Kawasaki Norihito, Matsuo Ichiro, Totani Kiichiro, Nawa Daisuke, Suzuki Noriko, Yamaguchi Daisuke, Matsumoto Naoki, Ito Yukishige, Yamamoto Kazuo
Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, 277-8562 Chiba, Japan.
J Biochem. 2007 Feb;141(2):221-9. doi: 10.1093/jb/mvm024. Epub 2006 Dec 14.
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-beta-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) GlcNAc(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 sugar-binding activity of VIP36 using less than 100 ng of the sVIP36-SA. This method is highly sensitive and suitable for detecting interactions between lectins and sugar chains of low affinity.
高甘露糖型聚糖-凝集素相互作用尤其在糖蛋白的质量控制中发挥重要作用。VIP36是一种在内腔区域与植物豆科凝集素具有同源性的受体。带有C端生物素化标签的VIP36内腔区域（sVIP36）在大肠杆菌中表达，并与R-藻红蛋白（PE）标记的链霉亲和素寡聚化。流式细胞术分析表明，PE标记的sVIP36-SA复合物（sVIP36-SA）与脱氧甘露基野尻霉素（DMJ）和衣霉素（KIF）处理的HeLaS3细胞结合。用内切β-N-乙酰葡糖胺糖苷酶H处理细胞后，sVIP36-SA与用DMJ或KIF处理的HeLaS3细胞的结合被消除。此外，sVIP36-SA与细胞的结合受到高甘露糖型聚糖尤其是Man(7-9)GlcNAc(2) 的抑制，这表明sVIP36-SA与细胞表面的结合是由高甘露糖型聚糖介导的。尽管VIP36对配体的亲和力低于典型的同源植物凝集素，但我们能够使用少于100 ng的sVIP36-SA监测VIP36的糖结合活性。这种方法高度灵敏，适用于检测凝集素与低亲和力糖链之间的相互作用。
