Detection of weak sugar binding activity of VIP36 using VIP36-streptavidin complex and membrane-based sugar chains
Detection of weak sugar binding activity of VIP36 using VIP36-streptavidin complex and membrane-based sugar chains
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DOI：
10.1093/jh/mvm024
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发表时间：
2007-02-01
影响因子：
2.7
通讯作者：
Yamamoto, Kazuo
中科院分区：
文献类型：
--
作者：
Kawasaki, Norihito;Matsuo, Ichiro;Yamamoto, Kazuo
高甘露糖型糖链与凝集素的相互作用在糖蛋白的质量控制中发挥着重要作用。VIP36是一种与豆科植物凝集素在其管腔区域具有同源性的受体。在大肠杆菌中表达了带有C-末端生物素化标签的VIP36的管状区(SVIP36)，并用R-藻红蛋白(PE)标记的链霉亲和素进行寡聚反应。流式细胞仪分析显示PE标记的sVIP36-SA复合体(sVIP36-SA)可与脱氧甘露糖霉素(DMJ)和吉非新碱(KIF)处理的HeLaS3细胞结合。经DMJ或KIF处理的HeLaS3细胞经内切-对-N-乙酰氨基葡萄糖苷酶H处理后，sVIP36-SA与HeLaS3细胞的结合消失。此外，高甘露糖型糖链尤其是Man(7-9)GIcNAc(2)抑制了sVIP36-SA与细胞的结合，表明sVIP36-SA与细胞表面的结合是由高甘露糖型糖链介导的。虽然VIP36对配体的亲和力低于典型的同源植物凝集素，但我们能够使用不到100 ng的sVIP36-SA来监测VIP36的糖结合活性。该方法灵敏度高，适用于检测凝集素与低亲和力糖链之间的相互作用。
High mannose-type glycan-lectin interactions play important roles especially in quality control of glycoproteins. VIP36 is a receptor with homology to plant leguminous lectins in its luminal region. The luminal region of VIP36 with a C-terminal biotinylation-tag (sVIP36) was expressed in Escherichia coli and oligomerized with R-phycoerythrin (PE)-labelled streptavidin. Flow cytometric analysis revealed that PE-labelled sVIP36-SA complex (sVIP36-SA) bound to deoxymannojirimycin (DMJ)- and kifunensine (KIF)-treated HeLaS3 cells. The binding of sVIP36-SA to HeLaS3 cells treated with DMJ or KIF was abolished by endo-p-N-acetylglucosaminidase H treatment of the cells. Furthermore, the binding of sVIP36-SA to the cells was inhibited by high mannose-type glycans especially Man(7-9) GIcNAC(2), indicating that the binding of sVIP36-SA to cell surfaces was mediated by high mannose-type glycans. Although VIP36 has the lower affinity for ligands than typical homologous plant lectins, we were able to monitor the sugarbinding activity of VIP36 using less than 100ng of the sVIP36-SA. This method is highly sensitive and suitable for detecting interactions between lectins and sugar chains of low affinity.