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. 2012 Apr 15;443(2):369-78.
doi: 10.1042/BJ20112061.

A novel lectin from Agrocybe aegerita shows high binding selectivity for terminal N-acetylglucosamine

Shuai Jiang  1 , Yijie Chen, Man Wang, Yalin Yin, Yongfu Pan, Bianli Gu, Guojun Yu, Yamu Li, Barry Hon Cheung Wong, Yi Liang, Hui Sun
Affiliations

A novel lectin from Agrocybe aegerita shows high binding selectivity for terminal N-acetylglucosamine

Shuai Jiang et al. Biochem J. .

Abstract

A novel lectin was isolated from the mushroom Agrocybe aegerita (designated AAL-2) by affinity chromatography with GlcNAc (N-acetylglucosamine)-coupled Sepharose 6B after ammonium sulfate precipitation. The AAL-2 coding sequence (1224 bp) was identified by performing a homologous search of the five tryptic peptides identified by MS against the translated transcriptome of A. aegerita. The molecular mass of AAL-2 was calculated to be 43.175 kDa from MS, which was consistent with the data calculated from the amino acid sequence. To analyse the carbohydrate-binding properties of AAL-2, a glycan array composed of 465 glycan candidates was employed, and the result showed that AAL-2 bound with high selectivity to terminal non-reducing GlcNAc residues, and further analysis revealed that AAL-2 bound to terminal non-reducing GlcNAc residues with higher affinity than previously well-known GlcNAc-binding lectins such as WGA (wheatgerm agglutinin) and GSL-II (Griffonia simplicifolia lectin-II). ITC (isothermal titration calorimetry) showed further that GlcNAc bound to AAL-2 in a sequential manner with moderate affinity. In the present study, we also evaluated the anti-tumour activity of AAL-2. The results showed that AAL-2 could bind to the surface of hepatoma cells, leading to induced cell apoptosis in vitro. Furthermore, AAL-2 exerted an anti-hepatoma effect via inhibition of tumour growth and prolongation of survival time of tumour-bearing mice in vivo.

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Figures

Figure 1
Figure 1. Purification of AAL-2 and the amino acid sequences of tryptic peptides identified by MS
(A) Crude extract was applied to a GlcNAc monomer-coupled epoxy-activated Sepharose 6B column (1 cm×10 cm) and equilibrated with TBS (pH 7.2). After washing with TBS, AAL-2 was eluted with TBS containing 0.2 M GlcNAc (indicated by the arrow). The plot was generated by ÄKTAprime (GE Healthcare). (B) SDS/polyacrylamide gel of AAL-2 was stained with Coomassie Brilliant Blue. Molecular masses are indicated in kDa. (C) Amino acid sequence data for AAL-2. The five tryptic peptides identified by MS/MS are shown.
Figure 2
Figure 2. Identification of AAL-2 coding sequence
(A) The AAL-2 coding sequence is located on the complementary strand of UniGene 4198. (B) The AAL-2 coding sequence consists of 1224 bp, which is computationally translated into 407 amino acids. The deduced amino acid sequence is shown under the AAL-2 coding sequence, and the five tryptic peptides (underlined) of AAL-2 identified by MS are aligned under the deduced amino acid sequence. MALDI–TOF/TOF analysis revealed 69% sequence coverage of the full-length AAL-2 (grey box). The initiation codon ATG is boxed and the stop codon tag is marked with an asterisk. (C) Determination of AAL-2 molecular mass by MALDI–TOF-MS to be 43175.72 Da. Intens., intensity (in arbitrary units, a.u.).
Figure 3
Figure 3. Cloning, expression and purification of AAL-2
(A) Total RNA was isolated from fresh A. aegerita fruiting body and amplified by reverse transcription–PCR. The AAL-2 coding sequence (~1.2 kb) was amplified by nested PCR. (B) The AAL-2 coding sequence was cloned into pET-30a plasmids followed by transformation into E. coli BL21 cells. A strong band appeared at approximately 43 kDa after IPTG-induction for 5 h compared with non-induction. (C) nAAL-2 and rAAL-2 showed the same electrophoretic mobility on SDS/PAGE and shared the same immunogenicity after immunoblotting with the anti-nAAL-2 polyclonal antibody. Molecular masses are indicated in kDa.
Figure 4
Figure 4. Binding properties of AAL-2 for GlcNAc assessed by glycan array analysis and ITC profiling
AAL-2-binding specificity was determined by glycan screening on a printed array (Version 4.1 of the Consortium for Functional Glycomics). (A) Upper panel: the top ten glycans with highest binding affinity to AAL-2 (200 μg/ml). The binding glycan GlcNAc is shown and labelled with a red asterisk. Results are means±S.E.M. The glycan number at the bottom indicates the various glycans used in the glycan array. The entire glycan array is available at http://www.functionalglycomics.org/static/consortium/resources/resourcecoreh14.shtml. Lower panel: the heat map shows the binding selectivity of AAL-2 at different concentrations (0.5, 1, 50 and 200 μg/ml). The log (base 2) of RFU values is represented on the bar on the right-hand side. Warmer colours indicate higher binding affinity. (B) ITC results for GlcNAc monomer (7 mM) binding to AAL-2 (0.05 mM) in PBS at 25°C. Upper panel: data obtained from 28 automatic injections of 10 μl of GlcNAc monomer each into the AAL-2-containing cell. Lower panel: plot of the total heat released as a function of ligand concentration for the titration shown above (squares). The continuous line represented the best least-squares fit to the obtained data. All thermodynamic measurements of AAL-2 binding to GlcNAc were performed at 25°C.
Figure 5
Figure 5. Comparison of carbohydrate-binding specificity of AAL-2 with other GlcNAc-binding lectins
The lectin carbohydrate specificities are as summarized from the Consortium for Functional Glycomics (http://functionalglycomics.org). See also [–49].
Figure 6
Figure 6. AAL-2 induces apoptosis in hepatoma cells in vitro
AAL-2 could bind to H22 (A) and Huh7 (B) hepatoma cells. H22 cells and Huh7 cells were untreated (green histograms), incubated with the anti-AAL-2 antibody alone (pink histograms), or with both AAL-2 and anti-AAL-2 antibody (blue histograms), and analysed by flow cytometry. The mean fluorescence intensity (MFI) values of the different incubation conditions are indicated in each histogram. Apoptotic H22 (C) and Huh7 (E) cells were detected by Annexin V/PI staining after 24 and 36 h of incubation respectively with different concentrations of AAL-2. The percentages of pro-apoptotic H22 cells (D) and Huh7 cells (F) gated on Annexin V+/PI− were calculated. Results are means±S.E.M. (n=3); *P<0.05, **P<0.01, ***P<0.001.
Figure 7
Figure 7. AAL-2 inhibits hepatocellular carcinoma growth and prolongs survival time of tumour-bearing mice in vivo
A total of 107 H22 cells were injected subcutaneously into the right flank of BALB/c mice. On days 7, 9, 11, 13, 15, 17 and 19 after tumour inoculation, mice were injected at the tumour site with 5 mg of AAL-2/kg in 100 μl of PBS or diluent control. Tumour-bearing mice treated with AAL-2 and PBS were monitored for changes in body weight (A), tumour growth (B) and survival time (C). Results are means±S.D. (n=10); *P<0.05.
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