VIPL has sugar-binding activity specific for high-mannose-type N-glycans, and glucosylation of the alpha1,2 mannotriosyl branch blocks its binding.
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 beta-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 Manalpha1-2Manalpha1-2Man 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.
Yamaguchi
Daisuke
D
Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Bioscience BLD 602, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan.
Kawasaki
Norihito
N
Matsuo
Ichiro
I
Totani
Kiichiro
K
Tozawa
Hideto
H
Matsumoto
Naoki
N
Ito
Yukishige
Y
Yamamoto
Kazuo
K
D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D005434:Flow Cytometry; D006023:Glycoproteins / Q000378:metabolism; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D008358:Mannose; D037241:Mannose-Binding Lectins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010799:Phycoerythrin / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019809:Streptavidin / Q000378:metabolism*; D020349:Surface Plasmon Resonance
D006023:Glycoproteins; C086797:LMAN2 protein, human; D037241:Mannose-Binding Lectins; D026901:Membrane Transport Proteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D010799:Phycoerythrin; D019809:Streptavidin; D008358:Mannose