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

Byrne, Shaina L. and Leverence, Rachael and Klein, Joshua S. and Giannetti, Anthony M. and Smith, Valerie C. and MacGillivray, Ross T. A. and Kaltashov, Igor A. and Mason, Anne B. (2006) Effect of Glycosylation on the Function of a Soluble, Recombinant Form of the Transferrin Receptor. Biochemistry, 45 (21). pp. 6663-6673. ISSN 0006-2960. doi:10.1021/bi0600695.

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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 (∼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 (∼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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Mason, Anne B.0000-0003-3097-4589
Additional Information:© 2006 American Chemical Society. Received 12 January 2006. Published online 5 May 2006. Published in print 1 May 2006. This work was supported by USPHS Grants R01 DK21739 (A.B.M.), R01 GM061666 (I.A.K.), and R01 DK60770 (P.J.B.) and the Howard Hughes Medical Institute (to Pamela J. Bjorkman). S.L.B. was supported by a predoctoral fellowship from the NRSA Hemostasis & Thrombosis Training Grant. J.S.K. was supported by biology funds from the Lawrence Ferguson Endowment. We thank Dr. Caroline A. Enns for the full-length human TFR cDNA clone. We also thank Julia R. Larouche and Caroline George (SURE Program) for able technical assistance. We are very grateful to the College of Medicine at the University of Vermont for a grant to purchase the Applied Photophysics (AP) SX.18MV stopped-flow spectrofluorometer and to Dr. Iwona A. Buskiewicz for showing us how to use it.
Funding AgencyGrant Number
NIHR01 DK21739
NIHR01 GM061666
NIHR01 DK60770
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
NIH Predoctoral FellowshipUNSPECIFIED
Lawrence Ferguson EndowmentUNSPECIFIED
Issue or Number:21
Record Number:CaltechAUTHORS:20170131-140942441
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Official Citation:Effect of Glycosylation on the Function of a Soluble, Recombinant Form of the Transferrin Receptor Shaina L. Byrne, Rachael Leverence, Joshua S. Klein, Anthony M. Giannetti, Valerie C. Smith, Ross T. A. MacGillivray, Igor A. Kaltashov, and Anne B. Mason Biochemistry 2006 45 (21), 6663-6673 DOI: 10.1021/bi0600695
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:73895
Deposited By: Ruth Sustaita
Deposited On:01 Feb 2017 17:09
Last Modified:11 Nov 2021 05:23

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