The g277s mutation in transferrin does not disturb function

We read with interest the paper by Lee et al (2001) that reported an association between iron deficiency anaemia in menstruating women and a G277S mutation in transferrin (G258S in the mature protein), which suggested that the glycine in amino acid position 277 is important for the maintenance of the biological activity and/or structure of transferrin. To explore this suggestion, we constructed the mutation in a human transferrin expression vector, verified the construct by DNA sequencing and expressed the mutant in the non-glycosylated form in BHK21 cells (Zak et al, 1997). Further evidence of the mutation was provided by mass spectrometry (predicted molecular weight: 75 173 Da; identified protein: 75 175 Da; wild-type transferring: 75 143 Da). Optical and electron paramagnetic resonance spectroscopies showed no difference between the mutant and native transferrins. Using 100 mmol/l pyrophosphate, which is the active moiety of the physiological iron binder ATP, iron release rates from mutant and native proteins that bore iron only in the mutated N-lobe, at pH 7·4, were found to differ only slightly to: 9·1 ± 1·1 s−1 (n = 3; mean ± standard deviation) and 12·3 ± 0·4 s−1 respectively. More importantly, mutant and native proteins were equally efficient at donating iron to K562 cells (Fig 1), a cell type often used as a model of immature red cells. The absence of glycan moieties does not affect the interaction of transferrin with cells (Mason et al, 1993). The uptake of iron by K562 cells from wild-type transferrin (●) and the G277S mutant (▪), each at a concentration of 2·5 mmol/l and saturated with iron. The iron uptake is shown as 1 × 107 (1e + 7) to 7 × 107 (7e + 7) atoms/cell. It is therefore unlikely that the biological activity of transferrin is perturbed by the G277S mutation, and that the iron-binding sites of the mutant are altered from those in native transferrin. The cause of the iron deficiency observed in the subjects studied by Lee et al (2001) should be sought elsewhere.