Characterization of transferrin receptor-mediated endocytosis and cellular iron delivery of recombinant human serum transferrin from rice (Oryza sativa L.)

Abstract

Background: Transferrin (TF) plays a critical physiological role in cellular iron delivery via the transferrin receptor (TFR)-mediated endocytosis pathway in nearly all eukaryotic organisms. Human serum TF (hTF) is extensively used as an iron-delivery vehicle in various mammalian cell cultures for production of therapeutic proteins, and is also being explored for use as a drug carrier to treat a number of diseases by employing its unique TFR-mediated endocytosis pathway. With the increasing concerns over the risk of transmission of infectious pathogenic agents of human plasma-derived TF, recombinant hTF is preferred to use for these applications. Here, we carry out comparative studies of the TFR binding, TFR-mediated endocytosis and cellular iron delivery of recombinant hTF from rice (rhTF), and evaluate its suitability for biopharmaceutical applications.

Result: Through a TFR competition binding affinity assay with HeLa human cervic carcinoma cells (CCL-2) and Caco-2 human colon carcinoma cells (HTB-37), we show that rhTF competes similarly as hTF to bind TFR, and both the TFR binding capacity and dissociation constant of rhTF are comparable to that of hTF. The endocytosis assay confirms that rhTF behaves similarly as hTF in the slow accumulation in enterocyte-like Caco-2 cells and the rapid recycling pathway in HeLa cells. The pulse-chase assay of rhTF in Caco-2 and HeLa cells further illustrates that rice-derived rhTF possesses the similar endocytosis and intracellular processing compared to hTF. The cell culture assays show that rhTF is functionally similar to hTF in the delivery of iron to two diverse mammalian cell lines, HL-60 human promyelocytic leukemia cells (CCL-240) and murine hybridoma cells derived from a Sp2/0-Ag14 myeloma fusion partner (HB-72), for supporting their proliferation, differentiation, and physiological function of antibody production.

Conclusion: The functional similarity between rice derived rhTF and native hTF in their cellular iron delivery, TFR binding, and TFR-mediated endocytosis and intracellular processing support that rice-derived rhTF can be used as a safe and animal-free alternative to serum hTF for bioprocessing and biopharmaceutical applications.

Figure 1

TFR-competition binding assay of rhTF and hTF in Caco-2 cells. Different concentrations of rhTF or hTF mixed with 1 μg/ml of ¹²⁵I-hTF were added to confluent Caco-2 cells and incubated at 4°C for 1 hr. Cells were then washed with cold PBS, solubilized with 1 N NaOH, and the amount of radiolabelled hTF in cell lysate was determined. The total cellular protein in the cell lysate was measured, and used to normalize the data to ng of hTF per mg cell protein (“ng/mg cell protein”). Data is represented as average with error bars indicating standard deviation, n = 3.

Figure 2

TFR-binding affinity of rhTF and hTF in HeLa cells. Different concentrations of ¹²⁵I-labeled hTF or rhTF were added to confluent HeLa cells and incubated at 4°C for 2 hr. Cells were then washed with cold PBS, solubilized with 1 N NaOH, and the amount of radiolabelled hTF or rhTF in cell lysate was determined. The total cellular protein in the cell lysate was measured, and used to normalize the data to ng of hTF per mg cell protein (“ng/mg cell protein”). Data is represented as average with error bars indicating standard deviation, n = 3.

Figure 3

Comparison of uptake kinetics of rhTF and hTF in HeLa and Caco-2 cells. A. Uptake kinetics of rhTF and hTF in HeLa cells. One μg/ml of ¹²⁵I-labeled rhTF was added to confluent HeLa cells, and incubated at 37°C for 0.5, 1, 2, and 4 hr. In a comparison assay, 1 μg/ml of ¹²⁵I-hTF was added to confluent HeLa cells, and incubated at 37°C for 0.5 and 3 hr. Cells were then washed with cold PBS, solubilized with 1 N NaOH, and radioactivity in cell lysate was determined. B. Uptake kinetics of rhTF and hTF in Caco-2 cells. One μg/ml of ¹²⁵I-labeled hTF or rhTF was added to confluent Caco-2 cells and incubated at 37°C for 0.5, 1, 2, and 4 hr. Cells were then washed with cold PBS, solubilized with 1 N NaOH, and radioactivity in cell lysate was determined. The total cellular protein in the cell lysate was measured, and used to normalize the data to ng of hTF per mg cell protein (“ng/mg cell protein”). Data is represented as average with error bars indicating standard deviation, n = 3.

Figure 4

Pulse Chase Assay of rhTF in Caco-2 and HeLa Cells. Confluent Caco-2 or HeLa cells were pulsed for 1 hr by incubation with pulse medium containing 3 μg/ml ¹²⁵I-labelled hTF or rhTF at 37°C. Then, the pulse medium with radiolabeled TF was removed by aspiration of the medium followed by washing cell monolayers with cold DMEM medium supplemented with 0.1% BSA. Caco-2 and HeLa cells were then chased by incubation with excess molar (0.3 mg/ml) of unlabeled hTF or rhTF in chase medium at 37°C. After chasing for 1 or 3 hr, the percentage of cell-associated (A) and released (B) hTF or rhTF was determined. Data is represented as average with error bars indicating standard deviation, n = 3.

Figure 5

Pulse Chase Assay comparing hTF and rhTF in Caco-2 cells. Confluent Caco-2 cells were pulsed for 1 hr by incubation with pulse medium containing 3 μg/ml ¹²⁵I-labelled hTF or rhTF at 37°C. Then, the pulse medium with radiolabeled TF was removed by aspiration of medium followed by washing cell monolayers with cold DMEM medium supplemented with 0.1% BSA. Caco-2 cells were then chased by incubation with excess molar (300 μg/ml) of unlabeled hTF or rhTF in chase medium at 37°C. After chasing for 1 or 3 hr, the percentage of cell-associated (A) and released (B) hTF or rhTF were determined. Data is represented as average with error bars indicating standard deviation, n = 3.

Figure 6

Effect of rhTF on the proliferation of HL-60 cells. The viable cell concentration of HL-60 cells after three days culture in serum-free medium supplemented with no hTF, 0.005, 0.05, 0.5, 5 and 50 mg/L of hTF (holo-form, from Sigma) or rhTF (Partial; partially iron saturated) was determined by fluorescence assay. Data is represented as average with error bars indicating standard deviation, n = 3.

Figure 7

The growth kinetics of hybridoma cells in serum-free medium supplemented with hTF or rhTF at 5 mg/L. The hTF and rhTF were compared at different degrees of iron saturation at 5 mg/L concentration. Apo, iron-free; Partial, partially iron saturated; Holo, iron saturated. Error bars denote the standard deviation of triplicate cultures.

Figure 8

Comparison of rhTF and hTF for supporting hybridoma cell proliferation, cumulative cell density, and antibody production. Sp2/0 hybridoma cells were cultured in serum-free medium supplemented with hTF or rhTF at 0.03, 0.1, 0.3, 1, 5, or 30 mg/L. The hTF and rhTF were compared at different degrees of iron saturation: Apo, iron-free; Partial, partially iron saturated; Holo, iron saturated. A. Comparison of hTF to rhTF for cell proliferation during log phase growth (day0 through day3). B. Comparison of hTF to rhTF for cumulative cell density. Cumulative cell density is the estimated area under the growth curve and is indicative of the total cell mass generated by a culture system. Units are given in cell-days/ml. C. Comparison of hTF to rhTF for antibody production. Error bars denote the standard deviation of triplicate cultures.