Glycobiology Advance Access published online on July 9, 2007
Glycobiology, doi:10.1093/glycob/cwm074
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VIPL has sugar-binding activity specific for high mannose-type N-glycans, and glucosylation of the 
1,2 mannotriosyl branch blocks its binding
1 Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, 277-8562 Chiba, Japan
2 RIKEN, Saitama, Japan
3 CREST, JST, Saitama, Japan
Corresponding author: Kazuo Yamamoto, Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Bioscience BLD 602, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan. Phone: +81-4-7136-3614; Fax: +81-4-7136-3619; E-mail: yamamoto{at}k.u-tokyo.ac.jp
Received on February 9, 2007; accepted on July 1, 2007
VIP36-like protein (VIPL) was identified as an endoplasmic reticulum resident protein with homology to VIP36, a cargo receptor involved in the transport of glycoproteins within cells. Although VIPL is structurally similar to VIP36, VIPL is thought not to be a lectin, because its sugar-binding activity has not been detected in several experiments. Here, recombinant soluble VIPL proteins (sVIPL) were expressed in E. coli, biotinylated with biotin ligase, and oligomerized with R-phycoerythrin (PE)-labeled streptavidin (SA). As measured with flow cytometry, PE-labeled sVIPL-SA bound to deoxymannojirimycin (DMJ)- or kifunensine (KIF)- but not swainsonine (SW)-treated HeLaS3 cells in the presence of calcium. A surface plasmon resonance analysis showed the avidity of sVIPL was enhanced after it formed a complex with SA. The binding of PE-labeled sVIPL-SA was abrogated by endo ß-N-acetylglucosaminidase H treatment of the DMJ- or KIF-treated cells. Competition with several high mannose-type N-glycans inhibited VIPL binding, and indicated that VIPL recognizes the Man
1-2Man1-2Man sequence. Glucosylation of the outer mannose residue of this portion decreased the binding. Although the biochemical characteristics of VIPL are similar to those of VIP36, the sugar-binding activity of VIPL was stronger at neural pH, corresponding to the pH in the lumen of the ER, than under acidic conditions.
Key words: high mannose-type / lectin / N-glycan / VIPL
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