重组含糖识别结构域的人源半乳糖凝集素-3在糖蛋白/糖肽富集中的应用  

Recombination of human galectin-3 with the carbohydrate recognition domain to achieve glycoprotein/glycopeptide enrichment

作　　者：赵洋 张勇 王明超 孟波 应万涛 钱小红[1,2] ZHAO Yang;ZHANG Yong;WANG Mingchao;MENG Bo;YING Wantao;QIAN Xiaohong(College of Life Science and Bioengineering,Beijing University of Technology,Beijing,100124,China;State Key Laboratory of Proteomics,Beijing Proteome Research Center,National Center for Protein Sciences(Beijing),Beijing Institute of Lifeomics,Beijing,102206,China)

机构地区：[1]北京工业大学生命科学与生物工程学院,北京100124 [2]军事科学院军事医学研究院生命组学研究所,蛋白质组学国家重点实验室,北京蛋白质组研究中心,国家蛋白质科学中心-北京,北京102206

出　　处：《色谱》2018年第12期1197-1205,共9页Chinese Journal of Chromatography

基　　金：国家重点研发计划(2017YFF0205400,2016YFA0501300);国家自然科学基金重点项目(81530021);军事医学创新工程专项(16CXZ027).

摘　　要：植物凝集素是广泛使用的糖蛋白富集和识别材料,动物凝集素则较少被尝试用于糖蛋白富集。基于人源半乳糖凝集素-3的糖识别结构域(CRD),设计了两种重组凝集素:Gal3C(一个CRD)和Tetra-Gal3C(四重串联CRD)。通过将两种凝集素固定于链霉亲和素琼脂糖小球上,构造了富集糖蛋白的重组凝集素亲和柱。使用凝胶电泳、免疫印迹以及生物质谱技术对重组凝集素的生物特征及其糖蛋白富集能力进行了表征与评价,发现两种类型的重组凝集素对糖蛋白/糖肽都有良好的富集效果,并具有较高的特异性和灵敏度。相对于Gal3C而言,TetraGal3C由于具有四重串联的CRD结构域,表现出更高的糖蛋白/糖肽富集能力。该凝集素亲和柱成功用于人肝癌细胞系HepG2的糖蛋白富集,表明重组凝集素具有从复杂生物样本中选择性识别和富集糖蛋白/糖肽的能力。Though plant lectins have been widely used for the enrichment of glycoproteins/glycopeptides,mammalian lectins have rarely been explored for this purpose.Using the conserved sequence of the carbohydrate recognition domain(CRD)of the human galectin-3 protein,we engineered the following two novel human galectins:Gal3C(containing one CRD)and Tetra-Gal3C(containing four tandem CRDs).Moreover,we designed a galectin affinity column that could be used for glycoprotein/glycopeptide enrichment,by taking advantage of the ability of these recombinant galectins to become immobilized onto streptavidin agarose beads.SDS-PAGE,western blotting,and mass spectrometry were used to characterize the biological features and glycoprotein/glycopeptide enrichment abilities of both these recombinant galectins.Our results showed that both Gal3C and Tetra-Gal3C predominantly enrich glycoproteins/glycopeptides and that this enrichment process has a high specificity and sensitivity.Because Tetra-Gal3C contains three more CRD structural domains,it exhibited a better ability to achieve enrichment than Gal3C.Moreover,the immobilized Gal3C and Tetra-Gal3C were successfully used for the enrichment of glycoproteins from HepG2 cells,which indicated the selective glycoprotein/glycopeptide enrichment potential of recombinant galectins in complex samples.

关 键 词：液相色谱-质谱 半乳糖凝集素-3 糖识别结构域 糖蛋白富集 

分 类 号：O658[理学—分析化学]