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

Abstract

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,

Iron metabolism in trypanosomatids, and its crucial role in infection

“…Unlike the mammalian transferrin receptor, which remains tightly bound to its cargo throughout, ESAG6/7 loses affinity for apotransferrin at low pH. The apotransferrin is then degraded by the cathepsin B-like protein, TbcatB in the lysosome (Maier and Steverding, 1996 ;O'Brien et al 2008), while the receptor is recycled back to the flagellar pocket via TbRab11 positive vesicles (Steverding et al 1995 ;Jeffries et al 2001). The degraded transferrin fragments are recycled to the surface and exocytosed, also by a Rab11-dependent mechanism (Pal et al 2003 ;Hall et al 2005).…”

Low affinity of Trypanosoma brucei transferrin receptor to apotransferrin at pH 5 explains the fate of the ligand during endocytosis

“…Holo-Tf was labeled with 3H by reductive methylation using sodium boro[3H]hydride as described previously [2]. Tf-Sepharose was prepared by coupling holo-Tf to CNBr-activated Sepharose 4B (Pharmacia, Freiburg, Germany) as described by the manufacturer.…”

“…In contrast, Tf is delivered in trypanosomes to lysosomes ~vhere it is proteolytically degraded [2,9]. While the degradation products are released from the cells, iron remains cell associated [2]. As the sequence of events leading to ligand clissociation and degradation in T. brucei is undefined, we have studied the affinity of Tf for the trypanosome TfR at different pH values by Triton X-114 phase separation [10] and by determination of Ka values using membranes of trypanosomes.…”

“…The trypalosome TfR is a heterodimeric complex of very low abunlance (about 3000 molecules/cell) encoded by two expression rite-associated genes (ESAGs), ESAG6 and ESAG7 [1][2][3][4][5]. The ESAG6 product (pESAG6) is a heterogeneously glycosylated 9rotein of 50-60 kDa modified by a glycosyl-phosphatidylnositol membrane anchor, while the ESAG7 product (pEgAG7) is a 42 kDa glycoprotein carrying an unmodified COOH-terminus [1].…”

“…The iron-free l'f (apo-Tf) remains bound to its receptor and is recycled back ~.o the cell surface where it dissociates from the receptor [6,8]. In contrast, Tf is delivered in trypanosomes to lysosomes ~vhere it is proteolytically degraded [2,9]. While the degradation products are released from the cells, iron remains cell associated [2].…”