Human serum transferrin: a tale of two lobes. Urea gel and steady state fluorescence analysis of recombinant transferrins as a function of pH, time, and the soluble portion of the transferrin receptor

Abstract

Iron release from human serum transferrin (hTF) has been studied extensively; however, the molecular details of the mechanism(s) remain incomplete. This is in part due to the complexity of this process, which is influenced by lobe-lobe interactions, the transferrin receptor (TFR), the salt effect, the presence of a chelator, and acidification within the endosome, resulting in iron release. The present work brings together many of the concepts and assertions derived from previous studies in a methodical, uniform, and visual manner. Examination of earlier work reveals some uncertainty due to sample and technical limitations. We have used a combination of steady-state fluorescence and urea gels to evaluate the effect of conformation, pH, time, and the soluble portion of the TFR (sTFR) on iron release from each lobe of hTF. The use of authentic recombinant monoferric and locked species removes any possibility of cross-contamination by acquisition of iron. Elimination of detergent by use of the sTFR provides a further technical advantage. We find that iron release from the N-lobe is very sensitive to the conformation of the C-lobe, but is insensitive to the presence of the sTFR or to changes in pH (between 5.6 and 6.4). Specifically, when the cleft of the C-lobe is locked, the urea gels indicate that only about half of the iron is completely removed from the cleft of the N-lobe. Iron release from the C-lobe is most affected by the presence of the sTFR and changes in pH, but is unaffected by the conformation of the N-lobe. A model for iron release from diferric hTF is provided to delineate our findings.

Fig. 1

Pathways available to transition from Fe₂ hTF to apo hTF. Recombinant constructs used to specifically monitor each route are indicated above/below the arrows

Fig. 2

Effect of lowering pH on iron release from Fe₂ hTF. a 6 M urea gel. All samples were incubated for 15 min in iron-removal buffer before being loaded on the gel (2.5 µg per lane). b Bar graph of steady-state emission intensity at λ_max for each spectrum. The emission spectra from which the end points shown in the bar graph were obtained are provided as electronic supplementary material. All samples were incubated for 15 min in iron removal buffer [100 mM MES pH 5.6, 300 mM KCl, 4 mM EDTA], before the emission was monitored. Samples were excited at 280 nm and emission was monitored between 300 and 400 nm using a 320-nm cut-on filter. As an important control, we analyzed a construct with iron locked in both lobes and observed that at pH 5.6 no iron was removed and that the fluorescence intensity was equal to the intensity at pH 7.4, i.e., no iron was removed (data not shown)

Fig. 3

Effect of lowering pH, and the conformation of the C-lobe, on iron release from the N-lobe of hTF. a 6 M urea gel of Fe_N hTF, b bar graph of steady-state emission intensity, c 6 M urea gel of Lock_C hTF, d bar graph of steady-state emission intensity. All samples were prepared as described in the legend to Fig. 2. The appearance of a double band in c is ascribed to the extreme sensitivity of urea gels to charge heterogeneity. We have observed that this heterogeneity seems to increase as a function of the age of the sample and most likely results from oxidation and/or deamination of amino acid side chains

Fig. 4

Effect of lowering pH, and the conformation of the N-lobe, on iron release from the C-lobe of hTF. a 6 M urea gel of Fe_c hTF, b bar graph of steady-state emission intensity, c 6 M urea gel of Lock_N hTF, d bar graph of steady-state emission intensity. All samples were prepared as described in the legend to Fig. 2. See the legend to Fig. 3 for explanation of the appearance of the double band in c in the bands corresponding to the monoferric N-lobe and the diferric species (Lock_N hTF)

Fig. 5

Influence of the soluble portion of the transferrin receptor (sTFR) on time-based iron release from Fe₂ hTF at pH 5.6. a Fe₂ hTF alone, b Fe₂ hTF/sTFR complex. All samples were incubated for the designated time courses in 100 mM MES pH 5.6, 300 mM KCl, 4 mM EDTA. Iron release was quenched by the addition of sample buffer

Fig. 6

Influence of the sTFR on time-based iron release from the N-lobe at pH 5.6. a Fe_N hTF, b Fe_N hTF/sTFR complex, c Lock_C hTF, d Lock_C hTF/sTFR complex. All samples were incubated for the designated time courses in iron-removal buffer (100 mM MES pH 5.6, 300 mM KCl, 4 mM EDTA). Iron release was quenched by the addition of sample buffer. See the legend to Fig. 3 for explanation of the appearance of the double band in b and c

Fig. 7

Influence of the sTFR on time-based iron release from the C-lobe at pH 5.6. a Fe_C hTF, b Fe_C hTF/sTFR complex, c Lock_N hTF, d Lock_N hTF/sTFR complex. All samples were incubated for the designated time courses in 100 mM MES pH 5.6, 300 mM KCl, 4 mM EDTA. Iron release was quenched by the addition of sample buffer. See the legend to Fig. 3 for explanation of the appearance of the double band in c and d

Fig. 8

Model of iron release from Fe₂ hTF. See “Discussion” for full details