The subcellular distribution of terminal N-acetylglucosamine moieties. Localization of a novel protein-saccharide linkage, O-linked GlcNAc.
The subcellular distribution of terminal N-acetylglucosamine moieties. Localization of a novel protein-saccharide linkage, O-linked GlcNAc.
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DOI：
10.1016/s0021-9258(19)57510-x
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发表时间：
1986-06
期刊：
影响因子：
--
通讯作者：
G. Holt;G. Hart
中科院分区：
文献类型：
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作者：
G. Holt;G. Hart
以前我们的实验室报道了发现一种新的蛋白质-糖键，其中单个N-乙酰葡糖胺（GlcNAc）残基以O-键连接到蛋白质上（托雷斯，C. R.，和哈特，G. W.（1984）J.Biol.Chem.259，3308-3317）。这种连接首先通过牛乳半乳糖基转移酶的半乳糖基化作用在活细胞的质膜蛋白上发现。在这里，我们报告的O-连接GlcNAc在高度富集的大鼠肝脏亚细胞器中的分布。通过半乳糖基转移酶和UDP-[3 H]半乳糖标记Nonidet P-40溶解的细胞器，并通过其对β-消除的敏感性和对内切-β-N-乙酰氨基葡萄糖苷酶F去糖基化的抗性评估O-键中GlcNAc残基上发生的放射性标记量。半乳糖标记的O-连接的GlcNAc残基的存在下，通过高压纸电泳确认。在各种细胞器中发现的半乳糖基化末端GlcNAc残基的量为每mg蛋白质的17倍范围，以及细胞器的O-连接的GlcNAc残基的表观含量的宽范围。大鼠肝细胞的细胞核和可溶性部分特别富含具有O-连接GlcNAc残基的蛋白质，尽管这些残基在几乎所有测试的细胞器中都是可证明的。此外，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳的检查显示，许多不同的细胞器特异性蛋白质被O-连接的GlcNAc残基糖基化。由于这种独特的连接的广泛发生，这些数据表明，糖基化与O-连接的GlcNAc残基是不是一个特定的细胞器的排他性标志物。此外，我们还调查了细胞器中含有GlcNAc终止的N-连接寡糖的糖蛋白的含量。我们的数据表明，有大量的这些寡糖粗糙和剥离微粒体，细胞核和核膜。鉴于末端GlcNAc转移酶定位于高尔基复合体的证据，这些数据表明有糖蛋白进入高尔基体进行加工，然后被转运回粗面内质网，并且可能转运回细胞核。
Previously our laboratory reported the discovery of a novel protein-saccharide linkage in which single N-acetylglucosamine (GlcNAc) residues are attached in O-linkages to protein (Torres, C. R., and Hart, G. W. (1984) J. Biol. Chem. 259, 3308-3317). This linkage was first found on plasma membrane proteins of living cells by galactosylation with bovine milk galactosyltransferase. Here we report the distribution of O-linked GlcNAc in highly enriched rat liver subcellular organelles. Nonidet P-40 solubilized organelles were labeled by galactosyltransferase with UDP-[3H]galactose, and the amount of radiolabel occurring on GlcNAc residues in O-linkages was assessed by its sensitivity to beta-elimination and by its resistance to deglycosylation with endo-beta-N-acetylglucosaminidase F. The presence of galactose-labeled O-linked GlcNAc residues was confirmed by high voltage paper electrophoresis. There is a 17-fold range per mg of protein in the amount of galactosylatable terminal GlcNAc residues found in the various organelles, as well as a wide range in the organelles' apparent content of O-linked GlcNAc residues. Nuclei and the soluble fraction of rat liver cells are particularly enriched with proteins bearing O-linked GlcNAc residues, although these residues are demonstrable in virtually all organelles tested. Furthermore, examination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that many different organelle-specific proteins are glycosylated with O-linked GlcNAc residues. Because of the wide occurrence of this unique linkage, these data suggest that glycosylation with O-linked GlcNAc residues is not an exclusive marker for a particular organelle. In addition, we have surveyed the organelles for their content of glycoproteins bearing GlcNAc-terminated N-linked oligosaccharides. Our data demonstrate that there are significant amounts of these oligosaccharides in rough and stripped microsomes, nuclei, and nuclear envelopes. In light of evidence that terminal GlcNAc transferases are localized to the Golgi complex, these data suggest that there are glycoproteins which enter into the Golgi for processing and then are transported back into the rough endoplasmic reticulum, and possibly the nucleus.