**3. Results and discussion**

Ferritin is probably the most used protein in bionanotechnology. This is due to its well-known structural features, high stability, capability to mineralize metals in its cavity, self-assembly and possibility to redesign its interior and exterior by protein engineering [6, 7]. Hepcidin, a 25 amino acid peptide belonging to the β-defensins family, was isolated for the first time from plasma and human urine respectively by Krause *et al*. [8] and Park *et al*. [9]. It is a cysteine-rich cationic peptide, engaged with four disulfide bridges, which plays a major role in innate immunity and iron homeostasis [10]. It is induced by iron and inflammation and is suppressed by iron deficiency and hypoxia. It binds and inhibits ferroportin1, the only cellular iron exporter, thus, high hepcidin reduces while low hepcidin increases systemic iron availability. This hormone acts via its N-terminal part and more precisely the 7–9 amino acids including a single thiol cysteine, comprised the minimal structure that retained hepcidin activity [11] and can be refolded *in-vitro*. The 3D structure of human hepcidin is known [3]. Recombinant human and mouse hepcidins were expressed in *E. coli* in fusion with a Thioredoxin and treated by two sequential proteolytic enzymes to obtain functional hepcidin, in low yield <10% [12]. We used this procedure to clone and express camel hepcidin, in collaboration with Prof. Marie-Agnès Sari in the laboratory of Chemistry and Pharmacological and Toxicological Biochemistry (UMR 8601) at the University Paris Descartes. We showed that this hepcidin is functionally equivalent to human one in binding and inhibiting ferroportin using mouse monocyte–macrophage cell line J774 treated with Fe-nitrilotriacetate. Camel hepcidin differs from human hepcidin in 2 residues, which make it more stable [13]. Thus, we planned to use camel hepcidin to make hybrid molecules with ferritin. For this purpose, we have approached the laboratory of Prof. Paolo Arosio in Brescia that has a long experience in the production of recombinant proteins and is working on ferritin and hepcidin. The aim of this work was to fuse the full length hepcidin to the N-terminus of ferritin-H chain, which is exposed on the surface in the assembled protein. The chimeric subunit was assembled in the 24-mer shells together with various proportion of H or L chains to produce hybrid heteropolymers. These latter that can carry 1 to 24 hepcidin moieties and other functions that can be engineered and can include the electron dense iron core as tracer.

#### *The Use of Ferritin as a Carrier of Peptides and Its Application for Hepcidin DOI: http://dx.doi.org/10.5772/intechopen.94408*

We cloned the gene encoding camel hepcidin in fusion with the 5′ of the cDNA for heavy chain of human ferritin into the pASK-IBA 43plus vector (**Figure 1A, B**) for high expression in *E. coli*. The clone was verified and expressed the chimeric peptide in high amount but it was insoluble and could not be easily refolded in a soluble and active 24-mer shell. Thus, we planned to carry out this project with different strategies:

The construct was expressed in the prokaryotic system *E.coli* BL21 (DE3) pLysS, and the peptides were initially characterized on polyacrylamide gel in denaturing conditions (**Figure 2A**), western blotting (**Figure 2B**) and MALDI-TOF mass spectrometry. Hepcidin carries 8 cysteines that form 4 disulfide bridges in the folded molecule, while FTH has three cysteines, which do not form bonds. Thus, the presence of free -SH groups was also quantified throughout all the purification and refolding processes. We planned to produce bicistronic constructs that express the chimeric hepcidin-FTH and the FTH-WT, with an approach that has been found successful for the production of insoluble ferritin mutants [14]. We obtained ferritin shells with low proportion of chimeric hepcidin (**Figure 1C, D**). In parallel, we have performed co-renaturation together with ferritin-H or L chains. We obtained molecules with various amounts of hepcidin. To this aim the *in-vitro* refolding of the fusion protein hepcH was done with different molar ratio of H and L chains, in order to optimize the assembly of the hybrid nanoparticles [2]. Purified fusion hepcidin–ferritin H subunit (**Figure 3D**) assembled together with H- or L-chains at a ratio of 1:5, produced a stable and functional 24-mer ferritin exposing about 4 hepcidin per shell (**Figure 1C, D**; **Figure 3C**). HepcH-FTH heteropolymer was purified and characterized by analysis on polyacrylamide gel in non-denaturing condition and by western blotting (**Figure 3E, F, G, H**). MALDI-TOF spectra of the final oxidized HepcH-FTH heteropolymer exhibited, average mass peaks at m/z 21135.97 and at 243775.51 (**Figure 4A**) which corresponds to the theoretical average mass [M + H] + of 21225.64 of FTH (183 amino acids), and of 24410.50 to HepcH (213 amino acids).

These procedures have produced ferritin shells that expose on the surface the hepcidin moiety. In order to control the correct folding, the cysteines of the

#### **Figure 1.**

*(A) Construction of the recombinant HepcH pASK-IBA 43 plus vector. (B) Amino acid sequence of the fusion HepcH protein. In this recombinant protein, the coding sequence of camel hepcidin, in red and italic, was fused directly to the 5′ end of human H-ferritin, in green, and cloned into pASK-IBA43 plus vector. (C) Schematic representation (left) and space-filling diagram (right) of the assembled HepcH-FTH heteropolymer (with molar ratio hepcidin/ferritin = 1:5).*

#### **Figure 2.**

*(A) SDS-PAGE analysis of the induced and uninduced E.coli transformed by the recombinant HepcH-FTH pASK-IBA 43 plus vector. Lane 1 and 2, induced and uninduced pellet respectively (insoluble fraction). Lane 3 and 4, induced and uninduced supernatant (soluble fraction). Lane 5 and 6, induced and uninduced total sonicate (total protein). (B) Western blot analysis of the recombinant HepcH monomer (in denaturing conditions). Lane 1, rabbit hepcidin antibodies recognized the human hepcidin-25 (control). Lane2, rabbit hepcidin antibodies recognized the recombinant HepcH. Lane3, rH02 antibodies recognized the recombinant HepcH. Lane4, rH02 antibodies recognized the recombinant human H-ferritin (control).*

#### **Figure 3.**

*Renaturation of the insoluble HepcH monomer in absence and in presence of FTH. (A) Purified FTH (line1) and solubilized HepcH monomer (line 2 and 3) analyzed on denaturing conditions. (B) FTH homopolymer (line1) and renatured HepcH, in absence of FTH, analyzed on non-denaturing conditions (line 2 and 3). (C) FTH homopolymer (line1) and renatured HepcH in presence of FTH with molar ratio of hepcidin/ ferritin = 1:5 (lane 2 and 3) analyzed on non-denaturing conditions. (D) SDS-PAGE analysis of the purified HepcH, from the insoluble fraction (IS), by gel filtration on a Sepharose 6B column (GE Healthcare, life sciences). (E) Fast protein liquid chromatography chromatogram showing the purification of HepcH-FTH heteropolymer using a gel filtration on a Sepharose 6B column and analyzer on non-denaturing conditions (F). (G) Western blot analysis of the purified HepcH-FTH heteropolymer using the anti-rabbit hepcidine-25 and the human ferritin-H antibodies, rH02, as control (H).*

molecule was reduced and then slowly oxidized under controlled conditions (to allow the formation of the correct four disulfide bridges constituting the hormone). In case the cysteine of the ferritin interferes with the process or its monitoring by spectroscopic techniques, they could be removed by site-directed mutagenesis.

The functionality of the assembled heteropolymer was analyzed by its capacity to bind with high affinity the ferroportin, which is the natural hepcidin receptor. To this goal, we used the macrophagic cell line J774 that expose evident ferroportin after treatment with iron. The cells were incubated with the hybrid molecules and the binding analyzed directly with traced anti-ferritin antibodies. Binding specificity was analyzed by adding ferritin and synthetic hepcidin. This process was repeated with the various heteropolymer types. Next, we studied the biological activity of the chimera,

*The Use of Ferritin as a Carrier of Peptides and Its Application for Hepcidin DOI: http://dx.doi.org/10.5772/intechopen.94408*

#### **Figure 4.**

*(A) High-resolution MALDI-TOF/TOF mass analysis of the recombinant assembled HepcH-FTH heteropolymer and the recombinant human ferritin FTH homopolymer (B).*

in particularly if it is taken up and degraded together with ferroportin, as it occurs with the hepcidin. Hybrid and native ferritin binding to murine J774 cells were monitored using monoclonal anti-human FTH antibody rH02 that does not cross-react with the mouse ferritins [15]. This was confirmed by treating J774 cells with mouse ferritin alone or together with HepcH-FTH. The obtained results (**Figure 5A**) showed that these antibodies are specific only to human H-ferritin. J774 cells treated with 100 μM FAC showed to be able to internalize HepcH-FTH heteropolymers after 30 min to 2 h of incubation at 37°C (**Figure 5B**). Hepcidin exerts it function by binding and then inducing ferroportin degradation, and in fact we observed that after 2 h incubation, with a final concentration of 0.2 μM, more efficiently than human hepcidin-25 used as control

#### **Figure 5.**

*(A) Western blot analysis of the J774 cells treated with mouse ferritin (Mo-Ft), FTH, human hepcidin-25 and HepcH-FTH heteropolymer using anti-FTH antibodies (rH02). Lane 1, cells treatment with 0.5* μ*M Mo-Ft and HepcH-FTH; lane 2, cells treatment with 0.5* μ*M FTH; lane 3, cells treatment with 0.5* μ*M Mo-Ft; lane 4, untreated cells. (B) Western blot analysis of the J774 cells treated with 0.5* μ*M HepcH-FTH heteropolymer and FTH, using anti-FTH antibodies (rH02). Lane 1, non-treated cells; lane2, cells treated with 6 M guanidine; lane 3, cells treated with 0.5* μ*M FTH for 30′; lane 4, cells treated with 0.5* μ*M HepcH-FTH for 30′; lane 5, cells treated with human hepcidin for 30 min; lane 6, cells treated with FTH for 2 h; lane 7, cells treated with HepcH-FTH for 2 h; lane 8, cells treated with human hepcidin for 2 h. (C) Western blotting analysis of the SDS-PAGE of cell lysates using polyclonal anti-rabbit ferroportin antibody. CTR: Untreated cells. FTH: Cells treatment with 0.5* μ*M native FTH for 2 h. HepcH-FTH: Cells treatment with 0.2* μ*M HepcH-FTH (with molar ration of 1:5) for 2 h. Hepc: Cells treatment with 0.5* μ*M synthetic human hepcidin-25 for 2 h. nonadjacent bands, from the same blot, were denoted by vertical black lines.*

(**Figure 5C**). Indeed, the level of ferroportin in the J774 cells decreased, as it occurred in the cells treated with the synthetic hepcidin, while the incubation with FTH had no evident effect. This indicates that heteropolymer is biologically functional. Consequently, folded camel hepcidin activity against FPN1 could be enhanced thanks to its exposition, through its N-terminal part, at the H-ferritin surface nanocage [16]. This will probably offer a tool for a detailed study of the events after ferroportin binding the hepcidin. The heavy ferritin iron core and the fate of iron will facilitate the monitoring of the process.

As perspectives, the examination of the chronic effect of the purified heteropolymer injections on liver iron accumulation in a mouse model of hereditary hemochromatosis could be monitored. Indeed, hepcidin-1 knockout mice (hepc1−/−) can be used, as a model, to eliminate any possibility of endogenous hepcidin contributing to the regulation of iron loading. This strategy can also be developed by producing ferritin subunits carrying other functionalities or epitopes, in order to have multifunctional complexes. Moreover, involving the approach can be applied for other peptides/hormones for the design of 'smart' molecular systems programmed to allow the transport in the body of potent anticancer agents in an innocuous manner toward safe tissues. Thus, these hybrid molecules will be useful for future therapeutic applications to improve health and life quality of a great number of patients with iron disorders or cancer diseases.
