Glycosylation plays important roles in many cellular processes, such as signal transduction, cell cycle progression and transcriptional regulation. However, the identification and analysis of glycosylation are severely hampered by the low specificity or avidity of antiglycan antibodies and lectins. We have reported that a lectin AANL, which has high specificity for terminal GlcNAc glycans and contains six carbohydrate binding sites (CBSs), was used to enrich O-GlcNAcylated peptides. To further improve AANL binding specificity, we designed a CBS-homogenization strategy and restructured six mutant lectins, known as AANL1-AANL6. Affinity chromatography with GlcNAc and isothermal titration calorimetry analysis indicated that the two mutants (AANL3 and AANL6) all maintained GlcNAc binding activity. AANL6 and AANL3 showed higher specificity for terminal GlcNAc glycans than AANL, as shown by the hemagglutination assay, cell binding assays and glycan microarray analysis, and AANL6 exhibited the highest specificity. The binding activity of AANL6 for O-GlcNAcylated peptides was shown by surface plasmon resonance assays. By AANL6 affinity chromatography enrichment and mass spectrometry analysis, 79 high-confidence and 21 putative O-GlcNAcylated sites were identified on 85 peptides mapped onto 54 proteins. Most of these sites were new sites compared with reported data. These results indicate that the enrichment capacity of AANL6 is higher than that of wild-type AANL. In conclusion, the CBS-homogenization mutation strategy was successful, and AANL6 was identified as a powerful tool for O-GlcNAcylation enrichment. Our research suggests that the CBS-homogenization strategy is valuable for improving the specificity of lectins with multiple CBSs.

中文翻译：

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CBS均质化突变策略可缩小GlcNAc特异性凝集素AANL的聚糖结合谱。

糖基化在许多细胞过程中起重要作用，例如信号转导，细胞周期进程和转录调控。然而，抗糖基化抗体和凝集素的低特异性或亲和力严重阻碍了糖基化的鉴定和分析。我们已经报道，对末端GlcNAc聚糖具有高度特异性并含有六个碳水化合物结合位点（CBS）的凝集素AANL被用于富集O-GlcNAcylated肽。为了进一步提高AANL结合特异性，我们设计了CBS均质化策略并重组了六个突变型凝集素，称为AANL1-AANL6。用GlcNAc进行的亲和层析和等温滴定热分析表明，这两个突变体（AANL3和AANL6）均保持了GlcNAc的结合活性。血凝测定，细胞结合测定和聚糖微阵列分析显示，AANL6和AANL3对末端GlcNAc聚糖的特异性高于AANL，AANL6表现出最高的特异性。通过表面等离振子共振测定法显示了AANL6对O-GlcNA酰化肽的结合活性。通过AANL6亲和色谱富集和质谱分析，在映射到54种蛋白质上的85个肽段上，鉴定出79个高可信度和21个推定的O-GlcNAcy酰化位点。与报告的数据相比，这些站点中的大多数都是新站点。这些结果表明AANL6的富集能力高于野生型AANL。总之，CBS均质化突变策略是成功的，并且AANL6被确定为O-GlcNAcylation富集的强大工具。