A novel core fucose-specific lectin from the mushroom Pholiota squarrosa

Abstract

Fucα1-6 oligosaccharide has a variety of biological functions and serves as a biomarker for hepatocellular carcinoma because of the elevated presence of fucosylated α-fetoprotein (AFP) in this type of cancer. In this study we purified a novel Fucα1-6-specific lectin from the mushroom Pholiota squarrosa by ion-exchange chromatography and affinity chromatography on thyroglobulin-agarose. The purified lectin was designated as PhoSL (P. squarrosa lectin). SDS-PAGE, MALDI-TOF mass spectrometry, and N-terminal amino acid sequencing indicate that PhoSL has a molecular mass of 4.5 kDa and consists of 40 amino acids (NH(2)-APVPVTKLVCDGDTYKCTAYLDFGDGRWVAQWDTNVFHTG-OH). Isoelectric focusing of the lectin showed bands near pI 4.0. The lectin activity was stable between pH 2.0 and 11.0 and at temperatures ranging from 0 to 100 °C for incubation times of 30 min. When PhoSL was investigated with frontal affinity chromatography using 132 pyridylaminated oligosaccharides, it was found that the lectin binds only to core α1-6-fucosylated N-glycans and not to other types of fucosylated oligosaccharides, such as α1-2-, α1-3-, and α1-4-fucosylated glycans. Furthermore, PhoSL bound to α1-6-fucosylated AFP but not to non-fucosylated AFP. In addition, PhoSL was able to demonstrate the differential expression of α1-6 fucosylation between primary and metastatic colon cancer tissues. 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 1.

Characterization of PhoSL. A, shown is an SDS-PAGE linear gradient gel (10–15%). Lane M indicates marker proteins; lane 1, purified PhoSL under reducing conditions with 2-mercaptoethanol. B, shown is a high density SDS-PAGE gel. Lane pM indicates marker peptides; lane 1, purified PhoSL under reducing conditions with 2-mercaptoethanol; lane 2, purified PhoSL non-reduced; lane M, marker proteins. C, shown is isoelectric focusing of PhoSL. Lane M indicates marker proteins; lane 1, PhoSL.

FIGURE 2.

Multiple alignment of PhoSL and RSL. The residues in the first row describe the amino acid sequence of PhoSL, and those in the second row describe the amino acid sequence of RSL. Shown in gray shading are the amino acid residues that are identical between PhoSL and RSL.

FIGURE 3.

The stability of PhoSL over a range of temperature and pHs. The stability of PhoSL was investigated over a broad range of temperatures for 30 min (A), long incubation times for four different temperatures (B), and incubation in different pH buffers (C).

FIGURE 4.

FAC analysis of PhoSL. A, shown is the elusion pattern of several types of PA-oligosaccharides on the PhoSL-immobilized column. B, shown are association constants (K_a) values of the purified PhoSL to various types of PA-glycans.

FIGURE 5.

Comparative analysis of glycan binding specificity of PhoSL and LCA by the GRYP code. A, shown are definitions of the GRYP code for representing the branch positions and non-reducing end residues. The non-reducing end sugars and the core fucose are shown in different colors in the left panel. Each branch is numbered from I to VI corresponding to GlcNAc transferases, as shown in the middle panel. B, shown are bar graph representations of the association constants (K_a) of PhoSL toward core-fucosylated N-glycans. Numbers at the bottom of the bar graphs correspond to the sugar numbers indicated in supplemental Fig. S2. C, shown are bar graph representations of the association constants (K_a) of LCA.

FIGURE 6.

Binding of PhoSL and LCA to immobilized glycoproteins by ELISA. A, shown is binding activity of biotin-labeled PhoSL and various immobilized glycoproteins. B, shown is binding activity of biotin-labeled LCA and various immobilized glycoproteins. The immobilized glycoproteins are: HAS, human serum albumin; Fib, fibrinogen IgA Immunoglobulin A; α2M, α2-macroglobulin; A1AT, α1-antitrypsin; C3, third components of complement; HP, haptoglobin; AGP, α1-acid glycoprotein; PSA, prostate specific antigen; AFP-L3, 1–6-fucosylated fetoprotein; LF, lactoferrin; FT, fetuin; BSM, bovine submaxillary gland mucin; PSM, porcine stomach mucin; Inv, invertase; OVA, ovalbumin.

FIGURE 7.

Antibody-lectin sandwich ELISA using purified AFP-L3 and the sera of HCC patients and normal volunteers. ELISA with Anti-AFP (A), PhoSL (B), and LCA (C).

FIGURE 8.

Immunohistochemical analysis of human colon cancer tissues with PhoSL and AAL. Human colon cancer tissue arrays comprising normal colon tissues (A and B), primary colon cancers (C and D), and metastatic colon cancers (E and F) were subjected to immunohistochemical analysis with PhoSL and AAL. G and H show the ratio of the numbers in each staining-intensity group to the total number is shown.