O-linked N-acetylglucosamine (O-GlcNAcylation) is an important post-translational modification on serine or threonine of proteins, mainly observed in nucleus or cytoplasm. O-GlcNAcylation regulates many cell processes, including transcription, cell cycle, neural development and nascent polypeptide chains stabilization. However, the facile identification of O-GlcNAc is a major bottleneck in O-GlcNAcylation research. Herein, we report that a lectin, Agrocybe aegerita GlcNAc-specific lectin (AANL), also reported as AAL2, can be used as a powerful probe for O-GlcNAc identification. Glycan array analyses and surface plasmon resonance (SPR) assays show that AANL binds to GlcNAc with a dissociation constant (KD) of 94.6 μM, which is consistent with the result tested through isothiocyanate (ITC) assay reported before (Jiang S, Chen Y, Wang M, Yin Y, Pan Y, Gu B, Yu G, Li Y, Wong BH, Liang Y, et al. 2012. A novel lectin from Agrocybe aegerita shows high binding selectivity for terminal N-acetylglucosamine. Biochem J. 443:369–378.). Confocal imaging shows that AANL co-localizes extensively with NUP62, a heavily O-GlcNAcylated and abundant nuclear pore glycoprotein. Furthermore, O-GlcNAc-modified peptides could be effectively enriched in the late flow-through peak from simple samples by using affinity columns Sepharose 4B-_AANL or POROS-_AANL. Therefore, using AANL affinity column, we identified 28 high-confidence O-linked HexNAc-modified peptides mapped on 17 proteins involving diverse cellular progresses, including transcription, hydrolysis progress, urea cycle, alcohol metabolism and cell cycle. And most importantly, major proteins and sites were not annotated in the dbOGAP database. These results suggest that the AANL lectin is a new useful tool for enrichment and identification of O-GlcNAcylated proteins and peptides.

中文翻译：

---

AANL（Agrocybe aegerita 凝集素 2）是一种新的简便工具，用于探测 O-GlcNAcylation


O-连接的N-乙酰氨基葡萄糖( O -GlcNAcylation)是蛋白质丝氨酸或苏氨酸的重要翻译后修饰，主要在细胞核或细胞质中观察到。 O -GlcNAc 酰化调节许多细胞过程，包括转录、细胞周期、神经发育和新生多肽链稳定。然而， O -GlcNAc的简便鉴定是O -GlcNAc酰化研究的主要瓶颈。在此，我们报道了一种凝集素， Agrocybe aegerita GlcNAc 特异性凝集素（AANL），也被报道为 AAL2，可用作O -GlcNAc 鉴定的强大探针。聚糖阵列分析和表面等离子共振（SPR）分析表明，AANL 与 GlcNAc 结合的解离常数（KD）为 94.6 μM，这与之前报道的异硫氰酸酯（ITC）分析测试的结果一致（Jiang S，Chen Y， Wang M、Yin Y、Pan Y、Gu B、Yu G、Li Y、Wong BH、Liang Y 等，2012。来自杨树菇的新型凝集素对末端N-乙酰氨基葡萄糖显示出高结合选择性。 Biochem J. 443： 369–378。）。共聚焦成像显示 AANL 与 NUP62 广泛共定位，NUP62 是一种重度O -GlcNA 酰化且丰富的核孔糖蛋白。此外，通过使用亲和柱 Sepharose 4B- _AANL或 POROS- _AANL ， O -GlcNAc 修饰的肽可以在简单样品的后期流通峰中有效富集。 因此，使用 AANL 亲和柱，我们鉴定了 28 个高置信度 O 连接 HexNAc 修饰的肽，这些肽映射在涉及不同细胞进程（包括转录、水解进程、尿素循环、酒精代谢和细胞周期）的 17 种蛋白质上。最重要的是，主要蛋白质和位点在 dbOGAP 数据库中没有注释。这些结果表明，AANL 凝集素是富集和鉴定O -GlcNA 酰化蛋白和肽的新的有用工具。