Expression and binding assay of recombinant Siglecs
Siglec family is the largest known group of vertebrate lectins that recognize sialylated glycans (Varki and Angata 2006; Crocker et al. 2007). Humans have more than 10 Siglecs, most of which are expressed on the cells in the immune system. Most Siglecs also interact with proteins involved in intracellular signaling. Different species have different set of Siglecs (Angata 2006), and the glycan binding specificity of each Siglec differ markedly (Blixt et al. 2003). Glycan recognition is essential for Siglec function (Poe et al. 2004). Therefore, it is essential to know glycan-binding specificity of each Siglec to understand its higher-order function.
Recombinant proteins fused with Fc portion of IgG have been used extensively in biological research. Protocols for Siglec-Fc fusion protein production and glycan binding assays using the fusion protein are explained in this section.
This protocol is an enhanced and updated version of the one published in "Experimental Glycoscience - Glycobiology" ("Siglec family", pp.136-140, Springer Japan KK. 2008). The original publication is available at http://www.springerlink.com/content/m36g797j85327813/.
1. Production of Siglec-Fc fusion protein
2. Glycan binding analysis by ELISA-like assay
3. Glycan binding analysis by flow cytometry
Cell culture facility (CO2 incubator, safety cabinet, tabletop centrifuge, microscope, etc.)
Production of Siglec-Fc fusion protein
Seed 293T cells to a T75 flask at ~50 % confluence a day before the transfection. The flask should be near-confluent the next day.
Filter-sterilize the plasmid (30 μg) solution using Spin-X (12,000 g, 5 min). Mix 3 ml Opti-MEM and 75 ml LipofectAMINE 2000 in a 15 ml tube and leave for 5 min at room temperature. Add the filter-sterilized plasmid into the tube and mix well. Leave for 20 min at room temperature, and add evenly to the semi-confluent flask of 293T cells. Culture overnight in CO2 incubator.
Remove medium, gently wash once with 5 ml Opti-MEM or PBS, and add 15 ml of Opti-MEM + 2% low IgG-FCS. Culture for 3 days in CO2 incubator.
Transfer culture sup from the flask (by decantation) to 50 ml tube. Add 15 ml fresh Opti-MEM + 2% low IgG-FCS to the flask and put back into CO2 incubator, and culture for 3 days. Meanwhile, centrifuge the collected culture sup (at 1,000-1,500 g, 5 min) to remove debris. Store at 4˚C.
Collect culture sup from the flask into 50 ml tube. Centrifuge the collected culture sup (1,000-1,500 g, 5 min) to remove debris. Culture sup harvested on day 3 and day 6 may be combined.
Add 1/50 vol. of 1 M Tris-HCl buffer to the clarified culture sup. Add 0.1 ml (as packed gel) protein A-Sepharose, prepared as instructed by the manufacturer. Incubate overnight at 4˚C, with gentle agitation.
Prepare disposable column assembly. Pour the culture sup + protein A-Sepharose into the column. Wait until all culture sup pass through the column.
Add 2 ml Tris-buffered saline to the column and drain (= "wash the column"). Wash the column once again with 2 ml Tris-buffered saline.
Wash the column with 1 ml of 20 mM HEPES-NaOH buffer. Cap the column tip and add the same buffer (final volume should be ~ 500 μl). Add 10 mU of sialidase and incubate at 37˚C for 1 h. Remove the bottom cap and wash the column 5 times with 1 ml Tris-buffered saline, to remove sialidase.
Wash the column twice with 1 ml of 0.1 M citrate-NaOH buffer, pH 5.8.
Elute Siglec-Fc protein from the column twice with 1 ml 0.1 M glycine-HCl buffer, pH 3.0, and collect the eluate. Immediately add 0.2 ml of 1 M Tris-HCl buffer to neutralize. The protein A-Sepharose column can be regenerated by washing with 1 ml of 6 M guanidine HCl, followed by wash with Tris-buffered saline.
Transfer the neutralized eluate to ultrafiltraion device and concentrate. Discard flow-through. Replace to a desired buffer by repeated concentration and buffer addition, if necessary. Recover the Siglec-Fc fusion protein and analyze quantity (by protein assay) and quality (by SDS-PAGE). Store the protein at –20˚C.
Glycan binding analysis by ELISA-like assay
Prepare 5 μg/ml solution of protein A in immobilization buffer, and add 100 μl/well to the wells of 96-well plate. Each Siglec – probe combination should be assayed in triplicate wells. Incubate overnight at 4˚C.
Remove protein A solution. Pat the plate lightly against paper towel to remove excess solution lingering to the wells. Add 150 μl ELISA buffer to each well, then revert to discard the solution, and pat lightly against paper towel (= "wash the plate"). Wash the plate once again.
Add 150 μl ELISA buffer to each well and incubate at room temperature for 1 h. Meanwhile, prepare 1-5 μg/ml solution of Siglec-Fc or human IgG in ELISA buffer.
Remove the ELISA buffer and lightly pat the plate against paper towel. Add 100 μl Siglec-Fc (or human IgG) solution in each well. Incubate at room temperature for 2 h.
Remove Siglec-Fc solution by aspiration, and wash the plate 3 times with 150 μl/well ELISA buffer. Add 100 μl/well of 1-10 μg/ml glycan-PAA-Bio probes, diluted in ELISA buffer. Incubate at room temperature for 2 h.
Remove probe solution by aspiration, and wash the plate 3 times with 150 μl/well ELISA buffer. Dilute streptavidin-alkaline phosphatase at 1:1,000 in ELISA buffer. Add 100 μl/well. Incubate at room temperature for 1 h.
Remove streptavidin-alkaline phosphatase solution, and wash the plate 3 times with 150 μl/well ELISA buffer.
Add 100 μl/well alkaline phosphatase substrate and incubate at room temperature for 5 min to overnight. Measure absorbance at 405 nm using a plate reader. Check the plate at 5 min, 15 min, 30 min, 1 h, and 2 h after addition of the substrate.
Glycan binding analysis by flow cytometry
Harvest mammalian cells (e.g., BEAS-2B) to be stained. Trypsinization may be better avoided. Approximately 0.1-1 x 106 cells/sample should be used. Wash cells with PBS.
Suspend cells in sialidase buffer (at 1-5 x 106/ml), aliquote to 2 tubes and add sialidase to one of the tubes (at 50 mU/ml). Incubate at 37˚C for 30 min. Centrifuge to remove supernatant, and wash with PBS for 3 to 5 times to remove sialidase.
Suspend cells in staining buffer (at 1-5 x 106/ml) and aliquote at 0.1 ml/tube. Add 1 μg Siglec-Fc and 0.5 μg biotinylated anti-FLAG antibody per tube. Mix and incubate on ice for 1 h.
Add 0.9 ml of staining buffer, centrifuge and remove supernatant.
Suspend cells in 0.1 ml staining buffer containing 1 μl of streptavidin-conjugated R-phycoerythrin (= 1:100 dilution). Mix and incubate on ice for 30 min.
Suspend in 0.5 ml of staining buffer, filter through Cell-Strainer or nylon mesh if necessary (e.g., if the cells are clumped), and analyze with a flow cytometer.
1. Production of recombinant Siglec-Fc fusion protein
Yield differs markedly depending on the construct used. Expect 0-1 mg of Siglec-Fc per T75 flask. Protein purity generally exceeds >90 %; however, lower yield results in lower purity. A major contaminant (if any) is bovine IgG.
If the yield of Siglec-Fc is not satisfactory, consider using other cells (e.g., CHO cells). If both 293T and CHO cell lines fail to produce Siglec-Fc of your interest, then consider using S2 insect (Drosophila) cell line. Choice of culture media is critically important if you use S2 cell line, and a serum-free medium (HyQ SFX-Insect from HyClone) gave the best result in my experience. A construct for production of recombinant Siglec-Fc in S2 cells is available from the author upon request.
Some Siglecs (e.g., CD22/Siglec-2 and myelin-associated glycoprotein/Siglec-4) show strong binding signals to the preferred probes within a few minutes, while others show weak binding signals even after extended incubation. There is no strong correlation between the Siglec-Fc protein yield and the probe binding signal, although a Siglec-Fc that produce extremely poorly tends to bind glycan probes very poorly.
Sialidase treatment of Siglec-Fc is essential to expose glycan binding ability of Siglec-Fc. The choice of 96-well plate also affects assay outcome, and in my experience NUNC's plate (catalog #269620) has worked consistently well.
This protocol works only if the recombinant Siglec contains FLAG tag. (The author's constructs contain a FLAG tag.) If no tag is available, use anti-Fc antibody conjugated with fluorescence chromophore instead. Although it is necessary to empirically determine optimal amount of the secondary antibody, 0.5 μg secondary antibody per 1 mg recombinant Siglec-Fc would be a good starting point for optimization.
Human Siglec-9 consistently gives a good binding signal to many types of human cells, so it should be included as a positive control. An appropriate negative control, such as Fc-fusion of other immunoglobulin superfamily protein (e.g., TREM1), may be included as a negative control.
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Blixt O, Collins BE, van den Nieuwenhof IM, Crocker PR, Paulson JC (2003) Sialoside specificity of the siglec family assessed using novel multivalent probes: identification of potent inhibitors of myelin-associated glycoprotein. J Biol Chem 278:31007-31019. [PMID : 12773526]
Crocker PR, Paulson JC, Varki A. (2007) Siglecs and their roles in the immune system. Nat Rev Immunol 7:255-266. [PMID : 17380156]
Poe JC, Fujimoto Y, Hasegawa M, Haas KM, Miller AS, Sanford IG, Bock CB, Fujimoto M, Tedder TF (2004) CD22 regulates B lymphocyte function in vivo through both ligand-dependent and ligand-independent mechanisms. Nat Immunol 5:1078-1087. [PMID : 15378059]
Varki A, Angata T (2006) Siglecs – the major subfamily of I-type lectins. Glycobiology 16:1R-27R. [PMID : 16014749]