Journal of Biochemistry Advance Access originally published online on December 14, 2006
Journal of Biochemistry 2007 141(2):221-229; doi:10.1093/jb/mvm024
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© 2006 The Japanese Biochemical Society.
Detection of Weak Sugar Binding Activity of VIP36 using VIP36–streptavidin Complex and Membrane-based Sugar Chains
1Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, 277-8562 Chiba, Japan; 2RIKEN, The Institute of Physical and Chemical Research, Saitama, Japan; and 3CREST, JST, Saitama, Japan
*To whom correspondence should be addressed. Tel: +81-4-7136-3614, Fax: +81-4-7136-3619, E-mail: yamamoto{at}k.u-tokyo.ac.jp
Received September 12, 2006; Accepted December 3, 2006
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Abstract |
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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-ß-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.
Key Words: flow cytometry, membrane-based ligand, sugar-binding, VIP36, weak interaction
Abbreviations:
CNX, calnexin; CRT, calreticulin; DMJ, deoxymannojirimycin; DSA, Datura stramonium agglutinin; EDEM, ER degradation enhancing 
-mannosidase-like protein; Endo H, endo-ß-N-acetylglucosaminidase H; ER, endoplasmic reticulum; ERGIC-53, ER-Golgi compartment protein of 53 kDa; FCS, fetal calf serum; GNA, Galanthus nivalis agglutinin; KIF, kifunensine; MFI, mean fluorescence intensity; PA, pyridylamino; PE, R-phycoerythrin; PE-SA, PE-conjugated streptavidin; PI, propidium iodide; SA, streptavidin; SW, swainsonine; VIP36, vesicular integral protein of 36 kDa
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