Structural, functional, and tissue distribution analysis of human transferrin receptor-2 by murine monoclonal antibodies and a polyclonal antiserum

Blood ◽  
2002 ◽  
Vol 100 (10) ◽  
pp. 3782-3789 ◽  
Author(s):  
Silvia Deaglio ◽  
Andrea Capobianco ◽  
Angelita Calı̀ ◽  
Francesca Bellora ◽  
Federica Alberti ◽  
...  

Human transferrin receptor-2 (TFR-2) is a protein highly homologous to TFR-1/CD71 and is endowed with the ability to bind transferrin (TF) with low affinity. High levels of TFR-2 mRNA were found in the liver and in erythroid precursors. Mutations affecting the TFR-2gene led to hemochromatosis type 3, a form of inherited iron overload. Several issues on distribution and function of the receptor were answered by raising a panel of 9 monoclonal antibodies specific for TFR-2 by immunizing mice with murine fibroblasts transfected with the human TFR-2 cDNA. A polyclonal antiserum was also produced in mice immunized with 3 peptides derived from the TFR-2 sequence, exploiting an innovative technique. The specificity of all the reagents produced was confirmed by reactivity with TFR-2+ target cells and simultaneous negativity with TFR-1+ cells. Western blot analyses showed a dominant chain of approximately 90 kDa in TFR-2 transfectants and HepG2 cell line. Analysis of distribution in normal tissues and in representative cell lines revealed that TFR-2 displays a restricted expression pattern—it is present at high levels in hepatocytes and in the epithelial cells of the small intestine, including the duodenal crypts. Exposure of human TFR-2+cells to TF-bound iron is followed by a significant up-regulation and relocalization of membrane TFR-2. The tissue distribution pattern, the behavior following exposure to iron-loaded TF, and the features of the disease resulting from TFR-2 inactivation support the hypothesis that TFR-2 contributes to body iron sensing.

1997 ◽  
Vol 324 (3) ◽  
pp. 815-822 ◽  
Author(s):  
Dharmendar RATHORE ◽  
Janendra K. BATRA

Restrictocin is a ribonucleolytic toxin produced by the fungus Aspergillus restrictus. Two chimaeric toxins containing restrictocin directed at the human transferrin receptor have been constructed. Anti-TFR(scFv)–restrictocin is encoded by a gene produced by fusing the DNA encoding a single-chain antigen-combining region (scFv) of a monoclonal antibody, directed at the human transferrin receptor, at the 5′ end of that encoding restrictocin. The other chimaeric toxin, restrictocin–anti-TFR(scFv), is encoded by a gene fusion containing the DNA encoding the single-chain antigen-combining region of antibody to human transferrin receptor at the 3′ end of the DNA encoding restrictocin. These gene fusions were expressed in Escherichia coli, and fusion proteins purified from the inclusion bodies by simple chromatography techniques to near-homogeneity. The two chimaeric toxins were found to be equally active in inhibiting protein synthesis in a cell-free in vitrotranslation assay system. The chimaeric toxins were selectively toxic to the target cells in culture with potent cytotoxic activities. However, restrictocin–anti-TFR(scFv) was more active than anti-TFR(scFv)–restrictocin on all cell lines studied. By using protease and metabolic inhibitors, it can be shown that, to manifest their cytotoxic activity, the restrictocin-containing chimaeric toxins need to be proteolytically processed intracellularly and the free toxin or a fragment thereof thus generated is translocated to the target via a route involving the Golgi apparatus.


FEBS Letters ◽  
1984 ◽  
Vol 175 (2) ◽  
pp. 279-283 ◽  
Author(s):  
Barbara Uchańska-Ziegler ◽  
Peter Wernet ◽  
Shi Liangru ◽  
Andreas Ziegler

1984 ◽  
Vol 20 (5) ◽  
pp. 441-447 ◽  
Author(s):  
B. NIKINMAA ◽  
C. A. ENNS ◽  
S. E. TONIK ◽  
H. H. SUSSMAN ◽  
J. SCHRODER

Structure ◽  
1998 ◽  
Vol 6 (10) ◽  
pp. 1235-1243 ◽  
Author(s):  
Hendrik Fuchs ◽  
Uwe Lücken ◽  
Rudolf Tauber ◽  
Andreas Engel ◽  
Reinhard Geßner

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