Journal
BIOCHEMISTRY
Volume 45, Issue 21, Pages 6663-6673Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi0600695
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Funding
- NIDDK NIH HHS [R01 DK21739, R01 DK60770] Funding Source: Medline
- NIGMS NIH HHS [R01 GM061666] Funding Source: Medline
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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 (similar to 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 (Fe-2 hTF), an authentic monoferric hTF with iron in the C-lobe (designated Fe-C hTF), and a mutant (designated Mut-Fe-C hTF that features a 30-fold slower iron release rate) was determined by surface plasmon resonance; a small (similar to 20%) but consistent difference is noted for the binding of Fe-C hTF and the Mut-Fe-C hTF to the sTFR N317D mutant. The rate of iron release from Fe-C hTF and Mut-Fe-C 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.
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