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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

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


Authors

Dania Martínez-Alarcón

Annabelle Varrot

John A. Gatehouse

Min Cao

Prashant Pyati

Alejandro Blanco-Labra

Teresa Garcia-Gasca



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., …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 Mar 28, 2024
Journal Biomolecules
Publisher MDPI
Peer Reviewed Peer Reviewed
Volume 10
Issue 4
Article Number 654
DOI https://doi.org/10.3390/biom10040654

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