C-mannosylation of human RNase 2 is an intracellular process performed by a variety of cultured cells
C-mannosylation of human RNase 2 is an intracellular process performed by a variety of cultured cells
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DOI：
10.1074/jbc.272.42.26687
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发表时间：
1997-10-17
影响因子：
4.8
通讯作者：
Hofsteenge, J
中科院分区：
文献类型：
--
作者：
Krieg, J;Glasner, W;Hofsteenge, J
C-2-α-甘露糖基色氨酸是在人尿中的核糖核酸酶2中发现的，代表了一种将碳水化合物连接到蛋白质上的新方法。在这里，我们解决了与这种修饰的生物合成相关的两个问题：（i）C-甘露糖基化是正常细胞内生物合成途径的一部分吗？（ii）它的普遍性如何，即哪些生物体进行这种糖基化？为了回答第一个问题，从培养的人HL-60细胞的细胞内储存中分离出与嗜酸性粒细胞衍生的神经毒素相同的RNA酶2。该酶在Trp-7处被C-甘露糖基化，表明该修饰发生在细胞内，在蛋白质分泌之前。第二个问题通过从来自不同生物体的瞬时转化细胞的上清液中纯化的RNA酶2的免疫学和化学分析来研究，这表明C-甘露糖基化发生在来自人、绿色猴、猪、小鼠和仓鼠的细胞中，观察到猪肾细胞含有人RNase 2的Trp-7的C-甘露糖基化的机制，但来自猪肾的同源RNase不是底物，因为它在7位不含色氨酸，这强烈表明存在RNase 2以外的C-甘露糖基化蛋白，从昆虫细胞、植物原生质体和大肠杆菌中分离的重组RNase 2不被C-甘露糖基化。这些结果不仅为进一步研究C-甘露糖基化的生物化学方面奠定了基础，而且对生产重组糖蛋白的细胞的选择也有一定的意义。
C-2-alpha-Mannosyltryptophan was discovered in RNase 2 from human urine, representing a novel way of attaching carbohydrate to a protein, Here, we have addressed two questions related to the biosynthesis of this modification: (i) is C-mannosylation part of the normal intracellular biosynthetic route, and (ii) how general is it, i.e. which organisms perform this kind of glycosylation? To answer the first question, RNase 2, which is identical to the eosinophil-derived neurotoxin, was isolated from intracellular stores of cultured human HL-60 cells. The enzyme was C-mannosylated at Trp-7, showing that the modification occurs intracellularly, before secretion of the protein, The second question was investigated by immunological and chemical analysis of RNase 2 purified from the supernatant of transiently transformed cells from different organisms, This revealed that C-mannosylation occurs in cells from man, green monkey, pig, mouse, and hamster, The observation that pig kidney cells contain the machinery for C-mannosylation of Trp-7 of human RNase 2 but that the homologous RNase from porcine kidney is not a substrate, since it does not contain a tryptophan at position 7, strongly suggests that C-mannosylated proteins other than RNase 2 exist, Recombinant RNase 2 isolated from insect cells, plant protoplasts, and Escherichia coli was not C-mannosylated, These results not only form the basis for further studies on the biochemical aspects of C-mannosylation but also have implications for the choice of cells for production of recombinant glycoproteins.