Identification of the epitope of a monoclonal antibody that disrupts binding of human transferrin to the human transferrin receptor.
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.
Teh
Evelyn M
EM
Department of Biochemistry and Molecular Biology and Centre for Blood Research, University of British Columbia, Vancouver, Canada.
Hewitt
Jeff
J
Ung
Karen C
KC
Griffiths
Tanya A M
TA
Nguyen
Vinh
V
Briggs
Sara K
SK
Mason
Anne B
AB
MacGillivray
Ross T A
RT
D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D001665:Binding Sites; D003198:Computer Simulation; D018604:Epitope Mapping; D000939:Epitopes; D006367:HeLa Cells; D006801:Humans; D011485:Protein Binding / Q000187:drug effects; D011990:Receptors, Transferrin / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000378:metabolism; D013045:Species Specificity; D014168:Transferrin / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011990:Receptors, Transferrin; D014168:Transferrin