Detection of weak sugar binding activity of VIP36 using VIP36-streptavidin complex and membrane-based sugar chains.
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.
Kawasaki
Norihito
N
Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, 277-8562 Chiba, Japan.
Matsuo
Ichiro
I
Totani
Kiichiro
K
Nawa
Daisuke
D
Suzuki
Noriko
N
Yamaguchi
Daisuke
D
Matsumoto
Naoki
N
Ito
Yukishige
Y
Yamamoto
Kazuo
K
D017485:1-Deoxynojirimycin / Q000494:pharmacology; D000470:Alkaloids / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D037241:Mannose-Binding Lectins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010799:Phycoerythrin / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D019809:Streptavidin / Q000378:metabolism*
D000470:Alkaloids; D004791:Enzyme Inhibitors; C086797:LMAN2 protein, human; D037241:Mannose-Binding Lectins; D026901:Membrane Transport Proteins; D011134:Polysaccharides; D011994:Recombinant Proteins; C065629:kifunensine; D010799:Phycoerythrin; D017485:1-Deoxynojirimycin; D019809:Streptavidin