CYTOLOGIC ASSESSMENT OF NUCLEAR AND CYTOPLASMIC O-LINKED N-ACETYLGLUCOSAMINE DISTRIBUTION BY USING ANTISTREPTOCOCCAL MONOCLONAL-ANTIBODIES
CYTOLOGIC ASSESSMENT OF NUCLEAR AND CYTOPLASMIC O-LINKED N-ACETYLGLUCOSAMINE DISTRIBUTION BY USING ANTISTREPTOCOCCAL MONOCLONAL-ANTIBODIES
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DOI：
10.1073/pnas.87.15.5608
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发表时间：
1990-08-01
影响因子：
11.1
通讯作者：
GREENSPAN, NS
中科院分区：
文献类型：
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作者：
TURNER, JR;TARTAKOFF, AM;GREENSPAN, NS
最近的研究表明，在细胞质和核糖蛋白上存在单一的O-连接N-乙酰氨基葡萄糖(O-GlcNAc)残基。凝集素标记和酶技术已被用来鉴定O-GlcNAc蛋白，但还没有描述一般与这种O-连接的GlcNAc部分反应的抗体。我们以前已经鉴定了针对A组链球菌糖类的GlcNAc残基的单抗，它是由带有GlcNAc侧链的多聚鼠李糖骨架组成的。我们现在报道，这些单抗识别O-GlcNAc蛋白。通过免疫荧光，单抗与哺乳动物细胞的核周和核质反应强烈，而对细胞质的染色较弱。其分布与内质网、高尔基复合体或质膜的标记不一致。此外，突变细胞系在许多N-连接的糖链上保留末端GlcNAc残基的染色模式与野生型细胞难以区分。游离的GlcNAc抑制细胞核和细胞质的染色，而末端的GlcNAc残基的半乳糖化则完全消除。间接酶联免疫吸附试验证明，单抗与已知含有O-GlcNAc的65 kDa人红细胞胞浆蛋白的结合是GlcNAc和半乳糖化抑制的。因此，这些单抗与O-GlcNAc反应，不受多肽决定簇的明显影响，不与N-或O-葡聚糖结合，因此是研究O-GlcNAc部分的有价值的工具。此外，这些单抗首次对O-GlcNAc残基在哺乳动物细胞的细胞核和细胞质中的分布进行了细胞学分析。
Recent studies have demonstrated the existence of single O-linked N-acetylglucosamine (O-GlcNAc) residues on cytoplasmic and nuclear glycoproteins. Labeled lectin and enzymatic techniques have been used to identify O-GlcNAc-bearing proteins, but no antibodies generally reactive with such O-linked GlcNAc moieties have been described. We have previously characterized monoclonal antibodies (mAbs) specific for the GlcNAc residues of streptococcal group A carbohydrate, which is composed of a polyrhamnose backbone with GlcNAc side chains. We now report that these mAbs recognize O-GlcNAc-bearing proteins. By immmunofluorescence, the mAbs reacted strongly with the nuclear periphery and nucleoplasm of mammalian cells and stained the cytoplasm less intensely. The distribution was not consistent with labeling of the endoplasmic reticulum, Golgi complex, or plasma membrane. Furthermore, the staining pattern of a mutant cell line, which retains terminal GlcNAc residues on many N-linked glycans, was indistinguishable from that of wild-type cells. Nuclear and cytoplasmic staining were inhibited by free GlcNAc and were completely abolished by galactosylation of terminal GlcNAc residues. Indirect ELISA demonstrated GlcNAc- and galactosylation-inhibitable binding of the mAbs to a 65-kDa human erythrocyte cytosolic protein known to contain O-GlcNAc. Thus, these mAbs react with O-GlcNAc without apparent influence of peptide determinants, do not show detectable binding to N- or O-glycans, and, therefore, represent a valuable tool for the study of O-GlcNAc moieties. In addition, these mAbs provide the first cytologic analysis of the distribution of O-GlcNAc residues throughout the nucleus and the cytoplasm of mammalian cells.