Abrin immunotoxin: targeted cytotoxicity and intracellular trafficking pathway

Abstract

Background: Immunotherapy is fast emerging as one of the leading modes of treatment of cancer, in combination with chemotherapy and radiation. Use of immunotoxins, proteins bearing a cell-surface receptor-specific antibody conjugated to a toxin, enhances the efficacy of cancer treatment. The toxin Abrin, isolated from the Abrus precatorius plant, is a type II ribosome inactivating protein, has a catalytic efficiency higher than any other toxin belonging to this class of proteins but has not been exploited much for use in targeted therapy.

Methods: Protein synthesis assay using (3)[H] L-leucine incorporation; construction and purification of immunotoxin; study of cell death using flow cytometry; confocal scanning microscopy and sub-cellular fractionation with immunoblot analysis of localization of proteins.

Results: We used the recombinant A chain of abrin to conjugate to antibodies raised against the human gonadotropin releasing hormone receptor. The conjugate inhibited protein synthesis and also induced cell death specifically in cells expressing the receptor. The conjugate exhibited differences in the kinetics of inhibition of protein synthesis, in comparison to abrin, and this was attributed to differences in internalization and trafficking of the conjugate within the cells. Moreover, observations of sequestration of the A chain into the nucleus of cells treated with abrin but not in cells treated with the conjugate reveal a novel pathway for the movement of the conjugate in the cells.

Conclusions: This is one of the first reports on nuclear localization of abrin, a type II RIP. The immunotoxin mAb F1G4-rABRa-A, generated in our laboratory, inhibits protein synthesis specifically on cells expressing the gonadotropin releasing hormone receptor and the pathway of internalization of the protein is distinct from that seen for abrin.

Figure 1. F1G4-IT inhibits protein synthesis in MCF-7 and HepG2 cells but not in KB cells.

Cells were treated with F1G4-IT or mAb VU1D9-rABRa-A for 8 h in leucine free RPMI. The cells were pulsed with ³[H] L-leucine for 2 h and total cellular protein precipitated using 5% TCA. The incorporated radioactivity was plotted against that of the control cells. A: MCF-7; B: HepG2; C: KB. D: Cells treated with F1G4-IT or F1G4-IT_R167L. Each bar represents the mean of three separate experiments carried out with duplicate samples. **P<0.05.

Figure 2. Kinetics of inhibition of protein synthesis by F1G4-IT is slower than that of abrin.

HepG2 cells (1×10⁶/ml) were treated with IC₉₀ of either abrin or F1G4-IT for different time intervals and the procedure followed as described in Figure 1. The Boltzmann curve was used to analyze the data. The graph represents the mean of three separate experiments carried out with duplicate samples.

Figure 3. Trigger of cell death by abrin-a A chain is independent of inhibition of protein synthesis.

HepG2 cells (1×10⁶/ml) were treated with 19.2 nM of either one of the immunoconjugates: F1G4-IT or F1G4-IT_R167L, or abrin (51.25 pM) for different time intervals. The cells were harvested, fixed with 70% ethanol at −20°C, stained with staining solution (20 µg/ml propidium iodide and 50 µg/ml RNase A in PBS) and analyzed by flow cytometry. The percentage of dead population was determined and plotted above control cells. Each bar represents the mean of at least three different experiments carried out with duplicate samples. **P<0.005.

Figure 4. Intracellular localization of F1G4-IT in HepG2 cells is different from that of abrin.

Cells (5×10⁶) treated with either abrin or F1G4-IT for different time intervals were fixed with 4% para-formaldehyde and stained with mAb D6F10-Alexa 488 for 2 h in the dark at RT. The cells were counter stained with 5 µg/ml of Hoechst 33342 for 10 min at RT, washed with PBS, mounted on slides and images acquired in the Zeiss confocal scanning microscope. The images were analysed using the Image J image browser. Confocal microscopy of, A: abrin treated cells; B: F1G4-IT treated cells.

Figure 5. A chain of abrin and F1G4-IT have unique destinations.

Cells treated with either 6 nM abrin or 50 nM F1G4-IT, for different intervals were subjected to sub-cellular fractionation. The nuclear (N), cytosolic (C) and organellar (O) fractions of each sample were electrophoresed on a 12.5% polyacrylamide SDS gel under reducing conditions and subjected to immunoblot analysis. A: Cells treated with abrin immunoblotted with mAb D6F10 for the A chain; Rabbit antibodies to acetylated histone, H3 (17 kDa), GAPDH (37 kDa) and Calnexin (67 kDa) were used as controls for nuclear, cytosolic and organellar fractions respectively. B: Cells treated with F1G4-IT immunoblotted with mAb D6F10; MAb to Lamin-A (70 kDa) and rabbit antibodies to GAPDH and Calnexin were used as controls for nuclear, cytosolic and organellar fractions respectively.

Figure 6. TrxR inhibitor rescues cells from F1G4-IT activity.

HepG2 cells (1×10⁶/ml) were treated with auranofin for 6 h and cultured in presence of IC₉₀ of either abrin (51.25 pM) or F1G4-IT (19.2 nM) for 6 h in RPMI minus leucine. The cells were then pulsed with ³[H] L-leucine for 2 h, total protein precipitated with 5% TCA, solubilized with 0.1 N NaOH containing 1% SDS and the incorporated radioactivity measured. The percent radioactivity above control was determined. The graph depicts the mean of at least three different experiments carried out with duplicate samples. **P<0.05.

Figure 7. Novel intracellular trafficking of abrin and its IT.

F1G4-IT binds to the GnRH receptor via the antibody, mAb F1G4, and internalized via receptor-mediated endocytosis through clathrin coated pits. The protein is then released from the vesicles into the cytosol where the S-S bond between rABRa-A and the cross-linker SMPT is cleaved by thioredoxin, releasing the recombinant A chain. The thioredoxin, on the other hand, gets oxidized which is reduced back by the enzyme, thioredoxin reductase, using protons donated by cytosolic NADPH. This pathway is different from that observed for abrin, shown in the right half of the figure, wherein the internalized protein follows the retrograde pathway to reach the ER. In the ER, the disulfide bond is cleaved, releasing the A chain to the cytosol through the ERAD pathway. Once in the cytosol, irrespective of the pathway followed, the A chain binds to the 60S ribosomal subunit, depurinating the 28S rRNA, thus inhibiting protein synthesis.