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Analysis of intracellular lectins using reporter cell expressing lectin on its surface
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Analysis of intracellular lectins using reporter cell expressing lectin on its surface

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Introduction Protocol References Credit lines
Category
Sugar binding proteins
Protocol Name

Analysis of intracellular lectins using reporter cell expressing lectin on its surface

Authors
 Yamamoto, Kazuo
Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo
KeyWords
Reagents
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reporter cells, BWZ.36 or 2B4.

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transfection reagent: ripofectamine 2000, transfectam etc.

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polyacrylamide-based sugar polymer (Glycotech, Gaithersburg, MD) or glycoproteins.

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mono, di, oligosaccharides, and glycopeptides

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96-well ELISA plate

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chlorophenol red-β-D-galactopyranoside (Wako Pure Chemical Industries Ltd., Osaka, Japan)

Instruments
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ELISA plate reader (Hitachi, Ltd., Tokyo, Japan)
Methods
1.

Preparation of reporter cells expressing lectin fusion protein on their surface

1) 

 Construct an expression plasmid containing cDNAs encoding the CD8β signal sequence followed by a Myc-tag, a lectin domain of desired protein, the NKp46 stalk domain, the CD8α transmembrane domain, and mouse CD3ζ.

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2) 

 Transform mouse reporter cells, BWZ.36 or 2B4, with above plasmid using transfection reagent.

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3) 

 Forty-eight hours after the transfection, harvest transfected cells and apply for the following reporter assay.

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2.

Reporter assay for sugar binding

1) 

 Coat each well of ELISA plate with 50 μL of 20 μg/mL polyacrylamide-based sugar polymers, glycoproteins, anti-Myc antibody and so on.

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2) 

 Culture reporter cells expressing lectin fusion protein (105 cells/well) on a sugar polymer-coated well at 37°C for 16 h in culture medium.

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3) 

 Remove culture medium and then add 2.5 mM chlorophenol red-β-D-galactopyranoside as the substrate.

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4) 

 Incubate for a few minutes at 37°C or a few hours at room temperature.

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5) 

 Measure the colorimetric absorbance at 574 nm using ELISA plate reader.

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3.

Inhibition assay using reporter cell

1) 

 Prepare several concentrations of inhibitory sugar solution.

Add inhibitory sugars in sugar polymer-coated wells and culture reporter cells at 37°C for 16 h.

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2) 

 Remove culture medium and then add 2.5 mM of chlorophenol red-β-D-galactopyranoside as the substrate.

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3) 

 Incubate for a few minutes at 37°C or a few hours at room temperature.

 Comment 1
Initial amount

One microgram of sugar polymer/well

105 transfected cells/well (when transfection efficiency is approximately 30%)

Discussion

To discuss about sugar-binding specificity of lectins or to rule out nonspecific binding, reporter assay should be performed under several control conditions. When sugar-binding ability of expressed lectin fusion protein is quite strong, β-galactosidase expression level will reach a plateau. In this case, sugar polymer concentration should be considered to minimize cross-linking signaling to reporter cells. In the case of GFP-expressing reporter cell, 2B4, expression of GFP is measured by flow cytometry using FACScalibur and accompanying CellQuest software (BD Biosciences, San Jose, CA) and values of mean fluorescent intensity are compared.

Figure & Legends

Figure & Legends

 

 

Fig. 1.  Schematic illustration of reporter cells expressing lectin fusion protein on their surface.

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Date of registration：2014-07-30 10:32:33
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