Effect of Glycosylation on the Function of a Soluble, Recombinant Form of the Transferrin Receptor

Production of the soluble portion of the transferrin receptor (sTFR) by baby hamster kidney (BHK) cells is described, and the effect of glycosylation on the biological function of sTFR is evaluated for the first time. The sTFR (residues 121−760) has three N-linked glycosylation sites (Asn251, Asn317, and Asn727). Although fully glycosylated sTFR is secreted into the tissue culture medium (∼40 mg/L), no nonglycosylated sTFR could be produced, suggesting that carbohydrate is critical to the folding, stability, and/or secretion of the receptor. Mutants in which glycosylation at positions 251 and 727 (N251D and N727D) is eliminated are well expressed, whereas production of the N317D mutant is poor. Analysis by electrospray ionization mass spectrometry confirms dimerization of the sTFR and the absence of the carbohydrate at the single site in each mutant. The effect of glycosylation on binding to diferric human transferrin (Fe2 hTF), an authentic monoferric hTF with iron in the C-lobe (designated FeC hTF), and a mutant (designated Mut-FeC hTF that features a 30-fold slower iron release rate) was determined by surface plasmon resonance; a small (∼20%) but consistent difference is noted for the binding of FeC hTF and the Mut-FeC hTF to the sTFR N317D mutant. The rate of iron release from FeC hTF and Mut-FeC hTF in complex with the sTFR and the sTFR mutants at pH 5.6 reveals that only the N317D mutant has a significant effect. The carbohydrate at position 317 lies close to a region of the TFR previously shown to interact with hTF.