Isolation and biochemical characterization of Apios tuber lectin

Abstract

Apios tuber lectin, named ATL, was isolated from Apios americana Medikus by two chromatography steps, hydrophobic chromatography and anion-exchange chromatography. The minimum concentration required for the hemagglutination activity toward rabbit erythrocytes of ATL was 4 μg/mL. ATL was composed of a homodimer of 28.4 kDa subunits. The amino acid sequence of ATL was similar to those of other legume lectins. The lectin showed moderate stability toward heating and acidic pH, and the binding affinity against several monosaccharides, such as D-glucosamine and D-galactosamine. ATL also bound to desialylated or agalactosylated glycoproteins such as asialo and agalacto transferrin. ATL decreased the transepithelial electrical resistance across human intestinal Caco-2 cell monolayers, suggesting the effect on the tight junction-mediated paracellular transport.

Figure 1

Purification of the Apios tuber lectin (ATL). (A) The protein fraction obtained by 40% saturated ammonium sulfate precipitation was separated by hydrophobic chromatography on a Toyopearl phenyl-650M column (2.8 × 16 cm) pre-equilibrated with 1.0 M ammonium sulfate in 50 mM Tris-HCl buffer (pH 8.0). Proteins were eluted with a decreasing linear gradient of ammonium sulfate (1.0–0 M) in the same buffer. Factions showing hemagglutination activity were collected and dialyzed against 50 mM Tris-HCl buffer (pH 8.0). (B) The dialysate was subjected to anion-exchange chromatography on a Toyopearl DEAE-650M column (2.8 × 32 cm) pre-equilibrated with 50 mM Tris-HCl (pH 8.0) and eluted with a linear gradient of NaCl (0 to 1.0 M) in the same buffer. Fractions A and B (ATL) were dialyzed against distilled water and lyophilized.

Figure 2

SDS-PAGE patterns of Apios tuber proteins in 15% acrylamide gel under reducing condition. Lanes M: Protein markers, E: Apios protein fraction obtained by ammonium sulfate precipitation, A: Fraction A; B: Fraction B (Apios tuber lectin).

Figure 3

Molecular mass determination of Apios tuber lectin. (A) Size exclusion chromatography on a PC200S (N) column (5 μm, 7.8 × 300 nm) in 50 mM HEPES (pH 6.9) containing 0.25 M NaCl and 5 mM CaCl₂ as the mobile phase. Flow rate: 0.8 mL/min; UV detection: 280 nm. (B) MALDI-TOF mass spectral analysis.

Figure 4

Effect of temperature and pH on the hemagglutination activity of Apios tuber lectin (ATL). (A) ATL was incubated at the indicated temperatures for 30 min; (B) ATL was incubated at various pH values overnight. After adjusting the pH to 7, the hemagglutination activity was measured. Solid line: ATL, broken line: Soybean lectin (SBA).

Figure 5

Peptide maps of Apios tuber lectin (ATL) and Fraction A. Each protein was reduced and S-carboxamidomethylated, and digested with arginylendpeptidase. Peptides were separated by reversed-phase HPLC on TSKgel ODS 120T (5 μm, 4.6 × 250 mm) using a linear gradient of acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 1.0 mL/min. (A) ATL; (B) Fraction A.

Figure 6

Sequence comparison of Apios tuber lectin (AAL) with soybean lectin (SBA). Identical amino acid residues are underlined beneath the sequence of SBA. The broken lines under the sequence of AAL are the sequence which was confirmed by direct sequencing of ATL using a protein sequencer in this study. Loops A-D are indicated by boldface letters. Asp (D) 240, Asp (D) 243, and Ser (S) 246 are putative truncating sites of SBA [24]. The amino acid sequence of AAL deduced from cDNA sequencing is cited from reference [22].

Figure 7

Specificity profiling of Apios tuber lectin (ATL). Scan images of ATL (12 ng/well) were analyzed with the Array Pro analyzer ver. 4.5. The net intensity value for each spot was determined as the signal intensity minus the background value. Data are the average ± S.D. of triplicate determinations. Detailed information on the glycans can be found in reference [26].

Figure 8

Effects of lectins on the TER values of Caco-2 monolayers. The cell monolayer TER value was measured after incubating for 2 h with a lectin (20–200 μg/mL). ATL: Apios tuber lectin, SBA: Soybean lectin, CGA: Japanese jack bean lectin, WGA: Wheat germ lectin, and AOL: Aspergillus oryzae lectin. Bovine serum albumin (BSA) (20–200 μg/mL) was used as a reference. Results are expressed as the percentage of the control value without a lectin at 0 h, and are the mean ± S.D. of three different determinations. * p < 0.05, compared with the control value.