重组茶树菇凝集素6二聚体的原核表达、纯化及糖结合活性鉴定

Prokaryotic Expression,Purification and Glycan Binding Activity Identification of the Dimer of the Recombinant Agrocybe Aegerita Lectin 6

N-连接糖基化的N-乙酰葡萄糖胺(N-acetylglucosamine,GlcNAc)末端修饰是一种缩短的糖基化修饰,与多种疾病密切相关,例如自身免疫性疾病、癌症和神经退行性疾病等。然而,因为缺乏有效的识别工具导致对这种修饰的功能研究仍然是一个挑战。之前的研究已经表明,一种识别GlcNAc的茶树菇凝集素6(Agrocybe aegerita lectin recognizing GlcNAc,AANL6)对末端的GlcNAc糖结构特异性识别,但是亲和力较低。为了改造更高效的末端GlcNAc糖识别工具,AANL6通过二聚化来提高单体的亲和力。将AANL6单体通过中间的接头序列(linker)进行分子水平的二聚化,然后克隆到pET-30a质粒上,转化至大肠杆菌表达菌BL21中。确立最优的表达条件,并大量表达纯化AANL6二聚体蛋白(根据linker命名为PA6和EAA6)。随后对AANL6二聚体进行纯度、分子量和糖结合活性研究。SDS-PAGE结果显示,AANL6二聚体纯度较高(92.5%),分子量介于66.2~116 kD之间,符合预期值88 kD的大小。等温滴定热量法(isothermal titration calorimetry,ITC)检测结果显示,二聚化的AANL6显著提高了对GlcNAc的结合能力(P<0.01)。糖结合活性检测显示,AANL6二聚体对末端的GlcNAc糖基化修饰与单体相比有着更高的特异性。这些结果显示,AANL6二聚体通过二聚化显著提高了对末端GlcNAc糖基化的亲和力和特异性,这不仅为未来的末端GlcNAc糖基化修饰的功能提供有效的识别工具,而且提供了一种新的改造凝集素的策略。

The terminal N-acetylglucosamine(GlcNAc)modification of N-linked glycosylation is a type of highly truncated glycosylation,which is closely related to many diseases such as autoimmune diseases,cancer and neurodegenerative diseases.However,it is still a challenge to make the function of this modification clear due to the lack of effective recognition tools.Previous studies have shown that Agrocybe aegerita lectin recognizing GlcNAc named AANL6,which specifically recognize terminal GlcNAc glycosylation,but its avidity is very weak.In order to improve efficiency of the tool binding to terminal GlcNAc glycosylation,the affinity of AANL6 was improved by dimerization.AANL6 monomer was dimerized through the linker sequence,then cloned into pET-30a plasmid and transformed into Escherichia coli BL21.After the optimal expression conditions were established,AANL6 dimer protein(named PA6 and EAA6 according to linker)was expressed and purified.Then,the purity,molecular weight and glycan binding activity of AANL6 dimer were studied.The SDS-PAGE analysis showed that the purity of AANL6 dimer was high(92.5%),and its molecular weight ranged from 66.2 to 116 kD,consistent with putative molecule weight 88 kD.Microcalorimeter results showed that dimerized AANL6 bound to terminal GlcNAc with higher avidity(P<0.01).The assay detecting glycan binding activity showed that the specificity of dimerized AANL6 binding to terminal GlcNAc glycosylation was improved compared with the monomer.These results showed that the affinity and specificity of AANL6 dimer were improved significantly through dimerization strategy.This study not only provided an effective recognition tool for the function of terminal GlcNAc glycosylation in the future,but also provided a new strategy for improving the efficiency of other lectins.