The uptake of iron into mammalian cells involves the binding of transferrin, the serum iron transport protein, to the tranferin receptor, followed by the internalization of the receptor-transferrin complex. Iron accumulation is essential for cell division. Rapidly proliferating cells have higher iron requirements than non-dividing cells and the receptor has been identified as a proliferation specific marker. The transferrin receptor contains three asparagine-linked oligosaccharides, which are a mixture of """"""""high mannose"""""""" and """"""""complex"""""""" structures and at least one O-linked (serine/threonine) oligosaccharide. Prior studies using tunicamycin-treated human cells have demonstrated that the unglycosylated from of the transferrin receptor is not functional. Thus, the goals of this proposal are to test the hypothesis that glycosylation is vital for the correct folding and transport of the transferrin receptor to the cell surface and in the binding or transferrin. Site-directed mutagenesis will be used to test the contribution of each of the asparagine-linked glycosylation sites to the folding and transport of the transferrin receptor. Combinations of single, double and triple mutations will be prepared and tested mouse cell lines. Evidence suggests that processing of oligosaccharides to complex forms enhances receptor function and the role of this processing will be examined in glycosylation deficient Chinese Hamster Ovary cell mutants. Such mutants will also be sued to investigate the role of O-linked glycosylation in receptor function. Transferrin receptor oligosaccharides have not been directly characterized and they will be analyzed with respect to structure (high mannose/complex) present on each of the asparagine-linked sites in the native transferrin receptor as well as the receptor expressed in murine and CHO cells following transfection. These analyses will be performed via carbohydrate and protein chemical techniques, including - reverse phase HPLC, peptides sequencing and analysis of oligosaccharides on lectin affinity and molecular sieve columns.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Physiological Chemistry Study Section (PC)
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Upstate Medical University
Schools of Medicine
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Hayes, G R; Williams, A M; Lucas, J J et al. (1997) Structure of human transferrin receptor oligosaccharides: conservation of site-specific processing. Biochemistry 36:5276-84
Hayes, G R; Williams, A; Costello, C E et al. (1995) The critical glycosylation site of human transferrin receptor contains a high-mannose oligosaccharide. Glycobiology 5:227-32
Rutledge, E A; Root, B J; Lucas, J J et al. (1994) Elimination of the O-linked glycosylation site at Thr 104 results in the generation of a soluble human-transferrin receptor. Blood 83:580-6
Hayes, G R; Himpler, B S; Weiner, K X et al. (1994) A chicken transferrin binding protein is heat shock protein 108. Biochem Biophys Res Commun 200:65-70
Rutledge, E A; Green, F A; Enns, C A (1994) Generation of the soluble transferrin receptor requires cycling through an endosomal compartment. J Biol Chem 269:31864-8
Hayes, G R; Enns, C A; Lucas, J J (1992) Identification of the O-linked glycosylation site of the human transferrin receptor. Glycobiology 2:355-9