Isolation of the rat transferrin receptor by affinity chromatography

Rudolph, John R.; Regoeczi, E.

doi:10.1016/s0021-9673(01)94076-2

Cited by 6 publications

(3 citation statements)

References 36 publications

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“…The same binding and dissociation behavior was observed for the TFBP complex from clone MITat 1.4. In addition, we confirmed the observation of Schell et al (1991b) that, in contrast to the mammalian system (Fernandez-Pol and Klos, 1980;Rudolph and Regoeczi, 1987;Turkewitz et al, 1988), the apo-T F -T F B P complex is not dissociated in the presence of the chaotropic SCN--ion (0.5 M in 100 mM Tris/0.2% Triton X-100, p H 8).…”

Section: Binding Characteristics Of Holo-tf and Apo-tf To The Tfbp Cosupporting

confidence: 90%

Transferrin-binding protein complex is the receptor for transferrin uptake in Trypanosoma brucei.

Steverding

Stierhof

Fuchs

et al. 1995

The Journal of Cell Biology

157

173

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Abstract. In Trypanosoma brucei, the products of two genes, ESAG 6 and ESAG 7, located upstream of the variant surface glycoprotein gene in a polycistronic expression site form a glycosylphosphatidylinositolanchored transferrin-binding protein (TFBP) complex. It is shown by gel filtration and membrane-binding experiments that the TFBP complex is heterodimeric and binds one molecule of transferrin with high affinity (2,300 binding sites per cell; K D = 2.1 nM for the dominant expression site from T. brucei strain 427 and KD = 131 nM for ES1.3A of the EATRO 1125 stock). The ternary transferrin-TFBP complexes with iron-loaded or iron-flee ligand are stable between pH 5 and 8. Cellular transferrin uptake can be inhibited by 90% with Fab fragments from anti-TFBP antibodies. After uptake, the TFBP complex and its ligand are routed to lysosomes where transferrin is proteolytically degraded. While the degradation products are released from the cells, iron remains cell associated and the TFBP complex is probably recycled to the membrane of the flagellar pocket, the only site for exo-and endocytosis in this organism. It is concluded that the TFBP complex serves as the receptor for the uptake of transferrin in T. brucei by a mechanism distinct from that in mammalian cells.T RANSFERRIN (TF) 1 is the major serum glycoprotein that transports iron to most tissues in mammals. Diferric-TF (holo-TF) binds to a specific cell-surface receptor, the ligand-receptor complex is endocytosed and delivered to endosomes (for reviews see Crichton and Charloteaux-Wauters, 1987;Huebers and Finch, 1987). The low pH of endosomes triggers the release of iron from holo-TF but the resulting iron-free TF (apo-TF) remains bound to the receptor and is recycled back to the cell surface where, at neutral pH, apo-TF dissociates from the receptor. The human TF receptor is a transmembrane glycoprotein composed of two identical disulphide-linked subunits of 90 kD; each monomer can bind one molecule of TF. The intracellular domain of the receptor subunits contains specific signals for uptake in clathrin-coated vesicles (Trowbridge et al., 1993).African trypanosomes, the causative agents of sleeping sickness in humans and Nagana in cattle, are unicellular, flagellated protozoa that live extracellularly in the blood Address all correspondence to P. Overath,

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Section: Binding Characteristics Of Holo-tf and Apo-tf To The Tfbp Cosupporting

confidence: 90%

Transferrin-binding protein complex is the receptor for transferrin uptake in Trypanosoma brucei.

Steverding

Stierhof

Fuchs

et al. 1995

The Journal of Cell Biology

157

173

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“…Purification of a transferrin-binding membrane protein Before embarking on the purification of a putative trypanosomal transferrin receptor, we reproduced the purification of the mammalian receptor from human placenta. Affinity chromatography on human transferrin coupled to Sepharose readily confirmed that the human receptor was released from its ligand by the chaotropic SCNion (Fernandez-Pol and Klos, 1980;Rudolph and Regoeczi, 1987;Turkewitz et al, 1988). A detergent extract from trypanosome membranes of variant 117a bloodstream forms did not yield a protein under the same conditions.…”

Section: Resultsmentioning

confidence: 99%

“…A column (530 td bed volume) was packed with human diferric transferrin coupled to CH-Sepharose. The following solutions were applied to the column at a rate of 0.5 ml/min (0.2 ml/min for the detergent extract): 20 ml PBS, 0.2% Triton X-100; 5 ml 100 mM NH4HCO3, 1 mg/mi Fe3N+NH4-citrate (Serva, Heidelberg), 0.2% Triton X-100 (Van Driel et al, 1984); 10 ml PBS, 0.2% Triton X-100; 50 ml detergent extract: 20 ml PBS, 0.2% Triton X-100; 10 ml 100 mM citrate, 50 Ag/ml Desferal, 0.2% Triton X-100, pH 5.0 (Anderson et al 1986); 10 ml 100 mM Tris, 50 Ag/ml Desferal, 0.2% Triton X-100, pH 8.0; 10 ml 100 mM Tris, 500 mM NaSCN (Rudolph and Regoeczi, 1987;Turkewitz et a!., 1988), 50 ug/ml Desferal, 0.2% Triton X-100, pH 8.0; 10 ml 50 mM glycine, 0.2% Triton X-100, pH 2.0. The eluate of the last two buffers was collected in 1 ml fractions.…”

Section: Reagentsmentioning

confidence: 99%

A transferrin-binding protein of Trypanosoma brucei is encoded by one of the genes in the variant surface glycoprotein gene expression site.

et al. 1991

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Spemannstrasse 34, D7400 Tubingen and 'Max-Planck-Institut fiir Biochemie, D8033 Martinsried, FRG Communicated by P.Overath A transferrin-binding protein (TFBP) with an apparent molecular weight of 42 kd was purified from detergentsoluble membrane proteins of bloodstream forms of Trypanosoma brucei. The protein is not expressed in the insect-borne stage of the parasite's life-cycle. Purified TFBP can be converted from an amphiphilic to a hydrophilic form by cleavage with T.brucei glycosylphosphatidylinositol (GPI)-specific phospholipase C, demonstrating that the C-terminus is modified by a GPImembrane anchor. The TFBP is encoded by an expression-site-associated gene [ESAG 6 in the nomenclature of Pays et al. (1989) Cell, 57,[835][836][837][838][839][840][841][842][843][844][845] which is under the control of the promoter transcribing the expressed variant surface glycoprotein gene. The possible function of TFBP as a receptor for the uptake of transferrin in bloodstream forms is discussed.

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A Transferrin-Binding Protein in Trypanosoma brucei: Does it Function in Iron Uptake?

Overath

Schell

Stierhof

et al. 1992

Dynamics of Membrane Assembly

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Isolation of the rat transferrin receptor by affinity chromatography

Cited by 6 publications

References 36 publications

Transferrin-binding protein complex is the receptor for transferrin uptake in Trypanosoma brucei.

Transferrin-binding protein complex is the receptor for transferrin uptake in Trypanosoma brucei.

A transferrin-binding protein of Trypanosoma brucei is encoded by one of the genes in the variant surface glycoprotein gene expression site.

A Transferrin-Binding Protein in Trypanosoma brucei: Does it Function in Iron Uptake?

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