A new glycome analysis by changing carbohydrate-binding specificities of cargo receptors in the cells

在真核细胞的分泌途径中，新合成的糖蛋白从内质网通过高尔基体通过运输小泡运输到细胞表面。在这个过程中，分拣事件由货物受体执行。ERIGIC-53和VIP36在管腔区域具有与豆科凝集素同源的碳水化合物结合结构域，是将特定蛋白定向到囊泡中的受体。构建了两种受体凝集素结构域的编码基因，并在大肠杆菌中进行了表达。在不同pH条件下，用表面等离子体共振技术分析了重组可溶性ERGIC-53和VIP36与糖蛋白的相互作用。在pH为6.5~7.2的范围内，ERGIC-53与IgY的相互作用差异不大。相比之下，在酸性条件下，可溶性VIP36与猪甲状腺球蛋白的结合比在中性条件下更强。在酸性条件下，可溶性VIP36的齐聚与配体的结合增强有关。然后将随机寡核苷酸引入到cDNA中，分别代替编码VP36和ERGIC-53糖结合环的核苷酸。突变的cDNAs在CHO细胞中得到表达，并用流式细胞仪对表面糖基不同的细胞进行了富集化。将突变的VIP36cDNAs导入后与PNA特异结合的浓缩细胞克隆后，建立了13种稳定的克隆。根据在细胞中表达的突变的cDNA将这些克隆分为两组，这些突变的VIP36 cDNA的碳水化合物结合环分别为DPDSNGGSF和DSSFNYVHA。

In secretory pathway of eucaryotic cells, newly synthesized glycoproteins are transported from the ER through the Golgi toward cell surface via transport vesicles. During this process, sorting events are performed by cargo receptors. ERGIC-53 and VIP36 having carbohydrate-binding domain homologous to leguminous lectins at lumenal region are receptors to direct specific proteins into vesicles. cDNA coding lectin domains of both receptors were constructed and expressed in E.coli. Interactions of these recombinant soluble ERGIC-53 and VIP36 with glycoproteins were analyzed using surface plasmon resonance with or without calcium ions under several pH conditions. Interaction of ERGIC-53 and IgY was not so different under the pH ranging from 6.5 to 7.2. By contrast, soluble VIP36 bound to porcine thyroglobulin stronger under the acidic condition than neutral pH. Oligomerization of soluble VIP36 occurred in relation to the enhanced association with the ligands under acidic condition. Then random oligonucleotides were introduced into cDNAs in place of the nucleotides encoding carbohydrate-binding loops of VP36 and ERGIC-53, respectively. The mutated cDNAs were expressed in CHO cells and the cells having different sugar moieties on the surfaces were enriched by flow cytometry. After cloning of the enriched cells, which have specifically changed to bind to PNA after the trasfection of mutated VIP36 cDNAs, 13 kinds of stable clones were established. These clones were classified into two groups based on the mutated cDNAs expressed in the cells and the carbohydrate-binding loops of these mutated VIP36 cDNAs were deduced to be DPDSNGGSF and DSSFNYVHA, respectively.