Lectins are powerful tools for determining the structure of glycans. Historically, their sugar-binding specificities have been used for qualitative analysis of sugar chains. The recently developed lectin array technology has become popular, but it requires lectins with specific sugar binding properties.
Pholiota squarrosa lectin (PhoSL) was recently purified from mushroom. PhoSL specifically binds a1-6 fucosylated sugar chains. This protocol describes the detection of fucose α1-6 sugar chains using PhoSL. This method is more accurate for detecting fucose α1-6 sugar chains than conventional assays, such as using Lens culinaris agglutinin (LCA) Pisum sativum agglutinin (PSA), Aleuria aurantia lectin (AAL) Aspergillus oryzae lectin (AOL). This protocol also describes an enzyme-linked immunosorbent assay (ELISA) using PhoSL and other fucose-specific lectins. |
| Category | Sugar binding proteins |
| Protocol Name | Binding assay of core fucose-specific lectin, Pholiota squarrosa lectin [2] ELISA |
Authors
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Kobayashi, Yuka
Biochemical Laboratory, J-Oil mills, Inc.
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| KeyWords |
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Reagents
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Biotin Pholiota squarrosa lectin (PhoSL) |
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Biotin Lens culinaris agglutinin (LCA) (J-Oil mills, Inc., Tokyo, Japan) |
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Biotin Aleuria aurantia lectin (AAL) (J-Oil mills, Inc.) |
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Horseradish peroxidase-streptavidin (Vector Laboratories, Inc., Burlingame, CA) |
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TMB microwell peroxidase substrate system (KPL, Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD) |
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Blocking buffer: 1 g of defatted bovine serum albumin (BSA) in 100 mL phosphate-buffered saline (PBS) |
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Washing buffer: PBS containing 0.05% Tween 20, pH 7.4 |
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Instruments
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Microplate (96-well MaxiSorp flat-bottom plates: Nunc, Roskilde, Denmark ) |
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Multichannel pipette (Finnpipette F2: Thermo Fisher Scientific Inc., Waltham, MA) |
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Microplate shaker (FINEPCR, MX2) |
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Incubator (CN-25C: As One Corporation, Osaka, Japan) |
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Plate reader (L580 POWERSCANⓇHT: DS Pharma Biomedical Co., Ltd., Osaka, Japan) |
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| Methods |
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Coat a microtiter plate with 50 μL of each diluted protein or glycoprotein (100 ng/mL) in 0.1 M carbonate buffer (pH 9.5) per well. |
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Incubate the plate overnight at 4°C. |
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Block with 250 μL blocking buffer (PBS containing 1% BSA) for 1.5 h at room temperature. |
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Rinse 3 times with washing buffer (PBS containing 0.05% Tween 20, pH 7.4). |
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Add 50 μL of each biotin-lectin in blocking buffer (1 μg/mL). |
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Incubate for 1 h at room temperature. |
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Wash the plate 3 times with washing buffer. |
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Add horseradish peroxidase-streptavidin (0.5 μg/mL). |
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Wash the plate 3 times with washing buffer. |
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Use the TMB microwell peroxidase substrate system for colorimetric analysis.
Reagents are used according to the manufacturer’s protocols. |
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Incubate the plate for 5–30 min at room temperature. |
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Add 10 μL of 1M phosphoric acid |
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Measure the absorbance at 450 nm by using a plate reader. |
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| Notes | Glycoproteins are as follows: HSA, human serum albumin; IgG, immunoglobulin G; TF, transferrin; Fib, fibrinogen IgA immunoglobulin A; a2M, alpha-2-macroglobulin; IgM, immunoglobulin M; alpha-1-antitrypsin; C3, third component of complement; HP, haptoglobin; AGP, alpha-1-acid glycoprotein; PSA, prostate-specific antigen; AFP, alpha-fetoprotein; AFP-L3, 1-6 fucosylated fetoprotein; TG, thyroglobulin; LF, lactoferrin; FT, fetuin; BSM, bovine submaxillary gland mucin; PSM, porcine stomach mucin; Inv, invertase; OVA, ovalbumin; BSA, bovine serum albumin. |
| Discussion | The binding of biotinylated lectins to immobilized proteins or glycoproteins was examined using ELISA. Among the major human serum glycoproteins (HSA, IgG, TF, Fib, IgA, a2M, IgM, A1AT, C3, HP, and AGP), the most potent binding to PhoSL was exhibited by IgG (Fig. 1). PhoSL bound to fucosylated AFP (AFP-L3), one of the 3 tested serum tumor markers (PSA, AFP, and AFP-L3). TG (bovine) and LF (human milk) also bound to PhoSL. All the glycoproteins that bound to PhoSL possessed a core fucose. Although the profile of LCA was similar to that of PhoSL, its binding to glycoproteins was much weaker than that of PhoSL (Fig. 2). The superiority of PhoSL over LCA was confirmed by ELISA using biotin-labeled PhoSL and LCA and immobilized glycoproteins. AAL bound to PSM which has fucose a1-2, 1-3 sugar chains. PhoSL very strongly and specifically binds to Fucα-oligosaccharides. These advantages indicate that PhoSL can become a powerful tool to analyze fucosylated glycoproteins. |
| Figure & Legends |
Figure & Legends
Fig. 1. Binding of PhoSL to immobilized glycoproteins by ELISA
Binding activity of biotin-labeled PhoSL and various immobilized glycoproteins.
This figure was originally published in J Biol Chem. Kobayashi Y. et al. "A Novel Core Fucose-specific Lectin from the Mushroom Philiota Sqiarrosa" 2012, 287(41): 33973–82.
Fig. 2. Binding of LCA to immobilized glycoproteins by ELISA
Binding activity of biotin-labeled LCA and various immobilized glycoproteins
This figure was originally published in J Biol Chem. Kobayashi Y. et al. "A Novel Core Fucose-specific Lectin from the Mushroom Philiota Sqiarrosa" 2012, 287(41): 33973–82.
Fig. 3. Binding of AAL to immobilized glycoproteins by ELISA
Binding activity of biotin-labeled LCA and various immobilized glycoproteins
The immobilized glycoproteins are HSA, human serum albumin; IgG, immunoglobulin G; TF, transferrin; Fib, fibrinogen IgA immunoglobulin A; a2M, alpha-2-macroglobulin; IgM, immunoglobulin M; alpha-1-antitrypsin; C3, third component of complement; HP, haptoglobin; AGP, alpha-1-acid glycoprotein; PSA, prostate-specific antigen; AFP, alpha--fetoprotein; AFP-L3, 1-6 fucosylated fetoprotein; TG, thyroglobulin; LF, lactoferrin; FT, fetuin; BSM, bovine submaxillary gland mucin; PSM, porcine stomach mucin; Inv, invertase; OVA, ovalbumin; BSA, bovine serum albumin. |
| Copyrights |
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This work is released underCreative Commons licenses
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| Date of registration：2017-01-23 15:54:16 |
- Kobayashi, Y., Tateno, H., Dohra, H., Moriwaki, K., Miyoshi, E., Hirabayashi, J., and Kawagishi, H. (2012) A novel core fucose-specific lectin from the mushroom Pholiota squarrosa. J. Biol. Chem. 287, 33973–33982. [PMID : 22872641]
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