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

Martínez-Alarcón, Dania; Varrot, Annabelle; Fitches, Elaine; Gatehouse, John A.; Cao, Min; Pyati, Prashant; Blanco-Labra, Alejandro; Garcia-Gasca, Teresa

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.

Citation

Martínez-Alarcón, D., Varrot, A., Fitches, E., Gatehouse, J. A., Cao, M., Pyati, P., Blanco-Labra, A., & Garcia-Gasca, T. (2020). Recombinant Lectin from Tepary Bean (Phaseolus acutifolius) with Specific Recognition for Cancer-Associated Glycans: Production, Structural Characterization, and Target Identification. Biomolecules, 10(4), Article 654. https://doi.org/10.3390/biom10040654

• Journal Article Type: Article
• Acceptance Date: Apr 20, 2020
• Online Publication Date: Apr 23, 2020
• Publication Date: Apr 30, 2020
• Deposit Date: May 12, 2020
• Publicly Available Date: May 12, 2020
• Journal: Biomolecules
• Publisher: MDPI
• Peer Reviewed: Peer Reviewed
• Volume: 10
• Issue: 4
• Article Number: 654
• DOI: https://doi.org/10.3390/biom10040654
• Public URL: https://durham-repository.worktribe.com/output/1271064