Characterization of a mouse monoclonal antibody specific for O-linked N-acetylglucosamine
Characterization of a mouse monoclonal antibody specific for O-linked N-acetylglucosamine
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DOI：
10.1006/abio.2001.5132
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发表时间：
2001-06-15
影响因子：
2.9
通讯作者：
Hart, GW
中科院分区：
文献类型：
--
作者：
Comer, FI;Vosseller, K;Hart, GW
β -O-linked N-acetylglucosamine （O-GlcNAc）是真核生物中大量的核蛋白和细胞质蛋白翻译后修饰物。越来越多的证据表明，O-GlcNAc在许多细胞过程中起调节作用。在这里，我们报道了一种高度特异性的小鼠单克隆抗体的生产和表征，MAb CTD110.6，特异性地与O-GlcNAc反应。该抗体识别β - o -糖苷连接丝氨酸和苏氨酸的O-GacNAc。我们可以检测到与α -连接的Ser/Thr-O-GrlcNAc， cu-连接的Ser- o -连接的n -乙酰半乳糖胺（O-GalNAc）或n -连接的寡糖在卵清蛋白和免疫球蛋白g上没有交叉反应性。单糖GlcNAc而不是GalNAc消除了免疫反应性，进一步证明了对O-GlcNAc的特异性，此外，半乳糖覆盖O-GlcNAc位点也抑制了CTD110.6的免疫反应性。利用凝集素小麦perm凝集素富集含glcnac的糖蛋白可显著富集ctd110.6反应蛋白。该抗体与细胞质和核提取物中的大量蛋白质反应，并易于检测O-GlcNAc修饰的体内变化。这些研究表明，CTD110.6对O-GlcNAc具有高度特异性，对类似的碳水化合物抗原或肽决定因子无交叉反应性。(C) 2001学术出版社。
beta -O-linked N-acetylglucosamine (O-GlcNAc) is an abundant posttranslational modification of resident nuclear and cytoplasmic proteins in eukaryotes. Increasing evidence suggests that O-GlcNAc plays a regulatory role in numerous cellular processes. Here we report on the production and characterization of a highly specific mouse monoclonal antibody, MAb CTD110.6, that specifically reacts with O-GlcNAc. The antibody recognizes O-GacNAc in beta -O-glycosidic linkage to both serine and threonine. We could detect no cross-reactivity with alpha -linked Ser/Thr-O-GrlcNAc, cu-linked Ser-O-linked N-acetylgalactosamine (O-GalNAc), or N-linked oligosaccharides on ovalbumin and immunoglobulin G. The monosaccharide GlcNAc, but not GalNAc, abolishes immunoreactivity, further demonstrating specificity toward O-GlcNAc, Furthermore, galactose capping of O-GlcNAc sites also inhibits CTD110.6 immunoreactivity. Enrichment of GlcNAc-containing glycoproteins using the lectin wheat perm agglutinin dramatically enriches for CTD110.6-reactive proteins. The antibody reacts with a large number of proteins from cytoplasmic and nuclear extracts and readily detects in vivo changes in O-GlcNAc modification. These studies demonstrate that CTD110.6 is highly specific toward O-GlcNAc, with no cross-reactivity toward similar carbohydrate antigens or toward peptide determinants. (C) 2001 Academic Press.