Structural insights into the recognition mechanism between an antitumor galectin AAL and the Thomsen-Friedenreich antigen

Abstract

Thomsen-Friedenreich (TF) antigen, which plays an important role in the regulation of cancer cell proliferation, occurs in ∼90% of all human cancers and precancerous conditions. Although TF antigen has been known for almost 80 yr as a pancarcinoma antigen, the recognition mechanism between TF antigen and target protein has not been structurally characterized. A number of studies indicated that TF disaccharide is a potential ligand of the galactoside-binding galectins. In this work, we identified the TF antigen as a potential ligand of the antitumor galectin AAL (Agrocybe aegerita lectin) through glycan array analysis and reported the crystal structure of AAL complexed with the TF antigen. The structure provides a first look at the recognition mode between AAL and TF antigen, which is unique in a conservative (Glu-water-Arg-water) structural motif-based hydrogen bond network. Structure-based mutagenesis analysis further revealed the residues responsible for recognition specificity and binding affinity. Crystal structures of AAL complexed with two other TF-containing glycans showed that the unique TF recognition mode is kept intact, which may be commonly adopted in some cancer-related galectins. The finding provided the new target and approach for the antitumor drug design and relative strategy based on the AAL-TF recognition mode as a prototype model.

Figure 1.

Sugar-binding specificities of AAL and mutant R85A. A) Binding of AAL to carbohydrates, from the CFG glycan array. Error bars = sd. Sulfate group of the sugar is shown as 3S. B) Glycan array result of mutant R85A.

Figure 2.

Overall structure of AAL-TF antigen complex. A) Dimer structure of AAL-TF antigen complex. TF antigen and water molecules bound to the CRD concave of AAL are shown in a ball-and-stick model. B) F_o-F_c omit electron density map around TF antigen and 2 conservative water molecules nearby is calculated without the ligand and contoured at 3σ. C) Carbohydrate recognition site showing a positively charged cavity bound to a TF antigen with 2 water molecules. D) Interactions between TF antigen and AAL. Residues Pro42, Asn43, His59, Arg63, Asn72, Trp80, and Glu83 are involved in galactose moiety recognition (blue), while Glu66 and Arg85 with 2 water molecules form a hydrogen bond network in recognition with the GalNAc moiety (magenta). Arg63 and Glu83 are attended to both galactose and GalNAc moieties. E) Topological diagram of AAL secondary structure.

Figure 3.

Recognition mode between AAL and TF antigen unique in a (Arg85-water-Glu66-water) motif-based hydrogen bond network. A, B) Stereo view of AAL-TF antigen (A) and AAL-TFN (B) complexes. Glycan ligands are shown as sticks; unique moiety GalNAc is highlighted in yellow. C) Special interactions between AAL and GalNAc moiety in the AAL-TF recognition site. D) Consensus recognition mode between galectin and TF antigen, unique in a water molecule-mediated hydrogen bond network.

Figure 4.

Recognition site in AAL-TF antigen (A) and CGL2-TF disaccharide (B) complexes, comparatively showing that the distinct interactions between the galectin and the GalNAc moiety occur in the AAL-TF antigen, which corresponds to a chair conformation of GalNAc (C), while missing in the CGL2-TF disaccharide, which otherwise corresponds to a twisted-boat conformation of GalNAc (D). Structural data for CGL2-TF disaccharide is from PDB 1ULG.

Figure 5.

Bioactivity and 3-D structure analyses of mutants E66A and R85A. A) Relative tumor cell apoptosis-inducing activities of E66A and R85A, assayed by flow cytometry using the PI staining method. Top panel: representative bivariate plots of the FACS analysis. Bottom panel: relative activities of E66A and R85A compared with AAL. B, C) Recognition sites relative to the GalNAc moiety observed in structures of R85A-TFN (B) and E66A-TFN (C). They both lose the (Arg85-water-Glu66-water) motif-based hydrogen bond network. D) More direct interactions between protein and ligand in the E66A-TFN complex. Residues and TFN ligand are shown as ball-and-stick model. Distance of hydrogen bonds is labeled on dashed lines.

Figure 6.

Conservation of the (Arg85-water-Glu66-water) motif-based recognition mode. A) Sequence alignment of CRD for AAL, CGL2, Gal 1, 2, 3, and 9. Triangles indicate critical residues (Glu66 and Arg85 in AAL), which are invariant in these galectins. Asterisks indicate galectins identified as being able to bind with TF antigen. Sequence data of CGL2 and Gal 1, 2, 3, and 9 are from the U.S. National Center for Biotechnology Information (NCBI) protein database. B) F_o-F_c omit electron density map for the TF-containing pentasaccharide observed in AAL-GM1 complex structure. C) Recognition sites between AAL and TF GalNAc moiety of GM1, showing that the distinct recognition mode is kept intact as in the AAL-TF antigen complex structure (see Fig. 3D).