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Link to original content: https://pubmed.ncbi.nlm.nih.gov/22916096
A synthetic peptide with the putative iron binding motif of amyloid precursor protein (APP) does not catalytically oxidize iron - PubMed Skip to main page content
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. 2012;7(8):e40287.
doi: 10.1371/journal.pone.0040287. Epub 2012 Aug 14.

A synthetic peptide with the putative iron binding motif of amyloid precursor protein (APP) does not catalytically oxidize iron

Kourosh Honarmand Ebrahimi  1 , Peter-Leon Hagedoorn, Wilfred R Hagen
Affiliations

A synthetic peptide with the putative iron binding motif of amyloid precursor protein (APP) does not catalytically oxidize iron

Kourosh Honarmand Ebrahimi et al. PLoS One. 2012.

Abstract

The β-amyloid precursor protein (APP), which is a key player in Alzheimer's disease, was recently reported to possess an Fe(II) binding site within its E2 domain which exhibits ferroxidase activity [Duce et al. 2010, Cell 142: 857]. The putative ligands of this site were compared to those in the ferroxidase site of ferritin. The activity was indirectly measured using transferrin, which scavenges the Fe(III) product of the reaction. A 22-residue synthetic peptide, named FD1, with the putative ferroxidase site of APP, and the E2 domain of APP were each reported to exhibit 40% of the ferroxidase activity of APP and of ceruloplasmin. It was also claimed that the ferroxidase activity of APP is inhibited by Zn(II) just as in ferritin. We measured the ferroxidase activity indirectly (i) by the incorporation of the Fe(III) product of the ferroxidase reaction into transferrin and directly (ii) by monitoring consumption of the substrate molecular oxygen. The results with the FD1 peptide were compared to the established ferroxidase activities of human H-chain ferritin and of ceruloplasmin. For FD1 we observed no activity above the background of non-enzymatic Fe(II) oxidation by molecular oxygen. Zn(II) binds to transferrin and diminishes its Fe(III) incorporation capacity and rate but it does not specifically bind to a putative ferroxidase site of FD1. Based on these results, and on comparison of the putative ligands of the ferroxidase site of APP with those of ferritin, we conclude that the previously reported results for ferroxidase activity of FD1 and - by implication - of APP should be re-evaluated.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Topology of β amyloid precursor protein (APP) and comparison of its putative ferroxidase site with the ferroxidase site of human H-chain ferritin.
(A) A schematic representation of APP and its metal binding domains (APP770 isoform). (B) Structure of the E2 domain of APP751 (PDB 1RW6) and (C) the putative ligands of the ferroxidase site (APP770 numbering). The residues in green show the section of the E2 domain that contains the putative ferroxidase site of APP and the residues that were used to synthesize the FD1 peptide. (D) The diiron catalytic center, the ferroxidase center, of human H-chain ferritin (PDB 1FHA).
Figure 2
Figure 2. Effect of concentration of Fe(II) on non-enzymatic oxidation of Fe(II) and incorporation of the Fe(III) product into transferrin.
Progress curves were recorded at 460 nm for formation of diferric-transferrin and the initial rates were plotted versus concentration of Fe(II). The error bars represent standard deviation of three independent measurements. Concentration of apo-transferrin was 100 µM. Buffer was 100 mM Hepes, 100 mM NaCl, pH 7.2. Measurements were performed at 37°C.
Figure 3
Figure 3. Use of transferrin to measure the ferroxidase activity of ferritin and ceruloplasmin.
(A) Effect of transferrin on Fe(II) oxidation by human H-chain ferritin (HuHF) measured at 650 nm for formation and decay of the blue intermediate, and at 460 nm for formation of diferric transferrin complex. Concentration of apo-HuHF was 1.7 µM (24-mer) and that of transferrin was 70 µM. Two Fe(II) per ferroxidase center, i.e. 48 Fe(II) per 24-mer which is equal to a final concentration of 81.6 µM Fe(II), were added and measurements were done at 34°C in 400 mM Mops buffer, 100 mM NaCl, pH 7.0. (B) Ferroxidase activity of ceruloplasmin was measured using the transferrin assay at 460 nm. Progress curves were recorded and initial rate of Fe(III) formation was plotted versus concentration of Fe(II). Concentration of ceruloplasmin was 0.1 µM. Measurements were performed at 37°C in triplicate. Buffer was 100 mM Mops, 100 mM NaCl, pH 7.1.
Figure 4
Figure 4. The FD1 peptide does not catalytically oxidize iron as measured by the transferrin assay.
(A) Non-enzymatic oxidation of Fe(II) and incorporation of Fe(III) into transferrin was compared with that of BSA, HuHF, ceruloplasmin (CP) and FD1. 52 µM of apo-transferrin was mixed with 2 µM BSA or 0.18 µM HuHF (24-mer) or 2 µM FD1 peptide, or 0.1 µM ceruloplasmin, and then an aliquot of 5 µl anaerobic solution of ferrous sulphate was added. Measurements were done in 100 mM Hepes, 100 mM NaCl, pH 7.2. Final concentration of Fe(II) was 40 µM. (B) Effect of pH on non-enzymatic oxidation of Fe(II) in the presence of transferrin was checked at three different pH values in the presence or absence of FD1 peptide (2 µM): pH 6.2, 100 mM Mes, 100 mM NaCl; pH 7.2, 100 mM Hepes, 100 mM NaCl, and pH 8.2, 100 mM Tris, 100 mM NaCl. Concentration of apo-transferrin was 52 µM. Final concentration of Fe(II) was 40 µM. Measurements were performed at 37°C in triplicate with two different batches of transferrin.
Figure 5
Figure 5. Oxygen consumption as a monitor of Fe(II) oxidation.
Figure 6
Figure 6. Fe(II) and Zn(II) binding to transferrin and to FD1 measured by isothermal titration calorimetry.
(A) Anaerobic Fe(II) titration of the FD1 peptide. Concentration of FD1 in the cell was 70 µM and that of Fe(II) in the syringe was 2.23 mM. The Fe(II) solution was prepared in 100 mM Mops, 100 mM NaCl pH 5.8, and FD1 was in 100 mM Mops, 100 mM NaCl, pH 7.0. The data for the integrated heat of binding were corrected for the heat of dilution due to titration of Fe(II) to buffer (control). Measurements were performed at 25°C. (B) Zn(II) binding to transferrin and (C) to the FD1 peptide. For experiments with transferrin concentration of protein in the cell was 126 µM and the concentration of Zn(II) in the syringe was 12 mM. For experiments with the FD1 peptide, concentration of peptide in the cell was 70 µM and that of Zn(II) in the syringe was 3.35 mM. Transferrin, FD1 peptide, and Zn(II) were prepared in 100 mM Mops, 100 mM NaCl, pH 7.0. Measurements were performed at 25°C. The data for the integrated heat of binding were corrected for the heat of dilution due to titration of Zn(II) to buffer (control).
Figure 7
Figure 7. Inhibitory effect of Zn(II) on diferric-transferrin complex formation.
(A) Initial rates of diferric-transferrin formation at 460 nm are plotted versus concentration of Zn(II). Concentration of apo-transferrin was 80 µM and that of Fe(II) was 200 µM. (B) Comparison of the inhibitory effect of Zn(II) on the diferric-transferrin complex formation for the non-enzymatic oxidation of Fe(II) by molecular oxygen in the presence of transferrin (Tf 70 µM) and for the ferroxidase activity of ceruloplasmin (CP 0.08 µM) measured with the transferrin assay. Final concentration of Fe(II) was 60 µM and that of Zn(II) was 70 µM. Measurements were done in 100 mM Hepes buffer, 100 mM NaCl, pH 7.2 in triplicate at 37°C.
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Grants and funding

This work was supported by a research grant from the Dutch National Research School Combination - Catalysis Controlled by Chemical Design (NRSC-Catalysis). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.