ERGIC-53 is one of cargo receptors, which transport glycoproteins in intracellular secretory pathway. This belongs to a new class of type I transmembrane sorting receptor of 53 kDa consisting of L-type lectin domain, stalk domain, transmembrane domain, and short cytoplasmic domain. ERGIC-53 associates with MCFD2 protein in calcium-dependent manner, and its interaction is essential for expressing carbohydrate-binding activity of lectin domain1). MCFD2 protein contains two EF-hand motifs near the C-terminus. Defect of the gene encoding either ERGIC-53 (LMAN1) or MCFD2 (LMAN2) causes autosomal recessive bleeding disorder, combined deficiency of coagulation factors V and VIII (F5F8D) 2) 3). ERGIC-53/MCFD2 preferentially binds to M8B high mannose-type glycan and transports glycoproteins form the endoplasmic reticulum to the Golgi. |
| Category | Sugar binding proteins |
| Protocol Name | Expression and binding assay of ERGIC-53 |
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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Preparation of soluble ERGIC-53 tetramer |
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Construct plasmid encoding a soluble lectin domain of ERGIC-53 with an enzymatic biotinylation sequence 4). |
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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 recovered 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. |
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Add biotin ligase BirA for biotinylation of soluble ERGIC-53 lectin domain. |
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Mix soluble ERGIC-53 lectin domain with PE-labeled streptavidin to make PE-labeled soluble ERGIC-53 tetramer. |
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Binding assay of soluble ERGIC-53 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. |
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Add PE-labeled soluble ERGIC-53 tetramer at a concentration of 10–100 μg/mL to 10 μL of the cell suspension in a 96-well plate. |
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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 ERGIC-53 tetramer at 575 nm by flow cytometry. |
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Other biochemical analysis of ERGIC-53 in cells |
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Construct plasmid encoding ERGIC-53 with FLAG-tag at N-terminus. |
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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. |
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Precipitate or stain ERGIC-53 using anti-FLAG antibody under appropriate 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 10:33:47 |
- Kawasaki, N., Ichikawa, Y., Matsuo, I., Totani, K., Matsumoto, N., Ito, Y., and Yamamoto, K. (2008) The sugar-binding ability of ERGIC-53 is enhanced by its interaction with MCFD2. Blood 111, 1972–1979 [PMID : 18056485]
- Nichols, W.C., Seligsohn U., Zivelin A., Terry, V.H., Wheatley, M.A., Moussalli, M.J., Hauri, H.P., Ciavarella, N., Kaufman, R.J., and Ginsburg, D. (1998) Mutations in the ER-Golgi intermediate compartment protein ERGIC-53 cause combined deficiency of coagulation factors V and VIII. Cell 93, 61–70 [PMID : 9546392]
- Zhang, B., Cunningham, M.A., Nichols, W.C., Bernat, J.A., Seligsohn, U., Pipe, S.W., McVey, J.H., Schulte-Overberg, U., de bosch, N.B., Ruiz-Saez, A., White, G.C., Tuddenham, E.G., Kaufman, R.J., Ginsburg, D. (2003) Bleeding due to disruption of a cargo-specific ER-to-Golgi transport complex. Nat Genet. 34, 220–225 [PMID : 12717434]
- Yamamoto, K. and Kawasaki, N. (2010) Detection of weak-binding sugar activity using membrane-based carbohydrates. Methods Enzymol. 478, 233–240 [PMID : 20816483]
- Pimpaneau, V., Midoux, P., Monsigny, M., and Roche, A.C. (1991) Characterization and isolation of an intracellular D-mannose-specific receptor from human promyelocytic HL60 cells. Carbohydr. Res. 213, 95–108 [PMID : 1933956]
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