VIP36 is one of cargo receptors and belongs to a type I transmembrane sorting receptor of 36 kDa consisting of L-type lectin domain, stalk domain, transmembrane domain, and short cytoplasmic domain. VIP36 localizes around the endoplasmic reticulum (ER), the Golgi, and trans Golgi network, and it seems to transport glycoproteins possessing unprocessed N-glycans from the Golgi to the ER. In the ER, it associates with a chaperone, BiP 1). VIPL is found as a homologue of VIP36 with high amino acid-sequence similarity but it localizes in the ER 2). VIP36 and VIPL have also similar sugar-binding specificity and preferentially binds to higher-molecular-weight high-mannose-type glycans with Manα1,2Manα1,2Man sequence, though optimum pH of sugar-binding activity is different each other 3) 4) 5). |
| Category | Sugar binding proteins |
| Protocol Name | Expression and binding assay of VIP36 |
Authors
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Yamamoto, Kazuo
Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo
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| KeyWords |
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Reagents
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Solubilization buffer: 50 mM Tris-HCl, pH 8.0, containing 6 M guanidine, 1 mM DTT, and 0.1 mM EDTA. |
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Refolding buffer: 100 mM Tris-HCl, pH 7.5, containing 0.4 M L-arginine, 5 mM reduced glutathione, 0.5 mM oxidized glutathione, and 0.5 mM phenylmethanesulfonyl fluoride. |
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Dialysis buffer: 20 mM Tris-HCl, pH 7.5, containing 25 mM NaCl and 0.1 mM EDTA. |
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Isopropyl β-thiogalactopyranoside |
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R-phycoerythrin (PE)-labeled streptavidin |
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HBS: 20 mM HEPES-NaOH, pH 7.4, containing 150 mM NaCl and 1 mM EDTA. |
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Instruments
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FACS Calibur (BD Biosciences, San Jose, CA) |
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CellQuest software (BD Biosciences) |
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| Methods |
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1. |
Preparation of soluble VIP36 tetramer |
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Construct plasmid encoding a soluble lectin domain of VIP36 with an enzymatic biotinylation sequence. |
Comment 1
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Transform E. coli cell with plasmid and induce expression by adding isopropyl β-thiogalactopyranoside. |
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Recover lectin domain as soluble proteins or inclusion bodies. |
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Solubilize inclusion bodies in solubilization buffer, diluted with refolding buffer to a protein concentration of 6 mM, and refolded in vitro by dialysis against dialysis buffer at 4°C for 24 h. |
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After removal of insoluble material by centrifugation, apply soluble fraction to anion-exchange chromatography and gel chromatography. |
Comment 0
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Add biotin ligase BirA for biotinylation of soluble VIP36 lectin domain. |
Comment 0
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Mix soluble VIP36 lectin domain with PE-labeled streptavidin to make PE-labeled soluble VIP36 tetramer. |
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Binding assay of soluble VIP36 tetramer to cells by flow cytometry |
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Harvest cultured mammalian cells, and suspend in HBS at a concentration of 2 × 107 cells/mL |
Comment 1
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Add PE-labeled soluble VIP36 tetramer at a concentration of 10–100 μg/mL to 10 μL of the cell suspension in a 96-well plate. |
Comment 1
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Allow to stand at 25°C for 30 min. |
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Suspend in 200 μL of HBS containing 1 μg/mL PI. |
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Measure the fluorescence intensity of PE-labeled VIP36 tetramer at 575 nm by flow cytometry. |
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3. |
Other biochemical analysis of VIP36 in cells |
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Construct plasmid encoding VIP36 with FLAG-tag at N-terminus. |
Comment 1
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Introduce plasmid into mammalian cells by lipofectamine 2000 according to manufacturer's protocol. |
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Culture transformed cells at 37°C for 24–48 h. |
Comment 0
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Precipitate or stain VIP36 using anti-FLAG antibody under apropriate conditions. |
Comment 0
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| Copyrights |
 Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
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| Date of registration：2014-07-30 10:33:01 |
- Nawa, D., Shimada, O., Kawasaki, N., Matsumoto, N., and Yamamoto, K. (2007) Stable interaction of the cargo receptor VIP36 with molecular chaperone BiP. Glycobiology 117, 913–921 [PMID : 17586539]
- Yamaguchi, D., Kawasaki, N., Matsuo, I., Totani, K., Tozawa, H., Matsumoto, N., Ito, Y., and Yamamoto, K. (2007) VIPL has sugar-binding activity speccific for high-mannnose-type N-glycans, and glucosylation of the a1,2 mannotriosyl branch blocks its binding. Glycobiology 17, 1061–1069 [PMID : 17621594]
- Kamiya, Y., Kamiya, D., Yamamoto, K., Nyfeler, B., Hauri, H.P., and Kato, K. (2008) Molecular basis of sugar recognition by the human L-type lectins ERGIC-53, VIPL and VIP36. J. Biol. Chem. 283,1857–1861 [PMID : 18025080]
- Kamiya, Y., Yamaguchi, Y., Takahashi, N., Arata, Y., Kasai, K., Ihara, Y., Matsuo, I., Ito, Y., Yamamoto, K., and Kato, K. (2005) Sugar-binding Properties of VIP36, an Intracellular Animal Lectin Operating as a Cargo Receptor. J. Biol. Chem. 280, 37178–37182 [PMID : 16129679]
- Kawasaki, N., Matsuo, I., Totani, K., Nawa, D., Suzuki, N., Yamaguchi, D., Matsumoto, N., Ito, Y., and Yamamoto, K. (2007) Detection of weak sugar binding activity of VIP36 using VIP36-streptavidin complex and membrane-based sugar chains. J. Biochem. 141, 221–229 [PMID : 17169971]
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Expression and binding assay of VIP36.
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