Teh Evelyn M, Hewitt Jeff, Ung Karen C, Griffiths Tanya A M, Nguyen Vinh, Briggs Sara K, Mason Anne B, MacGillivray Ross T A
Department of Biochemistry and Molecular Biology and Centre for Blood Research, University of British Columbia, Vancouver, Canada.
FEBS J. 2005 Dec;272(24):6344-53. doi: 10.1111/j.1742-4658.2005.05028.x.
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.
转铁蛋白（TF）与转铁蛋白受体（TFR）相互作用的分子基础尚不清楚。TF的C叶对于促进与TFR的结合是必需的，而N叶和C叶对于最大结合都是必要的。已经产生了几种针对人转铁蛋白（hTF）的单克隆抗体（mAb）。其中一种命名为F11的抗体，对hTF的C叶具有特异性，不识别小鼠或猪的TF。此外，单克隆抗体F11抑制TF与HeLa细胞上TFR的结合。为了绘制单克隆抗体F11的表位图谱，跨越hTF各个区域的构建体在大肠杆菌中表达为谷胱甘肽S-转移酶（GST）融合蛋白。使用单克隆抗体F11作为探针，通过免疫印迹以迭代方式分析重组融合蛋白。这个过程导致F11表位定位到hTF的C1结构域（残基365 - 401）。随后的计算机建模表明，该表位可能局限于hTF由残基365 - 385组成的表面区域。将hTF的F11表位突变为小鼠或猪TF的序列，证实了该表位的一致性，因为免疫反应性降低或丧失。与其他研究一致，这些表位图谱研究支持hTF的C1结构域中的残基在受体结合中的作用。
