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

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.

Keywords: branched N-glycans; cancer; recombinant lectins; tepary bean.

Figure 2

Characterization of the mutants. (A) Molecular size comparison between rTBL-1 and the mutants. SDS-PAGE of the electrophoretic profiles of rTBL-1, R103Q, and R130Q. Lane 1, ladder; lane 2, 5 µg of rTBL; lane 3, 5 µg of R103Q; lane 4, 5 µg of R130Q. (B) Analysis of glycosylation. Top panel shows a coomassie R-250-stained SDS-PAGE. N-Glycosidase F (PGNaseF), all O-glycosidases (β1,4-Galactosidase, endo-α-N-acetylgalactosaminidase, α2-3,6,8,9-Neuraminidase, and β-N-Acetylglucosaminidase), all N- and O-glycosidases (N-Glycosidase F, β1,4-Galactosidase, endo-α-N-acetylgalactosaminidase, α2-3,6,8,9-Neuraminidase, and β-N-Acetylglucosaminidase).

Figure 3

Cytotoxic effect of rTBL-1 and its mutants on HT-29 colon cancer cells. Cells were treated with rTBL-1, R103Q, or R130Q (0, 0.25, 0.5, 2.5, 5, 25, and 50 μg/mL) for 24 h and cell survival percentages were calculated from hemocytometer counts. Small letters show significant differences for each separate treatment at different concentrations (Tukey p < 0.05), where differences between treatments for rTBL-1 were pointed out with letters a–e, for R103Q with letters f–h, and for R130Q with letter i.

Figure 4

Molecular docking of the experimental N-glycans from the Mammalian Glycan Array 5.4 with rTBL-1 structures. The graphical 2D representation of N-glycans is shown according to the Symbol Nomenclature for Glycans (SNFG) [28]. The N-glycans from the mammalian glycan array “Version 5.4” are labeled as GA-##, where ## is the number described by Martínez-Alarcón et al. (2020) [13] and is related to the signal’s relative intensity of lectin recognition. Docking results are shown as topological representations of interactions at the same surface location between the N-glycans with the recombinant lectins: rTBL-1, R104Q, and with the R131Q lectin. The N-glycan in topological representations is presented as sticks colored according to the SNFG (as indicated by geometric codes), the protein surface in gray, highlighting the CBP in light blue and the arginine residues in dark blue. GA, glycan array.

Figure 6

Molecular docking results for EGFR-related N-glycans with rTBL-1 and mutants R104Q and R131Q. For each N-glycan a graphical 2D representation according to SNFG [28] is shown. Docking results are shown as topological representations of interactions at the same surface location between the N-glycans, and the recombinant lectins: rTBL-1, R104Q, and R131Q. The N-glycan in topological representations is presented as sticks colored according to the SNFG, the protein surface in gray, highlighting the CBP in light blue, the mutated residues in red, and the original arginine residues in dark blue.

Figure 7

Three-dimensional maps for the interactions between EGFR-related N-glycans to rTBL-1, and mutants R104Q and R131Q. For each N-glycan, a graphical 2D representation according to SNFG [28] is shown. The interaction maps are shown as topological representations of the interactions of the N-glycans and the recombinant lectins: rTBL-1, R104Q, and R131Q. The target N-glycans are shown as sticks, and the structure of N-glycan residues is colored according to SNFG. The docking-interacting residues of lectins are shown as orange sticks with their respective surface and relative sequence number as labels.

Figure 1

Mutants’ design and production. (A) Sequence alignment of rTBL-1 homologous lectins. PHAL_Phaseolus costaricensis (Q5ZF34_9FABA), PHAL_Phaseolus coccineus (Q84RP8_PHACN), PHAL_Phaseolus vulgaris (PHAL_PHAVU), PHAL_Phaseolus acutifolius (Q40750_PHAAT), rTBL-1 sequence [14]. White letters represent identical residues, semiconserved residues are displayed in red enclosed into empty boxes and non-conserved residues are depicted in black. Blue triangles show residues that correspond to the CBP similarly to PHA-L from P. Vulgaris; red triangles show residues interacting with metal cations; green triangle depicts sequence for N-glycosylation marked with a blue bracket; black stars show residues presumably responsible for the cytotoxic effect of rTBL-1 (R103 and R130). (B) Agarose gel electrophoresis showing pGAPαZB-rTBL-1 vector amplification. Lane 1, ladder; lanes 2–5, amplification products. (C) Western blot screening using anti-His antibodies for transformed colonies. (D,E) R103Q and R130Q SDS-PAGE electrophoretic profiles stained for total protein. Lane 1, ladder; lane 2, 25 µL of culture supernatant (CS); lane 3, 25 µL of flow through (FT); lane 4, 25 µL wash with 10 mM imidazole buffer (W); and lane 5, 25 µL elution with 200 mM imidazole buffer (E). PHAL, Leukocyte phytohemagglutinin.

Figure 5

EGFR-related N-glycans used in docking process with rTBL-1. Graphical 2D representation of the N-glycans was created according to SNFG [28]. All EGFR-related N-glycans were named according to the GlyConnect platform [30]. CBP, carbohydrate-binding pocket.