Directed Mutagenesis for Arginine Substitution of a Phaseolus acutifolius Recombinant Lectin Disrupts Its Cytotoxic Activity

Martínez-Alarcón, Dania; Castro-Guillén, José Luis; Fitches, Elaine; Gatehouse, John A.; Przyborski, Stefan; Moreno-Celis, Ulisses; Blanco-Labra, Alejandro; García-Gasca, Teresa

Abstract

Recently, we reported that a recombinant Tepary bean (Phaseolus acutifolius) lectin (rTBL-1) induces apoptosis in colon cancer cell lines and that cytotoxicity was related to differential recognition of β1-6 branched N-glycans. Sequencing analysis and resolution of the rTBL-1 3D structure suggest that glycan specificity could be strongly influenced by two arginine residues, R103 and R130, located in the carbohydrate binding pocket. The aim of this work was to determine the contribution of these residues towards cytotoxic activity. Two rTBL-1 mutants were produced in Pichia pastoris, biochemically characterized, and cytotoxic effects were evaluated on human colorectal cancer cells (HT-29). Substitution of either of the arginine residues with glutamines resulted in significant reductions in cytotoxic activity, with losses of 1.5 and 3 times for R103 and R130, respectively. Docking analysis showed that the mutations decreased lectin affinity binding to some Epidermal Growth Factor Receptor (EGFR)-related N-glycans. Together, these findings confirm that both of the selected arginine residues (R103 and R130) play a key role in the recognition of tumor cell glycoconjugates by rTBL-1.

Citation

Martínez-Alarcón, D., Castro-Guillén, J. L., Fitches, E., Gatehouse, J. A., Przyborski, S., Moreno-Celis, U., Blanco-Labra, A., & García-Gasca, T. (2024). Directed Mutagenesis for Arginine Substitution of a Phaseolus acutifolius Recombinant Lectin Disrupts Its Cytotoxic Activity. International Journal of Molecular Sciences, 25(24), Article 13258. https://doi.org/10.3390/ijms252413258

• Journal Article Type: Article
• Acceptance Date: Dec 6, 2024
• Online Publication Date: Dec 10, 2024
• Publication Date: 2024-12
• Deposit Date: Jan 16, 2025
• Publicly Available Date: Jan 16, 2025
• Journal: International Journal of Molecular Sciences
• Print ISSN: 1661-6596
• Electronic ISSN: 1422-0067
• Publisher: MDPI
• Peer Reviewed: Peer Reviewed
• Volume: 25
• Issue: 24
• Article Number: 13258
• DOI: https://doi.org/10.3390/ijms252413258
• Keywords: recombinant lectins, cancer, tepary bean, branched N-glycans
• Public URL: https://durham-repository.worktribe.com/output/3332385