' oligosaccharide precursor and a 'complex' carbohydrate product. The precursor-product relationship of these two forms of oligosaccharide chains was demonstrated by pulse-chase labeling experiments. The precursor glycopeptide had an apparent size (Mr 2100) comparable to (Man)9GlcNAc (Mr 2080), and was sensitive to endo-beta-N-acetylglucosaminidase H; nearly all of the labeled mannose incorporated in a 10 min pulse was released from fibronectin glycopeptides by this enzyme. During a 90 min chase period, the glycopeptides became larger and increasingly resistant to endo-beta-N-acetylglucosaminidase H cleavage. The final 'complex' or processed oligosaccharide structure contained approximately two-thirds less [3H]mannose, was insensitive to endo-beta-N-acetylglucosaminidase H and had an apparent Mr of 2300 as estimated by gel filtration. We conclude that the carbohydrate portion of fibronectin is synthesized as a 'high-mannose' intermediate and is subsequently processed to give the characteristic 'complex' oligosaccharide chains of fibronectin.
我们研究了培养的鸡胚成纤维细胞中细胞纤连蛋白寡糖的成熟或加工过程。用[2-³H]甘露糖或[³⁵S]甲硫氨酸对纤连蛋白进行脉冲标记，并比较免疫沉淀的纤连蛋白碳水化合物和多肽部分的周转速率。通过凝胶电泳分析纤连蛋白上的寡糖，以检测其对内切-β-N-乙酰葡糖胺糖苷酶H的敏感性变化，该酶特异性切割天冬酰胺连接寡糖的“高甘露糖”类别。仅在早期时间点掺入的甘露糖被去除，这表明纤连蛋白寡糖的结构因加工而改变。通过对蛋白酶彻底消化产生的糖肽进行分析，证实了这种可能性。检测到两种主要的糖肽结构；它们的性质对应于“高甘露糖”寡糖前体和“复杂”碳水化合物产物。通过脉冲追踪标记实验证明了这两种寡糖链形式的前体-产物关系。前体糖肽的表观大小（Mr 2100）与(Man)9GlcNAc（Mr 2080）相当，并且对内切-β-N-乙酰葡糖胺糖苷酶H敏感；在10分钟脉冲中掺入的几乎所有标记甘露糖都被该酶从纤连蛋白糖肽中释放出来。在90分钟的追踪期内，糖肽变得更大，并且对内切-β-N-乙酰葡糖胺糖苷酶H切割的抗性越来越强。最终的“复杂”或加工后的寡糖结构所含的[³H]甘露糖大约少三分之二，对内切-β-N-乙酰葡糖胺糖苷酶H不敏感，并且通过凝胶过滤估计其表观Mr为2300。我们得出结论，纤连蛋白的碳水化合物部分作为“高甘露糖”中间体合成，随后被加工成纤连蛋白特有的“复杂”寡糖链。
