VIPL has sugar-binding activity specific for high-mannose-type N-glycans, and glucosylation of the α1,2 mannotriosyl branch blocks its binding

• Published in: Glycobiology (Oxford) 2007-10, Vol.17 (10), p.1061-1069
• Main Authors: Yamaguchi, Daisuke, Kawasaki, Norihito, Matsuo, Ichiro, Totani, Kiichiro, Tozawa, Hideto, Matsumoto, Naoki, Ito, Yukishige, Yamamoto, Kazuo
• Format: Article
• Language: English
• Subjects: high mannose type; lectin; N-glycan; VIPL
• ISSN: 0959-6658; 1460-2423
• DOI: 10.1093/glycob/cwm074

VIP36-like protein (VIPL) was identified as an endoplasmic reticulum (ER) 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 Escherichia 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 to swainsonine (SW)-treated HeLaS3 cells in the presence of calcium. A surface plasmon resonance analysis showed that the avidity of sVIPL was enhanced after it formed a complex with SA. The binding of PE-labeled sVIPL-SA was abrogated by endo β-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 Manα1-2Manα1-2Man 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 neutral pH, corresponding to the pH in the lumen of the ER, than under acidic conditions.