Expression and characterization of soluble transferrin receptor in BHK cells: effect of mutations at the three asparagine glycosylation sites

Abstract

Transferrin (TF) is a bilobal iron transport protein that specifically binds to receptors (TFR) on the cell surface and delivers iron to cells by a process involving receptor mediated endocytosis and a pH change. The TF binding portion of the TFR (residues 121–760) containing a hexa-His tag has been expressed by baby hamster kidney (BHK) cells as a secreted entity. TFR has three Asn linked glycosylation sites at position 251, 317 and 727. To prevent glycosylation at each site, three single point mutants in which Asn is mutated to Asp have been constructed. For wild type (WT), N251D and N727D TFR a maximum of 40 mg/L is produced, whereas maximum production of the N317D TFR mutant is considerably lower. Characterization of the WT and the mutant TFR constructs includes analysis by mass spectrometry and estimation of binding constants for interaction with diferric TF, monoferric TF C-lobe (FeC-TF) and a FeC-TF mutant. Additionally the rate of iron release from FeC-TF in the presence and absence of the various TFR constructs has been determined. The N317D TFR mutant binds more weakly to FeC-TF and shows a reduced ability to stimulate the release of iron from FeC-TF at pH 5.6 compared to WT TFR and the other two mutants. A structural basis for these findings is presented. This work was supported by USPHS Grant R01 DK 21739 (ABM) and R01 GM061666 (IAK).