Recombinant Lectin from Tepary Bean (Phaseolus acutifolius) with Specific Recognition for Cancer-Associated Glycans: Production, Structural Characterization, and Target Identification

Abstract

Herein, we report the production of a recombinant Tepary bean lectin (rTBL-1), its three-dimensional (3D) structure, and its differential recognition for cancer-type glycoconjugates. rTBL-1 was expressed in Pichia pastoris, yielding 316 mg per liter of culture, and was purified by nickel affinity chromatography. Characterization of the protein showed that rTBL-1 is a stable 120 kDa homo-tetramer folded as a canonical leguminous lectin with two divalent cations (Ca2+ and Mn2+) attached to each subunit, confirmed in its 3D structure solved by X-ray diffraction at 1.9 Å resolution. Monomers also presented a ~2.5 kDa N-linked glycan located on the opposite face of the binding pocket. It does not participate in carbohydrate recognition but contributes to the stabilization of the interfaces between protomers. Screening for potential rTBL-1 targets by glycan array identified 14 positive binders, all of which correspond to β1-6 branched N-glycans’ characteristics of cancer cells. The presence of α1-6 core fucose, also tumor-associated, improved carbohydrate recognition. rTBL-1 affinity for a broad spectrum of mono- and disaccharides was evaluated by isothermal titration calorimetry (ITC); however, no interaction was detected, corroborating that carbohydrate recognition is highly specific and requires larger ligands for binding. This would explain the differential recognition between healthy and cancer cells by Tepary bean lectins.

Bioaccessibility and In Vitro Intestinal Permeability of a Recombinant Lectin from Tepary Bean (Phaseolus acutifolius) Using the Everted Intestine Assay

“…Later on, a polyclonal antibody against TBLF was developed. Recently, as it was necessary to produce a recombinant lectin from Tepary bean (rTBL-1) by using Pichia pastoris yeast [ 9 ], we developed a specific polyclonal antibody against the recombinant lectin for its detection in specific tissues, serum, and fluids throughout the gastrointestinal tract.…”

“…The rTBL-1 was subjected to an additional in silico digestion, but none of the generated peptides were big enough to conduct an interaction (peptide mass < 500 Da, data not shown). Previous studies using glycan arrays and isothermal titration calorimetry showed that rTBL-1 recognized 14 β1-6 branched N-glycans independently of their size, where β-D-galactose, β-D-N-acetyl-glucosamine, β-D-mannose, α-L-fucose, and sialic acid were found to be part of the recognized glycans [ 9 ]. Sialic acid and N-acetyl-glucosamine exhibited the highest binding affinity, as indicated by the lowest binding energies, suggesting that the rTBL-1 could directly interact with membrane components and its possible internalization as shown by Western blot, immunohistochemistry, and mass spectrometry analyses.…”

“…Due to the low yield, time-consuming, and high cost process to obtain the TBLF, the production of a recombinant lectin (rTBL-1) expressed in yeast ( Pichia pastoris ) was performed in our laboratory [ 9 ]. The structural analysis of the whole polypeptide sequence showed a 277 amino acid chain that displayed high identity with lectin A (LA) and lectin B (LB) [ 10 ] and with a P. acutifolius lectin previously deduced by Mirkov et al (1994) [ 11 ] (accession number: AAA82181.1, ).…”

“…rTBL-1 is approximately 2.5 kDa larger than the native lectin, mainly due to the addition of a 6XHis tag (~840.92 Da), the sequence EAEAAA at the N-terminal, derived from Kex2p cleavage of the a-factor (~561 Da), and possibly the presence of bigger glycosidic antennas than those on TBL-1 (~640 Da). The structure and size of these carbohydrates analyzed by glycosidases treatment, SDS-PAGE and periodic acid Schiff staining suggest that both proteins contain exclusively N-linked glycans [ 9 ]. Although the rTBL-1 lacks agglutination activity, it exhibits similar cytotoxic effects to the TBLF (unpublished data).…”

“…The rTBL-1 production process, at this time, resulted in a yield of 316 mg/mL; the rTBL-1 was stable up to 55 °C and pH 8.5 in Tris-HCl buffer. rTBL-1 was purified by nickel affinity chromatography, and SDS-PAGE electrophoresis showed the presence of a single band of ~30 kDa [ 9 ].…”