Structural analysis and unique molecular recognition properties of a Bauhinia forficata lectin that inhibits cancer cell growth

Abstract

Lectins have been used at length for basic research and clinical applications. New insights into the molecular recognition properties enhance our basic understanding of carbohydrate-protein interactions and aid in the design/development of new lectins. In this study, we used a combination of cell based assays, glycan microarrays, and X-ray crystallography to evaluate the structure and function of the recombinant Bauhinia forficata lectin (BfL). The lectin was shown to be cytostatic for several cancer cell lines included in the NCI-60 panel; in particular, it inhibited growth of melanoma cancer cells (LOX IMVI) by over 95%. BfL is dimeric in solution and highly specific for binding of oligosaccharides and glycopeptides with terminal N-acetylgalactosamine (GalNAc). BfL was found to have especially strong binding (apparent Kd = 0.5-1.0 nM) to the tumor associated Tn antigen. High-resolution crystal structures were determined for the ligand-free lectin, as well as for its complexes with three Tn glycopeptides, globotetraose, and the blood group A antigen. Extensive analysis of the eight crystal structures and comparison to structures of related lectins revealed several unique features of GalNAc recognition. Of special note, the carboxylate group of Glu126, lining the glycan-binding pocket, forms H-bonds with both the N-acetyl of GalNAc and the peptide amido group of Tn antigens. Stabilization provided by Glu126 is described here for the first time for any GalNAc-specific lectin. Taken together, the results provide new insights into the molecular recognition of carbohydrates and provide a structural understanding that will enable rational engineering of BfL for a variety of applications.

Anti-glycan IgM repertoires in newborn human cord blood

“…Analysis of reproducibility[24] and validation of the array with numerous antibodies and lectins have been previously published. [25–28] Representative images illustrating the quality of the data can be found in the Supporting Information (see Figure A in S1 File). Full microarray data can be found in the Supporting Excel file, S2 File.…”

Plant-Derived Lectins as Potential Cancer Therapeutics and Diagnostic Tools

“…BFL exerts cytotoxicity via caspase-9 suppression and subsequent G2/M phase arrest [99]. Additionally, BFL inhibits various malignant cells in the NCI-60 panel, with melanoma LOX IMVI cells showing highest susceptibility [114]. Two other lectins, Bauhinia variegata lectin (BVL) and Bauhinia ungulate lectin (BUL) detected in Bauhinia spp., have demonstrated antitumor properties.…”

Plant Lectins Targeting O-Glycans at the Cell Surface as Tools for Cancer Diagnosis, Prognosis and Therapy

“… ( A , C , E , G ) Network of hydrogen bonds and stacking interactions anchoring Tn antigen (Tn) to the monosaccharide-binding site of: Bauhinia forficata BfL lectin ( A ) (PDB code 5T5J) [ 46 ]; soybean lectin SBA ( C ) (PDB code 4D69) [ 89 ]; Sambucus nigra SNA-II lectin ( E ) (PDB code 3CA6) [ 74 ]; and Vicia villosa VVA-B 4 lectin ( G ) (PDB code 1N47) [ 90 ]. Amino acid residues involved in stacking interactions with the disaccharide are colored orange; ( B , D , F , H ) Docking of Tn antigen to the monosaccharide-binding cavity (green dashed circle) of: Bauhinia forficata BfL lectin ( B ); soybean lectin SBA ( D ); Sambucus nigra SNA-II lectin ( F ); and Vicia villosa VVA-B4 lectin ( H ).…”