Identification of the epitope of a monoclonal antibody that disrupts binding of human transferrin to the human transferrin receptor

Abstract

The transferrins (TF) are a group of metal-binding proteins that are involved in iron homeostasis [1]. Structural studies have revealed that the TFs consist of a single polypeptide chain of M r 80000 that folds into two halves called the N-and C-lobes, each of approximately 330 amino acids. In human transferrin (hTF), the lobes are connected by a short peptide of seven residues. Each lobe itself can be further subdivided into two domains separated by a deep cleft that forms the iron-binding site [2][3][4]. The N1 domain (residues 1-93 and 247-315), C1 domain (residues 340-424 and 583-679), N2 domain (residues 94-246) and C2 domain (residues 425-582) are composed of a similar a ⁄ b fold in which a number of helices are packed against a central mixed b-sheet [5]. These domains are connected by two extended b-strands running antiparallel to each other forming a 'hinge' that allows the domains to open and close upon metal binding and release [6]. Iron is bound in a distorted octahedral coordination involving four amino acid ligands and two oxygen atoms from a synergistically bound carbonate ion.The iron-TF complex enters the cell by binding with high affinity (K d 1-10 nm) to a specific TF receptor (TFR), a type-II membrane protein consisting of two identical M r 95000 subunits covalently linked by two disulfide bonds [7]. The N-terminal region of the receptor projects into the cytoplasm of the cell and is joined via the transmembrane region to a 671-residue extracellular domain that binds TF. A soluble form of the receptor can be released by trypsin [8] or produced by recombinant techniques [9,10]. Although these forms lack the two disulfide linkages, strong noncovalent The molecular basis of the transferrin (TF)-transferrin receptor (TFR) interaction is not known. The C-lobe of TF is required to facilitate binding to the TFR and both the N-and C-lobes are necessary for maximal binding. Several mAb have been raised against human transferrin (hTF). One of these, designated F11, is specific to the C-lobe of hTF and does not recognize mouse or pig TF. Furthermore, mAb F11 inhibits the binding of TF to TFR on HeLa cells. To map the epitope for mAb F11, constructs spanning various regions of hTF were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. The recombinant fusion proteins were analysed in an iterative fashion by immunoblotting using mAb F11 as the probe. This process resulted in the localization of the F11 epitope to the C1 domain (residues 365-401) of hTF. Subsequent computer modelling suggested that the epitope is probably restricted to a surface patch of hTF consisting of residues 365-385. Mutagenesis of the F11 epitope of hTF to the sequence of either mouse or pig TF confirmed the identity of the epitope as immunoreactivity was diminished or lost. In agreement with other studies, these epitope mapping studies support a role for residues in the C1 domain of hTF in receptor binding.Abbreviations TF, transferrin; TFR, transferrin receptor; GST, glutathione S-transferase; hTF(R),...

Loss of Ca2+/Calmodulin Dependent Protein Kinase Kinase 2 Leads to Aberrant Transferrin Phosphorylation and Trafficking: A Potential Biomarker for Alzheimer's Disease

“…D-411 residue is involved in iron binding. The purple and green highlighted residues indicate the TFR binding site identified by cryo-EM (Cheng et al, 2004 ) and epitope mapping (Teh et al, 2005 ). (F) Ribbon representation of hTF showing TFR binding site (Wally et al, 2006 ).…”

“…The Tyr257/333/338, Ser381/389/409/511, and Thr586 residues are conserved between different mammalian species (Supplementary Figure 1A ). The Ser381/389 and Thr392/393 residues positioned within the conserved TFR binding site identified by cryo-electron microscopy (residues 349-372) (Cheng et al, 2004 ), radiation footprinting (residues 381-401) (Liu et al, 2003 ; Xu et al, 2005 ) and epitope mapping (residues 365-401) (Teh et al, 2005 ) (Figures 1F,G , Supplementary Figures 1A , 4A ). Loss of phosphorylation at Ser381/389 and Thr392/393 may affect interaction of TF with TFR (Wally et al, 2006 ).…”

“…Color highlights showing residues in N and C subdomain of human TF identified as interacting with hTFR. Specific residues identified by cryo-EM (yellow rectangle) (Cheng et al, 2004 ) and epitope mapping (pink rectangle) (Teh et al, 2005 ) may interact with TFR. Orange: overlap between cryo-EM (yellow) and epitope mapping (pink), green highlighted residues in the N-lobe that may interact with the human TFR(Cheng et al, 2004 ).…”

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

“…The ability of such antibodies to block binding of hTF to the TFR on the cell surface combined with identification of the epitopes recognized by such antibodies provides important information. Using this approach, we bracketed the region of a sequential epitope lying in a region between residues 365–401 (most likely between 365 and 385) in the C -lobe of hTF for a mAb designated F11 (Teh et al ., 2005). …”