title: N-linked glycosylation is required for transferrin-induced stabilization of transferrin receptor 2, but not for transferrin binding or trafficking to the cell surface.
abstract: Transferrin receptor 2 (TfR2) is a member of the transferrin receptor-like family of proteins. Mutations in TfR2 can lead to a rare form of the iron overload disease, hereditary hemochromatosis. TfR2 is proposed to sense body iron levels and increase the level of expression of the iron regulatory hormone, hepcidin. Human TfR2 (hTfR2) contains four potential Asn-linked (N-linked) glycosylation sites on its ectodomain. The importance of glycosylation in TfR2 function has not been elucidated. In this study, by employing site-directed mutagenesis to remove glycosylation sites of hTfR2 individually or in combination, we found that hTfR2 was glycosylated at Asn 240, 339, and 754, while the consensus sequence for N-linked glycosylation at Asn 540 was not utilized. Cell surface protein biotinylation and biotin-labeled Tf indicated that in the absence of N-linked oligosaccharides, hTfR2 still moved to the plasma membrane and bound its ligand, holo-Tf. However, without N-linked glycosylation, hTfR2 did not form the intersubunit disulfide bonds as efficiently as the wild type (WT). Moreover, the unglycosylated form of hTfR2 could not be stabilized by holo-Tf. We further provide evidence that the unglycosylated hTfR2 behaved in manner different from that of the WT in response to holo-Tf treatment. Thus, the putative iron-sensing function of TfR2 could not be achieved in the absence of N-linked oligosaccharides. On the basis of our analyses, we conclude that unlike TfR1, N-linked glycosylation is dispensable for the cell surface expression and holo-Tf binding, but it is required for efficient intersubunit disulfide bond formation and holo-Tf-induced stabilization of TfR2.
authors.affiliation: Department of Cell and Developmental Biology, Oregon Health & Science University , Portland, Oregon 97239, United States.
authors.forename: Ningning
authors.initials: N
authors.lastname: Zhao
authors.forename: Caroline A
authors.initials: CA
authors.lastname: Enns
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D006031:Glycosylation; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D055503:Protein Multimerization; D055550:Protein Stability; D021381:Protein Transport; D011990:Receptors, Transferrin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014168:Transferrin / Q000378:metabolism*
chemical_list: D011990:Receptors, Transferrin; C503640:TFR2 protein, human; D014168:Transferrin
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