Introduction
Zinc (Zn) is an essential component for living organisms for components of structural and regulatory proteins, including transcription factors [1]. Zn ions play an important role in skeletal growth and bone homeostasis [1,2]. Zn is mostly distributed in skeletal muscle but is ubiquitous and found in tissues such as the prostate, hippocampus, pancreas, and kidney cortex [1,2]. The active centers of more than 300 enzymes are Zn-dependent, and there are over 2,000 transcriptional factors that are dependent on zinc in the gene expression [1,3,4]. In animals, Zn deficiency results in the loss of a more extensive impact in the immunomodulation system than other tissues and organs [4,5]. Human zinc deficiency is currently worldwide, and Zn supplements may provide therapeutic benefits in the management of diabetes type 2, atherosclerosis, neuro-degenerative disorders, and some cancers [4,5].
Transferrin (Tf) is a monomeric 75 kDa glycoprotein with two sites binding ferric iron [6]. The previous study showed that bovine apo-Tf binds zinc ions and that it inhibited Zn measurement [7]. Tf binds Fe3+ and various other metal ions such as Cu2+, Cd2+, Zn2+ and Ni2+ in order: Fe3+2+2+≤Zn2+2+ [8]. However, the binding analysis of bovine Tf with Zn has not been fully elucidated. In this study, we prepared beads immobilized with bovine apo-Tf to examine its binding with Zn ion, and examined the binding of the bovine holo-Tf with beads immobilized with Zn ions, and the iron binding after the binding of bovine apo-Tf with zinc ion-immobilized beads.
Materials and Methods
Chemicals
Bovine apo- and holo-Tf, and horse spleen ferritin were purchased from Sigma (St. Louis, MO, USA). CNBr-activated Sepharose 4B, Chelating SepharoseTM Fast Flow, and Sepharose 4B were purchased from GE Healthcare (Pickaway, OH, USA).
2-(5-Bromo-2-pyridylazo)-5-[N-n-propyl-N-(3-sulfopropyl)amino]phenol, disodium salt, dihydrate (5-Br-PAPS) was purchased from Dojindo Co. (Kumamoto, Japan). Assay plates were purchased from Iwaki Brand Div., Asahi Techno Glass (Funakoshi, Chiba, Japan). Other chemical compounds of analytical grade were from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Pure water (Elix water) was obtained using Millipore’s Elix Advantage Water Purification System (Billerica, MA, USA) from tap water.
The binding of bovine apo-Tf with Zn
Bovine apo-Tf was immobilized with CNBr-activated sepharose 4B beads (10 mg/ml beads) as described previously [9], and finally was suspended with 50% (v/v) using phosphate-buffered saline (PBS: 150 mM NaCl, 20 mM sodium phosphate, pH 7.2). ZnSO4 was added to 1 ml of PBS containing beads immobilized with bovine apo-Tf (TfB) or CB (net volume of beads per sample: 20 µl) at its final concentration of 5 µM. After incubation for 30 minutes at room temperature (RT), the mixture was centrifuged for 5 minutes at 14,000 × g, and the supernatant was obtained and subjected to Zn assay by the modification of the previous method [10]. Briefly, 200 µl of 10 % (w/v) trichloroacetic acid was added equally to the supernatant, and the mixture was kept the supernatant at RT. The mixture was then centrifuged for 5 minutes at 14,000 × g, and the supernatant (300 µl) obtained was equally added 1 M NaHCO3, and the mixture was kept for 10 minutes at RT. After incubation, 200 µl of the mixture was added to the well of the assay plate, and then 40 µl of 93 µM 5-Br-PAPS was added to each well. The mixture was kept at RT for 10 minutes, and the absorbance of the mixture was measured by using a spectrophotometer at a wavelength of 560 nm.
The binding of bovine apo- and holo-Tf with beads immobilized with Zn ion (ZnB) and iron incorporation of ZnB binding apo-Tf
ZnB was prepared as described previously using Chelating Sepharose Fast Flow beads [9]. Bovine apo- and holo-Tf were added to 1 ml of PBS containing ZnB or CB (25 µg each, net volume of beads per sample: 20 µl), and the mixture was incubated for 30 minutes at 4°C. After incubation, the mixture was incubated at 4°C for 30 minutes, and the mixture was then subjected to centrifugation at 14,000 × g for 5 minutes. The supernatant was obtained after the first centrifugation to separate beads. One ml of PBS was added to the precipitated beads for washing beads. The mixture was centrifuged at the same speed followed by removing the supernatant, and rotated. After 1 ml of PBS was added to the precipitated beads again, this procedure was three times repeated. The supernatant and the beads obtained were finally subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to the method of Laemmli [11] using slab gel consisting of a 4.5% polyacrylamide stacking gel and 10% running gel under reducing condition. Apo-Tf or holo-Tf was applied to SDS-PAGE at the same method as the supernatant.
Bovine apo- and holo-Tf (2 mg each) were added to PBS (1 ml) containing ZnB or CB (net volume of beads per sample: 20 µl). The mixture was incubated at 4°C for 30 minutes, and the mixture was then subjected to centrifugation at 14,000 × g for 5 minutes. The supernatant was discarded, and 1 ml of PBS was added to the precipitated beads for washing beads. The mixture was centrifuged at the same speed followed by removing the supernatant, and rotated. After 1 ml of PBS was added to the precipitated beads again, and this procedure was three times repeated. Finally, after completion of the washing procedure, the color was compared between apo- and holo-Tf.
This study confirmed whether Zn-binding apo-Tf incorporates iron or not as follows. Commercial horse spleen ferritin was removed iron for preparing its apoferritin with 100 mM thioglycollic acid in sodium acetate (pH 5.5) followed by dialysis with PBS as described previously [12]. After washing and centrifugation of ZnB binding apo-Tf as described above, 1 ml of PBS containing 2.7 mM ferrous ammonium sulfate and 0.15 µM horse spleen apoferritin was added to the resulting precipitated beads with modification previous method [13]. After incubation for 30 minutes at RT, the beads were washed three times as described above, and then the color change of the beads was observed. CB was also treated with the same method as described above.
Statistical analysis
All data are expressed as the mean ± SD of three measurements. Student’s t-tests were used to compare the values obtained from the two groups. A p-value below 0.05 was considered statistically significant.
Results and Discussion
Recovery (%) of Zn (81% ± 3%, n=3) ion, as added ZnSO4 to TfB, was significantly lower than CB showing almost Zn recovery (100% ± 1%, n=3), indicating that Tf directly bound Zn (Fig. 1). Previous study showed that beads immobilized with zinc ion (ZnB) were used to identify Zn-binding protein and proteins interact with ZnB-binding protein [9]. In this study, ZnB showed the both binding of bovine apo- and holo Tf regardless of iron-binding although holo-Tf showed non-specific binding with beads because the holo-Tf was detected in both supernatants of CB and ZnB (Fig. 2A and B). Bovine Tf was separated into two bands with molecular masses of 69.0 and 74.1 kDa bands under reducing conditions. This finding showed faster band resulted in cleavage of the disulfide band formed between the whole protein (69.0 kDa) and its C-terminal part derived from the cleavage of a peptide between residues 55 and 54 from the C-terminus of the slower band (whole protein) with 74.1 kDa [6]. Eventually, Zn-binding apo-Tf was colorless, but ZnB binding with holo-Tf was pink as called as “pink protein” [14] as shown in Figure 3. Additionally, ZnB-binding apo-Tf showed iron incorporation by change of color in the presence of ferrous ammonium sulfate and apoferritin with ferroxidase, although CB-treated apo-Tf did not show any change (Fig. 4). Although these results remain to be clarified whether apo-Tf binds Zn ion another iron-binding site in monoferric Tf because Tf has two iron-binding sites, holo-Tf mainly bound with ZnB. These results suggest that Tf directly binds Zn ion at different sites with iron-binding sites.
Figure 1.
Binding of TfBs to Zn ions. ZnSO4 was added to PBS (1 ml) containing TfBs or CBs (net volume of beads per sample: 20 µl) to a final concentration of 5 µM, and the mixture was rotated for 30 minutes at 4°C. After incubation, the mixture was centrifuged at 1,4000 × g for 5 minutes, and the resulting supernatant was subjected to Zn measurement as described in the experimental methods section. Zn recovery was calculated considering the measured Zn in the absence of any beads as 100%. Data represent mean ± SD of three duplicate experiments. *p < 0.05, compared with CBs.
Figure 2.
The binding of bovine apo- and holo-Tf to ZnBs. Bovine apo-Tf (A) and holo-Tf (B) were added to 1 ml of PBS containing ZnBs or CBs (25 µg each, net volume of beads per sample: 20 µl), and the mixture was incubated for 30 minutes at 4°C. After incubation, the beads were washed as described in the experimental methods section. The supernatant was obtained after the first centrifugation to separate the beads. The supernatant (S) and beads were finally subjected to SDS-PAGE according to the method of Laemmli (1970) using a slab gel consisting of a 4.5% polyacrylamide stacking gel and 10% running gel under reducing conditions. Apo-Tf or holo-Tf was subjected to SDS-PAGE using the same method as the supernatant on the same gel.
Figure 3.
Binding of bovine apo-Tf and holo-Tf to ZnBs. Bovine apo-Tf and holo-Tf (25 µg each) were added to PBS (1 ml) containing ZnBs or CBs (net volume of beads per sample: 20 µl), and the mixture was incubated for 30 minutes at 4°C. After incubation, the beads were washed as described in the experimental methods section. After washing, the precipitated beads were photographed.
Figure 4.
Iron incorporation by apo-Tf after its binding to ZnBs. Bovine apo-Tf was added to PBS (1 ml) containing ZnBs or CBs (net volume of beads per sample: 20 µl), and the mixture was incubated for 30 minutes at 4°C. After incubation, the beads were washed as described in the experimental methods section. After washing, the beads were re-suspended in 1 ml of PBS containing 2.7 mM ferrous ammonium sulfate and 0.15 µM horse spleen apoferritin, and the mixture was incubated for 30 minutes at RT. After incubation, the beads were washed as described in the experimental methods section. After washing, the precipitated beads were photographed.
Tf is likely to bind Zn at a higher affinity than albumin [7]. However, Zn in the circulation was mostly bound to albumin and apha-2-macroglobulin and also was slightly contributed to some other Zn-binding proteins such as Tf and immunoglobulin [7,15,16]. Zn binding with Tf may be of the lesser extent to contribute to the biological importance as compared with albumin and apha-2-macroglobulin. On the other hand, Zn supplement enhances the anti-tumor effect in combination with chemotherapeutic agents by resoring p53 function [17]. Tf-conjugated doxorubicin-loaded lipid-coated nanoparticles demonstrated an efficient targeted drug-delivery system for lung cancer therapy [18]. Tf receptor 1 (TfR1) is expressed in many kinds of cancers and correlates with tumorigenesis and cancer progression [19]. TfR1 causes ferroptosis in cancer cells by the uptake of holo-Tf by an unknown mechanism [19]. Recent data shows that Zn promotes the interaction between ceruloplasmin and apo-Tf for the efficient transfer of ferric iron after the oxidation of ferrous iron into ferric iron [20].
Conclusion
This study showed preliminary data that apo-Tf directly binds Zn at different sites from iron-binding sites. Although Zn delivery by Tf is much less sufficient than albumin and alpha-2-globulin, apo-Tf or holo-Tf binding Zn may create new possibilities for the treatment of tumor and cancer in combination with the devices of targeted drug delivery systems. In addition, Zn-binding apo-TF may enhance iron delivery due to mediate ceruloplasimin ferroxidase.
Author contributions
Conceptualization, M. K., K. O., and R. S.; methodology, M. K., K. O. and R. S.; validation, M. K., K. O., R. S., and Y. Y.; formal analysis, M. K., K. O. and R. S.; investigation, R. S. and T. K.; data curation, M. K., K. O. and R. S.; supervision, K. O.; writing-original draft, M. K. and R. S.; writing-review and editing, K. O. All authors have read and agreed to the published version of the manuscript.
Conflict of interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
