Glycobiology 2018-03-19

AANL (Agrocybe aegerita lectin 2) is a new facile tool to probe for O-GlcNAcylation

Wei Liu, Guanghui Han, Yalin Yin, Shuai Jiang, Guojun Yu, Qing Yang, Wenhui Yu, Xiangdong Ye, Yanting Su, Yajun Yang, Gerald W Hart, Hui Sun

文献索引：10.1093/glycob/cwy029

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摘要

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, AANL (Agrocybe aegerita GlcNAc specific lectin), also reported as AAL2, can be used as a powerful probe for O-GlcNAc identification. Glycan array analyses and SPR (surface plasmon resonance) assays show that AANL binds to GlcNAc with a dissociation constant (KD) of 94.6 μM, which is consistent with the result tested through ITC assay reported before (Jiang et al. 2012). 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 29 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.