tailieunhanh - Báo cáo khoa học: 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 bind-ing. 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. | iFEBS Journal Identification of the epitope of a monoclonal antibody that disrupts binding of human transferrin to the human transferrin receptor Evelyn M. Teh1 Jeff Hewitt1 Karen C. Ung1 Tanya A. M. Griffiths1 Vinh Nguyen1 Sara K. Briggs2 ft If Anne B. Mason2 and Ross T. A. MacGillivray1 1 Department of Biochemistry and Molecular Biology and Centre for Blood Research University of British Columbia Vancouver Canada 2 Department of Biochemistry University of Vermont College of Medicine Burlington Vermont USA Keywords transferrin C-lobe transferrin-transferrin receptor interaction epitope mapping monoclonal antibody Correspondence . MacGillivray Department of Biochemistry and Molecular Biology and Centre for Blood Research University of British Columbia Vancouver BC V6T 1Z3 Canada Tel 1 604 822 3027 Fax 1 604 822 4364 E-mail macg@ Received 9 July 2005 revised 7 October 2005 accepted 19 October 2005 doi 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-trans-ferase 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 .