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

Kazuo Yamamoto

Introduction

Sugar-binding ability of lectins, especially those from mammals, is weak and animal lectins usually have a K_d value ~10⁻⁴ M. Animal lectins, including lectin-like receptors, are far less abundantly expressed in cells compared with plant lectins. Thus, it is quite difficult to evaluate sugar-binding activity and specificity in nature. To overcome such a characteristic of sugar-protein interaction, we established the tetramerization of lectins by introducing biotin-tag sequence along with biotin ligase BirA (1). Alternatively, a lectin cell surface display method, wherein the lectin domain is expressed as a transmembrane fusion protein, is very useful, especially for lectins that could not be prepared as soluble recombinant proteins expressed in E. coli cell. When CD3ζ is used as a cytoplasmic tail of a fusion protein, binding of sugar ligands through the lectin domain causes phosphorylation of CD3ζ in the cytoplasm, which resulted in the transcription of a reporter gene (β-galactosidase, green fluorescence protein, etc.) under the control of interleukin-2-responsive element (Figure 1). Using a cell having such a reporter gene, sugar-binding activity of lectins expressed on the cell surface are sensitively detected without their purification. The reporter cell expressing a lectin domain on the cell surface also could be both an effective immunogen and a screening probe for the establishment of specific monoclonal antibodies (2). In this chapter, the preparation of a reporter cell expressing a lectin domain on cell surface and a reporter assay for measuring sugar-binding ability of cell surface-expressing lectins.

Protocol

The following protocol describes a method for examining the sugar-binding ability and specificity of lectins that are difficult to purify due to their low expression levels. In this method, the cDNA encoding the lectin of interest is expressed in reporter cells as a membrane-bound fusion protein with a CD3ζ signaling module in the cytoplasm. Cross-linking of the lectin domain by binding to glycans causes expression of reporter genes, such as β-galactosidase and GFP.

Materials

1.: Reporter cells, BWZ.36 or 2B4 (3, 4)
2.: Transfection reagent: Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA), Transfectam (Promega, Madison, WI), etc.
3.: Polyacrylamide-based sugar polymer (Glycotech, Gaithersburg, MD) or glycoproteins.
4.: Mono, di, oligosaccharides, and glycopeptides
5.: 96-well enzyme-linked immunosorbent assay (ELISA) plate
6.: Chlorophenol red-β-D-galactopyranoside (Sigma-Aldrich, St. Louis, MO).

Instruments

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

Methods

1.

Preparation of reporter cells expressing lectin fusion protein on their surface

a.: Construct an expression plasmid containing cDNAs encoding the CD8β signal sequence followed by a Myc-tag, a lectin domain of the desired protein, the NKp46 stalk domain, the CD8α transmembrane domain, and the mouse CD3ζ (Note 1).
b.: Transform mouse reporter cells, BWZ.36 or 2B4, with the above plasmid using a transfection reagent (Note 2).
c.: A total of 48 h after the transfection, harvest the transfected cells and apply on the following reporter assay.

2.

Reporter assay for sugar binding

a.: Coat each well of ELISA plate with 50 μL of 20 μg/mL polyacrylamide-based sugar polymers, glycoproteins, anti-Myc antibody, and so on (Note 3).
b.: 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.
c.: Remove culture medium and then add 2.5 mM of chlorophenol red-β-D-galactopyranoside as the substrate.
d.: Incubate for a few minutes at 37°C or a few hours at room temperature (Note 4).
e.: Measure the colorimetric absorbance at 574 nm using an ELISA plate reader.

3.

Inhibition assay using reporter cell.

a.: Prepare several concentrations of inhibitory sugar solution.
b.: Add inhibitory sugars in sugar polymer-coated wells and culture reporter cells at 37°C for 16 h.
c.: Remove culture medium and then add 2.5 mM of chlorophenol red-β-D-galactopyranoside as the substrate.
d.: Incubate for a few minutes at 37°C or a few hours at room temperature (Note 5).
e.: Measure the colorimetric absorbance at 574 nm using an ELISA plate reader.

Notes

1.: In case of type 2 transmembrane proteins containing a lectin domain, the order of domain structures should be changed.
2.: Both stable and transient expressing cells can be used for reporter assay.
3.: A total of 10 μg/mL of anti-Myc antibody is used as a positive control because lectin fusion protein expressed on the cell surface has Myc-tag at N-terminus. Instead of sugar polymers, cells treated with sugar-processing inhibitors could be used.
4.: Incubation time depends on the expression level of lectin fusion protein and the cell numbers used for assay.
5.: Cross-linking of lectin fusion proteins is needed for transduction of NF-AT mediated reporter gene transcription. Thus, soluble saccharides could be used for inhibition. A total of 5 mM of EDTA is used for the sugar-binding inhibition when calcium-dependent C-type lectin domains are expressed on the reporter cells.

References

Yamamoto K, Kawasaki N. Detection of weak-binding sugar activity using membrane-based carbohydrates. Methods Enzymol. 2010;478:233–40. [PubMed: 20816483] [CrossRef]

Qin S, Kawasaki N, Hu D, Tozawa H, Matsumoto N, Yamamoto K. Subcellular localization of ERGIC-53 under endoplasmic reticulum-stress condition. Glycobiology. 2012 Dec;22(12):1709–20. [PubMed: 22821029] [CrossRef]

Sanderson S, Shastri N. LacZ inducible, antigen/MHC-specific T cell hybrids. Int Immunol. 1994 Mar;6(3):369–76. [PubMed: 8186188] [CrossRef]

Hooijberg E, Bakker AQ, Ruizendaal JJ, Spits H. NFAT-controlled expression of GFP permits visualization and isolation of antigen-stimulated primary human T cells. Blood. 2000 Jul 15;96(2):459–66. [PubMed: 10887106]

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Bookshelf ID: NBK593960PMID: 37590693

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