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，新合成的糖蛋白通过转运囊泡从内质网经高尔基体转运到细胞表面。在此过程中，分拣事件由货物接收器执行。ERGIC-53和VIP-36在其内腔区域具有与豆科凝集素同源的糖结合结构域，是将特异性蛋白质引导到囊泡中的受体。两种受体的凝集素结构域的cDNA编码的构建和表达在大肠杆菌中。这些重组可溶性ERGIC-53和VIP 36与糖蛋白的相互作用在几种pH条件下使用有或没有钙离子的表面等离子体共振进行了分析。在pH6.5 ~ 7.2范围内，ERGIC-53与IgY的相互作用无明显差异。相比之下，可溶性VIP 36结合到猪甲状腺球蛋白强酸性条件下比中性pH值。寡聚化的可溶性VIP 36发生在与增强关联的配体在酸性条件下。然后将随机寡核苷酸引入cDNA中，分别代替编码VP 36和ERGIC-53的碳水化合物结合环的核苷酸。在CHO细胞中表达突变的cDNA，并通过流式细胞术富集在表面上具有不同糖部分的细胞。将突变的VIP 36 cDNA转染后特异性改变为与PNA结合的富集细胞克隆后，建立了13种稳定的克隆。这些克隆根据在细胞中表达的突变的cDNA被分为两组，并且这些突变的VIP 36 cDNA的碳水化合物结合环分别被推断为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.