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- Title
Detection of Weak Sugar Binding Activity of VIP36 using VIP36–streptavidin Complex and Membrane-based Sugar Chains.
- Authors
Kawasaki, Norihito; Matsuo, Ichiro; Totani, Kiichiro; Nawa, Daisuke; Suzuki, Noriko; Yamaguchi, Daisuke; Matsumoto, Naoki; Ito, Yukishige; Yamamoto, Kazuo
- Abstract
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-β-N-acetylglucosaminidase H treatment of the cells. Furthermore, the binding of sVIP36–SA to the cells was inhibited by high mannose-type glycans especially Man7–9 GlcNAc2, 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.
- Subjects
MANNOSE; LECTINS; GLYCOPROTEINS; GLYCOASPARAGINASE; BIOCHEMISTRY
- Publication
Journal of Biochemistry, 2007, Vol 141, Issue 2, p221
- ISSN
0021-924X
- Publication type
Article
- DOI
10.1093/jb/mvm024