title: A loop in the N-lobe of human serum transferrin is critical for binding to the transferrin receptor as revealed by mutagenesis, isothermal titration calorimetry, and epitope mapping.
abstract: Transferrin (TF) is a bilobal transport protein that acquires ferric iron from the diet and holds it tightly within the cleft of each lobe (thereby preventing its hydrolysis). The iron is delivered to actively dividing cells by receptor mediated endocytosis in which diferric TF preferentially binds to TF receptors (TFRs) on the cell surface and the entire complex is taken into an acidic endosome. A combination of lower pH, a chelator, inorganic anions, and the TFR leads to the efficient release of iron from each lobe. Identification of residues/regions within both TF and TFR required for high affinity binding has been an ongoing goal in the field. In the current study, we created human TF (hTF) mutants to identify a region critical to the interaction with the TFR which also constitutes part of an overlapping epitope for two monoclonal antibodies (mAbs) to the N-lobe, one of which was previously shown to block binding of hTF to the TFR. Four single point mutants, P142A, R143A, K144A, and P145A in the N-lobe, were placed into diferric hTF. Isothermal titration calorimetry (ITC) revealed that three of the four residues (Pro142, Lys144, and Pro145) in this loop are essential to TFR binding. Additionally, Lys144 is common to the recognition of both mAbs which show different sensitivities to the three other residues. Taken together these studies prove that this loop is required for binding of the N-lobe of hTF to the TFR, provide a more precise description of the role of each residue in the loop in the interaction with the TFR, and confirm that the N-lobe is essential to high affinity binding of diferric hTF to TFR.
authors.affiliation: Department of Biochemistry, College of Medicine, University of Vermont, 89 Beaumont Avenue, Burlington, VT 05405, USA. anne.mason@uvm.edu
authors.forename: Anne B
authors.initials: AB
authors.lastname: Mason
authors.forename: Shaina L
authors.initials: SL
authors.lastname: Byrne
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Everse
authors.forename: Samantha E
authors.initials: SE
authors.lastname: Roberts
authors.forename: N Dennis
authors.initials: ND
authors.lastname: Chasteen
authors.forename: Valerie C
authors.initials: VC
authors.lastname: Smith
authors.forename: Ross T A
authors.initials: RT
authors.lastname: MacGillivray
authors.forename: Banu
authors.initials: B
authors.lastname: Kandemir
authors.forename: Fadi
authors.initials: F
authors.lastname: Bou-Abdallah
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001483:Base Sequence; D002151:Calorimetry / Q000379:methods; D006224:Cricetinae; D011992:Endosomes / Q000378:metabolism; D018604:Epitope Mapping; D006639:Histidine / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011990:Receptors, Transferrin / Q000737:chemistry*; D014168:Transferrin / Q000096:biosynthesis* / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D011990:Receptors, Transferrin; D014168:Transferrin; D006639:Histidine
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