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Binding assay of core-fucose-specific lectin, Pholiota squarrosa lectin [1] Lectin affinity HPLC
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Binding assay of core-fucose-specific lectin, Pholiota squarrosa lectin [1] Lectin affinity HPLC

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Category
Sugar binding proteins
Protocol Name

Binding assay of core-fucose-specific lectin, Pholiota squarrosa lectin [1] Lectin affinity HPLC

Authors
Kobayashi, Yuka
Biochemical Laboratory, J-Oil mills, Inc.
KeyWords
Reagents

PhoSL column for lectin affinity HPLC (LA-PhoSL: 0.46 × 15 cm) ( J-Oil mills, Inc., Tokyo, Japan)

Lens culinaris agglutinin (LCA) column for lectin affinity HPLC (LA-LCA: 0.46 × 15 cm) (J-Oil mills, Inc.)

Aleuria aurantia lectin (AAL) column for lectin affinity HPLC (LA-AAL: 0.46 × 15 cm) (J-Oil mills, Inc.)

Pyridylaminated sugars (PA sugars) (Takara Bio Inc., Otsu, Japan)(Masuda Chemical Industries Co., Ltd., Kagawa, Japan)

10 mM Tris-HCl buffer

10 mM Tris-HCl buffer containing 0.15 M NaCl, pH 7.3 (TBS)

50 mM Tris-H2SO4, pH 7.3

50 mM Tris-H2SO4, pH 7.3 containing 1 mM methyl-α-d-mannoside

10 mM CH3COONH4

10 mM CH3COONH4 containing 5 mM l-Fucose

Instruments

HPLC system (Shimadzu SCL-6A, LC-6A, RF-535, CTO-6A: Shimadzu Corporation, Kyoto, Japan)

Methods
1.

Lectin affinity HPLC

1) 

 Connect the lectin column to the HPLC system.

Comment 0
2) 

 Equilibrate the column with Solvent A.

Comment 0
3) 

 Perform the HPLC analyses as follows:

a. LA-PhoSL: 0.46 × 15 cm

Solvent A : 10 mM Tris-HCl buffer

Solvent B : 10 mM Tris-HCl buffer containing 0.15 M NaCl, pH 7.3 (TBS)

Flow rate : 0.5 mL/min

Fluorescence detection : Excitatory wavelength (Ex) = 320 nm;

Emission wavelength (Em) = 400 nm

Temperature : 25°C

Sample : 10 pmol PA-Sugar Chain

b. LA-LCA: 0.46 × 15 cm

Solvent A : 50 mM Tris-H2SO4, pH 7.3

Solvent B : 1 mM methyl-α-d-mannoside/Solvent A

Gradient : 0(3 min)-100(10 min)-0% B Stepwise gradient

Flow rate : 0.5 mL/min

Fluorescence detection : Ex = 320 nm; Em = 400 nm

Temperature : 25°C

Sample :10 pmol PA-Sugar Chain

c. LA-AAL: 0.46 × 15 cm

Solvent A : 10 mM CH3COONH4

Solvent B : 5 mM L-Fucose/Solvent A

Gradient : 0(3 min)-100(10 min)-0% B Stepwise gradient

Flow rate : 0.5 mL/min

Fluorescence detection : Ex = 320 nm; Em = 400 nm

Temperature : 25°C

Sample : 10 pmol PA-Sugar Chain

Comment 0
4) 

 Inject PA sugar (10 pmol) into each column using an auto-sampling system.

Comment 0
5) 

 Determine the fluorescence using a detector set to excitation and emission wavelengths of 320 nm and 400 nm, respectively. The fluorescence peaks can be identified by comparing the retention time of the sample in each case with its standard retention time.

Comment 0
Notes

Pyridylaminated sugars(PA-sugars) are NA2(PA-Sugar Chain 001, Takara Bio Inc. / PA-012, Masuda Chemical Industries Co., Ltd.), NA2F(PA-Sugar Chain 009, Takara Bio Inc. / PA-004, Masuda Chemical Industries Co., Ltd.), LNT(PA-Sugar Chain 042, Takara Bio Inc.), LNFP-I(PA-Sugar Chain 043, Takara Bio Inc.), LNFP-II(PA-Sugar Chain 044, Takara Bio Inc.) LNFP-III(PA-Sugar Chain 045, Takara Bio Inc.) and M5(PA-Sugar Chain 017, Takara Bio Inc. / PA-030, Masuda Chemical Industries Co., Ltd.) (Fig. 1).

Discussion

Fucose1-6 oligosaccharides, non-fucosylated oligosaccharides, and non-fucose1-6 oligosaccharides could be separated by HPLC using a PhoSL column, and these compounds produced individual peaks (Fig. 1). The HPLC results indicate that LCA columns recognize α1-6 fucosyl linkages, and LCA could bind to the mannose-oligosaccharide, M5, as well (Fig. 2). However, nonfucosylated lacto-N-tetraose(LNT), and the monofucosylated isomers, lacto-N-fucopentaose (LNF-I, LNF-II, and LNF-III), could be separated by HPLC using an AAL column, and these compounds produced individual peaks (Fig. 3). The unique property of a PhoSL column is its strict specificity for α1-6Fuc (Fig. 1). One of the most important oligosaccharide modifications during carcinogenesis is α1-6 fucosylation; however, although many studies on fucosylation have been performed, an easy method to differentiate between α1-2, α1-3 and α1-4 fucosylation and α1-6 fucosylation has not been established. This differentiation has been difficult because of the absence of a tool for the specific detection of the α1-6 fucosyl linkage. AAL, used in many studies, recognizes all types of fucosyl linkages. The HPLC results indicate that PhoSL specifically recognizes α1-6 fucosyl linkages. Thus, PhoSL will be a promising tool for analyzing the biological functions of α1-6 fucosylation and evaluating Fucα1-6 oligosaccharides as cancer biomarkers.

Figure & Legends

Figure & Legends

 

Fig. 1. Oligosaccharide structures, and comparison of the elution patterns of PA sugars from the LA-PhoSL column

 

 

 

Fig. 2. Oligosaccharide structures, and comparison of the elution patterns of PA sugars from the LA-LCA column

 


 

Fig. 3. Oligosaccharide structures, and comparison of the elution patterns of PA sugars from the LA-AAL column

 

 

Fig. 4. Sugar structures

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