Related Experiment Video
Updated: Jun 11, 2026
Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope
Published on: March 24, 2017
Directed Mutagenesis for Arginine Substitution of a Phaseolus acutifolius Recombinant Lectin Disrupts Its Cytotoxic
Dania Martínez-Alarcón1, José Luis Castro-Guillén2, Elaine Fitches3
1Centro de Investigación y de Estudios Avanzados Unidad Irapuato, Departamento de Biotecnología y Bioquímica, Irapuato 36821, Guanajuato, Mexico.
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.
Insights
Recombinant Tepary bean lectin (rTBL-1) induces colon cancer cell death by recognizing specific sugar structures. Key arginine residues (R103, R130) are crucial for this lectin's cytotoxic activity and binding to tumor cell glycans.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Recombinant Tepary bean lectin (rTBL-1) exhibits apoptosis-inducing properties in colon cancer cell lines.
- Cytotoxicity is linked to the lectin's recognition of specific β1-6 branched N-glycans.
- Structural analysis suggests arginine residues R103 and R130 influence glycan binding specificity.
Purpose of the Study:
- To investigate the role of arginine residues R103 and R130 in rTBL-1's cytotoxic activity.
- To determine the contribution of these residues to the recognition of tumor-associated glycans.
Main Methods:
- Site-directed mutagenesis to create rTBL-1 mutants (R103Q, R130Q).
- Expression and biochemical characterization of rTBL-1 mutants in Pichia pastoris.
- Evaluation of cytotoxic effects on HT-29 human colorectal cancer cells.
- Molecular docking analysis to assess binding affinity to Epidermal Growth Factor Receptor (EGFR)-related N-glycans.
Main Results:
- Mutating R103Q and R130Q significantly reduced rTBL-1's cytotoxic activity (1.5-fold and 3-fold loss, respectively).
- Docking simulations indicated decreased binding affinity to EGFR-related N-glycans for the mutant lectins.
- These findings highlight the importance of R103 and R130 in rTBL-1's interaction with cancer cell surface glycoconjugates.
Conclusions:
- Arginine residues R103 and R130 are critical for the cytotoxic function of rTBL-1.
- These residues mediate the recognition of specific N-glycans on colorectal cancer cells.
- rTBL-1's therapeutic potential in cancer may be modulated by altering these key binding residues.
Related Concept Videos
01:16In-vitro Mutagenesis
02:53Conservative Site-specific Recombination and Phase Variation
The recognition sites for Cre recombinase called LoxP...