title: Effect of glycosylation on the function of a soluble, recombinant form of the transferrin receptor.
abstract: 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 ( approximately 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 ( approximately 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.
authors.affiliation: Department of Biochemistry, University of Vermont College of Medicine, Burlington, Vermont 05405-0068, USA.
authors.forename: Shaina L
authors.initials: SL
authors.lastname: Byrne
authors.forename: Rachael
authors.initials: R
authors.lastname: Leverence
authors.forename: Joshua S
authors.initials: JS
authors.lastname: Klein
authors.forename: Anthony M
authors.initials: AM
authors.lastname: Giannetti
authors.forename: Valerie C
authors.initials: VC
authors.lastname: Smith
authors.forename: Ross T A
authors.initials: RT
authors.lastname: MacGillivray
authors.forename: Igor A
authors.initials: IA
authors.lastname: Kaltashov
authors.forename: Anne B
authors.initials: AB
authors.lastname: Mason
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D019281:Dimerization; D006031:Glycosylation; D007700:Kinetics; D011485:Protein Binding; D011990:Receptors, Transferrin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D020349:Surface Plasmon Resonance; D014168:Transferrin / Q000378:metabolism
chemical_list: D017931:DNA Primers; D011990:Receptors, Transferrin; D011994:Recombinant Proteins; D014168:Transferrin