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The transferrin receptor of Trypanosoma brucei
Parasitology International 48 Ž2000. 191]198
Mini review
The transferrin receptor of Trypanosoma brucei Dietmar SteverdingU Abteilung Parasitologie, Hygiene-Institut der Ruprecht-Karls-Uni¨ ersitat, ¨ Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany Received 5 March 1999; accepted 5 July 1999
Abstract Bloodstream forms of Trypanosoma brucei, the causative agent of sleeping sickness in humans, require transferrin for growth. Uptake of host transferrin is mediated by a heterodimeric glycosylphosphatidylinositol-anchored receptor. The trypanosomal transferrin receptor is homologous to the N-terminal domain of the variant surface glycoprotein ŽVSG. and bears no structural similarity with the human transferrin receptor. In this review, the structure, biochemical properties and function of the transferrin receptor of T. brucei are summarized and compared to the transferrin receptor of mammalian cells. Q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Trypanosoma brucei; Transferrin receptor; Transferrin uptake
1. Introduction The flagellated protozoan parasite, Trypanosoma brucei, which is transmitted by the bite of the tsetse fly, is the causative agent of sleeping sickness in humans and Nagana in livestock. Within the mammalian host, trypanosomes live U
Corresponding author. Tel.: q49-6221-567855; fax: q496221-564643. E-mail address: Dietmar ] [email protected] ŽD. Steverding.
and multiply extracellulary in the blood and tissue fluids. Like all living organisms, bloodstream forms of T. brucei require iron for growth w1,2x although this life-cycle stage lacks cytochromes. All iron that bloodstream-form trypanosomes need for propagation is delivered by transferrin of the host w1,2x. The parasite internalizes host transferrin by receptor-mediated endocytosis; this process is 200 times faster than the rate of pinocytosis w3x. The trypanosomal receptor for transferrin uptake is an unusual protein which bears no structural similarity with its mammalian
1383-5769r00r$ - see front matter Q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 1 3 8 3 - 5 7 6 9 Ž 9 9 . 0 0 0 1 8 - 5
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D. Ste¨ erding r Parasitology International 48 (2000) 191]198
counterpart. In this review, the transferrin receptors of T. brucei and human cells are compared with regard to structure, biochemical properties and function.
2. Receptor identification In search of a receptor involved in transferrin uptake, a heterodimeric protein of very low abundance Žapprox. 3000 moleculesrcell. was isolated from bloodstream forms of T. brucei by affinity chromatography w4]9x. The receptor is encoded by two homologous expression site-associated genes, ESAG6 and ESAG7 w4]9x. These genes are located upstream of the variant surface glycoprotein ŽVSG. gene in a polycistronic telomeric expression site w10]13x. Like the VSG, ESAGs are expressed exclusively in the mammalian stage of the parasite w6x. In Table 1, the features of the trypanosomal and the human transferrin receptors are compared. The receptor of T. brucei differs in primary structure, subunit organization and mode of membrane anchorage from its human counterpart. The latter is a transmembrane glycoprotein composed of two identical disulphide-linked sub-
units of 90 kDa, which are phosphorylated and acylated w14]16x. The subunits of the T. brucei transferrin receptor ŽESAG6 and ESAG7. are heterogeneously glycosylated proteins of 50]60 kDa and 40]42 kDa, respectively w4]7x. These proteins are synthesized with N-terminal signal sequences which are absent from the mature products w13x. The amino acid sequences of the two subunits are almost identical over their Nterminal half but show differences in the C-terminal region w13x. The hydrophobic C-terminal stretch of the primary ESAG6 is replaced by a glycosylphosphatidylinositol ŽGPI. anchor which attaches the heterodimeric receptor to the plasma membrane w4x. However, a significant proportion of the receptor including the GPI anchor is also found in the soluble fraction of cell lysates w4x. When purified from a lysate of bloodstream forms, part of the receptor is free and part is loaded with transferrin w4x. The cellular uptake of transferrin can be inhibited with IgGs w7x and Fab fragments w8,9x of anti-ŽESAG6r7 heterodimer. antibodies. Conversely, transferrin prevents receptor-dependent binding and uptake of anti-ŽESAG6r7 heterodimer. antibodies w8x. According to these findings it is most likely that the ESAG6r7 heterodimer is indeed the transferrin receptor in
Table 1 Features of the transferrin receptors ŽTfR. of T. brucei and human cells Features
Trypanosome TfR
Human TfR
Subunit organization
Heterodimer of ESAG6 Ž50]60 kDa. and ESAG7 Ž40]42 kDa.
Homodimer of 90-kDa subunits
Post-translational modifications
ESAG6: 2]5 N-linked glycans ESAG7: 2]3 N-linked glycans
Per subunit: 3 N-linked glycans 1 Phosphorylation ŽSer 24. 1 Acylation ŽCys 62.
Membrane anchorage
GPI-anchor at the C-terminus of ESAG6
1 Transmembrane domain per subunit
Copy number per cell
3000
20 000]700 000
Ligandrreceptor stoichiometry
1 Transferrin molecule per heterodimer
1 Transferrin molecule per monomer
D. Ste¨ erding r Parasitology International 48 (2000) 191]198
bloodstream forms of T. brucei. This is corroborated by the fact that no other proteins operating in transferrin uptake have been found in bloodstream-form trypanosomes.
3. Receptor structure The three-dimensional structure of the human transferrin receptor remains to be established although a large fragment of the extracellular domain Žresidues 121]760. has been analyzed by preliminary X-ray crystallography w17x. In contrast, the putative structure of the trypanosomal transferrin receptor was deduced by homology modeling on the basis of known structure of VSG, the surface coat protein of the parasite w18,19x. Both VSG and transferrin receptor occur as dimers and are attached to the plasma membrane by GPI-anchors. Significant sequence similarity Ž60]76%. has been reported between the Nterminal domain of VSG molecules and both subunits of the transferrin receptor w19,20x. In addition, the patterns of heptad repeats and of four cysteine residues are conserved in different VSGs and in the transferrin receptor subunits w18,19,21,22x. Based on these findings it has been predicted that ESAG6 and ESAG7 are folded in a similar fashion as the VSG molecule w18,19x. As shown in Fig. 1, computer modeling revealed an elongated structure of ESAG6 and ESAG7 resembling that of the N-terminal domain of VSGs w23x.
4. Ligand binding Each monomer of the human receptor can bind a single transferrin molecule w26x. In contrast, association of both subunits of the trypanosomal receptor is required for binding of one ligand molecule ŽTable 1. w8x. This was shown by coexpression of ESAG6 and ESAG7 in insect cells w5x, in procyclic forms of T. brucei w6x, and in Xenopus oocytes w7x. The ligand-binding site of the human receptor has yet not been defined. However, functional analysis of chimeric humanr chicken transferrin receptors indicates that the
193
C-terminal 192 amino acids are critical for ligand binding w27x. In contrast, the ligand-binding site of the trypanosomal transferrin receptor was recently identified as two small sequence stretches comprising amino acids 205]215 and 223]238 of ESAG6 and ESAG7 w19x. These two stretches form surface-exposed loops ŽFig. 1. as it had previously been predicted from sequence homology between ESAG6r7 and the N-terminal domains of two different VSG molecules w19,23x.
5. Binding affinities Despite the profound difference in receptor structure, the transferrin receptors of the T. brucei strain 427 and human cells have similar high affinities for holotransferrin Ždiferric transferrin . at pH 7 and pH 5, the estimated apparent dissociation constants Ž K d . being in the nanomolar range Žsee Table 2.. However, higher K d-values Ž5.3]830 nM. have been reported for transferrin receptors from other T. brucei clones and for transferrins from a variety of mammals w7,8,28x. This can be explained by the large sequence diversity of mammalian transferrins w33x, and by variations in the amino acid sequences of ESAG6 and ESAG7 expressed from different expression sites w13,19x. There are up to 20 expression sites in the genome of T. brucei but only one is active at a given time w10,34x. Different expression sites encode similar but not identical transferrin receptors w35x. During natural transmission from one host species to another, trypanosomes have to deal with the diversity of mammalian transferrins. Expression of a receptor with low affinity for transferrin will lead to limited iron supply of the parasite. By switching between different expression sites, the parasite can express a transferrin receptor more suitable for that host. Thus, expression of different transferrin receptors allows T. brucei to utilize transferrin from various mammalian hosts and permits the parasite to grow in a large range of mammalian species as hosts w28x. The affinities of the transferrin receptors of the T. brucei strain 427 and human cells for apotransferrin Žiron-free transferrin . at pH 7 and pH 5 are
194
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Fig. 1.
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Table 2 K d-Values of ligand]receptor complexes for apo- and holotransferrin at pH 7 and pH 5 Kd-Value ŽnM.
Transferrin receptor
Transferrin
pH
T. brucei strain 427
Holo bovine
7 5 7 5
2.6]3.6 12 20 1100
7 5 7 5
1.9]7.7 13 ) 700 13]21
Apo bovine
Human cells
Holo human Apo human
diametrically opposite Žsee Table 2.. Whereas the trypanosomal receptor shows at pH 7 a high and at pH 5 a low affinity for apotransferrin, the mammalian receptor exhibits a low and a high affinity. On the basis of the different affinities between receptor and apotransferrin at pH 7 and pH 5, the different intracellular processing of the ligand in trypanosomes and mammalian cells can be explained Žsee Section 6 and Fig. 2..
6. Transferrin uptake The cellular uptake of transferrin in bloodstream forms of T. brucei occurs by a mechanism distinct from the well-established transferrin cell cycle in human cells w30,31x. Binding of holotransferrin to the receptor takes place at the cell surface in human cells w36x, whereas in T. brucei it occurs in the flagellar pocket ŽFig. 2. w4,6,8x. The flagellar pocket is an invagination of the plasma membrane which serves as the sole site for endoand exocytosis w37]39x. Transferrin bound to the GPI-anchored trypanosomal receptor is pre-
References w8,28x w29x w2x w29x w30]32x w31x w31x w30,31x
sumably translocated into the cell interior by bulk membrane flow while the human ligand]receptor complex is internalized by clathrin-coated vesicles w40x. In both trypanosomes and human cells, the ligand]receptor complex is delivered into endosomes where the acidic pH triggers the release of iron from transferrin w29,31,41x. Because of different affinities at pH 5 Žsee Table 2., the apotransferrin remains bound to the human receptor w30,31x but dissociates from the trypanosomal receptor ŽFig. 2. w29x. In human cells, apotransferrin is recycled back to the cell surface while iron is retained within the cell w31,41x. At the neutral pH of the extracellular fluid, apotransferrin has a low affinity for the human receptor and is released to mediate further cycles of iron uptake w30,31x. In contrast, within trypanosomes apotransferrin is delivered into lysosomes where it is proteolytically degraded ŽFig. 2. w8,42x. The resulting large peptide fragments are released from the trypanosomes while iron remains cell-associated w8x. The receptor is probably recycled to the membrane of the flagellar pocket ŽFig. 2.. This is suggested by the observation that internalisation
Fig. 1. Putative structure of T. brucei transferrin receptor subunits as deduced from homology with VSG MITat 1.2 using the Inside 2 program w24x. Ža. ESAG6; Žb. ESAG7. Shown are backbone models excluding uncertain loop regions. Open ends are indicated by the number of amino acids. The amino- and carboxy-termini are labeled with N and C, respectively. a-Helices are shown in green, b-strands in red, and loops in gray. The two stretches Žamino acids 205]215 and 223]238 w19x. of ESAG6 involved in ligand binding are shown in blue. The heterodimeric receptor is presumably formed by close association of the two long a-helices of each subunit into a four-helix bundle, similar to that previously determined for the VSG MITat 1.2 dimer w25x. In the models, the top is exposed to the extracellular environment while the bottom faces the trypanosomal plasma membrane.
196
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Fig. 2. Transferrin uptake in bloodstream forms of T. brucei. See text for details. E6rE7, heterodimeric transferrin receptor; ellipsoid with Fe, holotransferrin; empty ellipsoid, apotransferrin; FP, flagellar pocket; FL, flagellum; V, endo- and exocytotic vesicles; EN, endosome; LY, lysosome; CY, cytosol.
of the ligand exceeds the total receptor content w8x. It should be noted that the uptake of transferrin in bloodstream forms of T. brucei resembles the internalization of low-density lipoproteins w43x and asialoglycoproteins w44x by mammalian cells. The rate of transferrin uptake is approximately 4.5 moleculesrreceptorrh in trypanosomes w2,8x which is only four times less than in immature erythroid cells showing an uptake rate of 18 moleculesrreceptorrh w45x. This is surprising since uptake via a GPI-anchored receptor through bulk membrane flow is expected to be much less efficient than via a transmembrane receptor in clathrin coated vesicles. However, bloodstream forms of T. brucei compensate the low effectiveness of their GPI-anchored transferrin receptor by a very high turnover of the flagellar pocket membrane w3,46,47x.
would probably not. As the subunits of the transferrin receptor are approximately 25% smaller than a VSG molecule, the surface coat may shield the trypanosomal receptor from antibodies leaving only the ligand-binding site accessible w48x. Acknowledgements I would like to thank Prof. R. Heiner Schirmer, BZH, Ruprecht-Karls-Universitat ¨ Heidelberg, for reading the manuscript, and Dr Thomas Dandekar, EMBL, Heidelberg, for computer modeling of ESAG6 and ESAG7. This work was supported by the Deutsche Forschungsgemeinschaft ŽSTE 803r1-1 and 1-2. and by the Bundesministerium fur ¨ Forschung und Technologie, Schwerpunkt fur ¨ tropenmedizinische Forschung in Heidelberg Ž01 KA 9301r3.. References
7. Conclusion The transferrin receptor of T. brucei has to function within the surface of the parasite. It is reasonable to assume that the ESAG6r7 heterodimer with its VSG-like structure fits perfectly into the monolayer of densely packed VSG dimers while a protein similar to the human receptor
w1x Schell D, Borowy NK, Overath P. Transferrin is a growth factor for the bloodstream form of Trypanosoma brucei. Parasitol Res 1991;77:558]60. w2x Steverding D. Bloodstream forms of Trypanosoma brucei require only small amounts of iron for growth. Parasitol Res 1998;84:59]62. w3x Coppens I, Opperdoes FR, Courtoy PJ, Baudhuin P. Receptor-mediated endocytosis in the bloodstream form of Trypanosoma brucei. J Protozool 1987;34:465]73.
D. Ste¨ erding r Parasitology International 48 (2000) 191]198 w4x Steverding D, Stierhof Y-D, Chaudhri M, Ligtenberg M, Schell D, Beck-Sickinger AG, Overath P. ESAG 6 and 7 products of Trypanosoma brucei form a transferrin binding protein complex. Eur J Cell Biol 1994;64:78]87. w5x Chaudhri M, Steverding D, Kittelberger D, Tjia S, Overath P. Expression of a glycosylphosphatidylinositol-anchored Trypanosoma brucei transferrin-binding protein complex in insect cells. Proc Natl Acad Sci USA 1994;91:6443]7. w6x Ligtenberg MJL, Bitter W, Kieft R, Steverding D, Janssen H, Calafat J, Borst P. Reconstitution of a surface transferrin binding complex in insect form Trypanosoma brucei. EMBO J 1994;13:2565]73. w7x Salmon D, Geuskens M, Hanocq F, Hanocq-Quertier J, Nolan D, Ruben L, Pays E. A novel heterodimeric transferrin receptor encoded by a pair of VSG expression site-associated genes in T. brucei. Cell 1994;78: 75]86. w8x Steverding D, Stierhof Y-D, Fuchs H, Tauber R, Overath P. Transferrin-binding protein complex is the receptor for transferrin uptake in Trypanosoma brucei. J Cell Biol 1995;131:1173]82. w9x Steverding D, Overath P. Trypanosoma brucei with an active metacyclic variant surface gene expression site expresses a transferrin receptor derived from esag6 and esag7. Mol Biochem Parasitol 1996;78:285]8. w10x Cully DF, Ip HS, Cross GAM. Coordinate transcription of variant surface glycoprotein genes and an expression site associated gene family in Trypanosoma brucei. Cell 1985;42:173]82. w11x Kooter JM, van der Spek HJ, Wagter R, d’Oliveira CE, van der Hoeven F, Johnson PJ, Borst P. The anatomy and transcription of a telomeric expression site for variant-specific surface antigens in T. brucei. Cell 1987; 51:261]72. w12x Johnson PJ, Kooter JM, Borst P. Inactivation of transcription by UV irradiation of T. brucei provides evidence for a multicistronic transcription unit including a VSG gene. Cell 1987;51:273]81. w13x Pays E, Tebabi P, Pays A, Coquelet H, Revelard P, Salmon D, Steinert M. The genes and transcripts of an antigen gene expression site from T. brucei. Cell 1989; 57:835]45. w14x Newman R, Schneider C, Sutherland R, Vodinelich L, Greaves M. The transferrin receptor. Trends Biochem Sci 1982;7:397]400. w15x Davis RJ, Johnson GL, Kelleher DJ, Anderson JK, Mole JE, Czech MP. Identification of serine 24 as the unique site on the transferrin receptor phosphorylated by protein kinase C. J Biol Chem 1986;261:9034]41. w16x Jing S, Trowbridge IS. Identification of the intermolecular disulphide bonds of the human transferrin receptor and its lipid-attachment site. EMBO J 1987;6:327]31. w17x Borhani DW, Harrison SC. Crystallization and X-ray diffraction studies of a soluble form of the human transferrin receptor. J Mol Biol 1991;218:685]9. w18x Carrington M, Boothroyd J. Implications of conserved
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structural motifs in disparate trypanosome surface proteins. Mol Biochem Parasitol 1996;81:119]26. Salmon D, Hanocq-Quertier J, Paturiaux-Hanocq F, Pays A, Tebabi P, Nolan DP, Michel A, Pays E. Characterization of the ligand-binding site of the transferrin receptor in Trypanosoma brucei demonstrates a structural relationship with the N-terminal domain of the variant surface glycoprotein. EMBO J 1997;16: 7272]8. Hobbs MR, Boothroyd JC. An expression-site-associated gene family of trypanosomes is expressed in ¨ i¨ o and shows homology to a variant surface glycoprotein gene. Mol Biochem Parasitol 1990;43:1]16. Carrington M, Miller N, Blum M, Roditi I, Wiley D, Turner M. Variant specific glycoprotein of Trypanosoma brucei consists of two domains each having an independently conserved pattern of cysteine residues. J Mol Biol 1991;221:823]35. Cohen C, Reinhardt B, Parry DAD, Roelants GE, Hirsch W, Kanwe ´ B. a-Helical coiled-coil structures of Trypanosoma brucei variable surface glycoproteins. Nature 1984;311:169]71. Blum ML, Down JA, Gurnett AM, Carrington M, Turner MJ, Wiley DC. A structural motif in the variant surface glycoproteins of Trypanosoma brucei. Nature 1993;362: 603]9. Jacobs T, Bruchhaus I, Dandekar T, Tannich E, Leippe M. Isolation and molecular characterization of a surface-bound proteinase of Entamoeba histolytica. Mol Microbiol 1998;27:269]76. Freymann D, Down J, Carrington M, Roditi I, Turner ˚ M, Wiley D. 2.9 A-resolution structure of the N-terminal domain of a variant surface glycoprotein from Trypanosoma brucei. J Mol Biol 1990;216:141]60. Turkewitz AP, Amatruda JF, Borhani D, Harrison SC, Schwartz AL. A high yield purification of the human transferrin receptor and properties of its major extracellular fragment. J Biol Chem 1988;263:8318]25. Buchegger F, Trowbridge IS, Liu L-FS, White S, Collawn JF. Functional analysis of humanrchicken transferrin receptor chimeras indicates that the carboxy-terminal region is important for ligand binding. Eur J Biochem 1996;235:9]17. Bitter W, Gerrits H, Kieft R, Borst P. The role of transferrin-receptor variation in the host range of Trypanosoma brucei. Nature 1998;391:499]502. Maier A, Steverding D. Low affinity of Trypanosoma brucei transferrin receptor to apotransferrin at pH 5 explains the fate of the ligand during endocytosis. FEBS Lett 1996;396:87]9. Klausner RD, Ashwell G, van Renswoude J, Harford JB, Bridges KR. Binding of apotransferrin to K562 cells: explanation of the transferrin cycle. Proc Natl Acad Sci USA 1983;80:2263]6. Dautry-Varsat A, Ciechanover A, Lodish HF. pH and the recycling of transferrin during receptor-mediated endocytosis. Proc Natl Acad Sci USA 1983;80:2258]62.
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w32x Hunt RC, Riegler R, Davis AA. Changes in glycosylation alter the affinity of the human transferrin receptor for its ligand. J Biol Chem 1989;264:9643]8. w33x Baldwin GS. Comparison of transferrin sequences from different species. Comp Biochem Physiol 1993;106B: 203]18. w34x Zomerdijk JCBM, Ouellette M, ten Asbroek ALMA, Kieft R, Bommer AMM, Clayton CE, Borst P. The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei. EMBO J 1990;9: 2791]801. w35x Zomerdijk JCBM, Kieft R, Duyndam M, Shiels PG, Borst P. Antigenic variation in Trypanosoma brucei: a telomeric expression site for variant-specific surface glycoprotein genes with novel features. Nucleic Acids Res 1991;19:1359]68. w36x Jandl JH, Katz JH. The plasma-to-cell cycle of transferrin. J Clin Invest 1963;42:314]26. w37x Balber AE. The pellicle and the membrane of the flagellum, flagellar adhesion zone, and flagellar pocket: functionally discrete surface domains of the bloodstream form of African trypanosomes. Crit Rev Immunol 1990;10:177]201. w38x Webster P, Russell DG. The flagellar pocket of trypanosomatids. Parasitol Today 1993;9:201]6. w39x Overath P, Chaudhri M, Steverding D, Ziegelbauer K. Invariant surface proteins in bloodstream forms of Trypanosoma brucei. Parasitol Today 1994;10:53]8. w40x Hopkins CR, Trowbridge IS. Internalization and processing of transferrin and the transferrin receptor in human carcinoma A431 cells. J Cell Biol 1983;97: 508]21. w41x Van Renswoude J, Bridges KR, Harford JB, Klausner
w42x
w43x
w44x
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w48x
RD. Receptor-mediated endocytosis of transferrin and the uptake of Fe in K562 cells: identification of a nonlysosomal acidic compartment. Proc Natl Acad Sci USA 1982;79:6186]90. Grab DJ, Wells CW, Shaw MK, Webster P, Russo DCW. Endocytosed transferrin in African trypanosomes is delivered to lysosomes and may not be recycled. Eur J Cell Biol 1992;59:398]404. Goldstein JL, Brunschede GY, Brown MS. Inhibition of proteolytic degradation of low density lipoprotein in human fribroblasts by chloroquine, concanavalin A, and Triton WR 1339. J Biol Chem 1975;250:7854]62. Bridges K, Harford J, Ashwell G, Klausner RD. Fate of receptor and ligand during endocytosis of asialoglycoproteins by isolated hepatocytes. Proc Natl Acad Sci USA 1982;79:350]4. Iacopetta BJ, Morgan EH, Yeoh GCT. Transferrin receptors and iron uptake during erythroid cell development. Biochim Biophys Acta 1982;687:204]10. Webster P, Griffiths G. A novel method for mean cell volume estimation. J Microsc 1994;174:85]92. Fairlamb AH, Bowman IBR. Trypanosoma brucei: maintenance of concentrated suspensions of bloodstream trypomastigotes in ¨ itro using continuous dialysis for measurement of endocytosis. Exp Parasitol 1980;49: 366]80. Borst P, Bitter W, Blundell P, Cross M, McCulloch R, Rudenko G, Taylor MC, Van Leeuwen F. The expression sites for variant surface glycoproteins of Trypanosoma brucei. In: Hide G, Mottram JC, Coombs GH, Holmes PH, editors. Trypanosomiasis and Leishmaniasis: Biology and Control. Oxon: CAB International, 1997:109]31.
Mini review
The transferrin receptor of Trypanosoma brucei Dietmar SteverdingU Abteilung Parasitologie, Hygiene-Institut der Ruprecht-Karls-Uni¨ ersitat, ¨ Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany Received 5 March 1999; accepted 5 July 1999
Abstract Bloodstream forms of Trypanosoma brucei, the causative agent of sleeping sickness in humans, require transferrin for growth. Uptake of host transferrin is mediated by a heterodimeric glycosylphosphatidylinositol-anchored receptor. The trypanosomal transferrin receptor is homologous to the N-terminal domain of the variant surface glycoprotein ŽVSG. and bears no structural similarity with the human transferrin receptor. In this review, the structure, biochemical properties and function of the transferrin receptor of T. brucei are summarized and compared to the transferrin receptor of mammalian cells. Q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Trypanosoma brucei; Transferrin receptor; Transferrin uptake
1. Introduction The flagellated protozoan parasite, Trypanosoma brucei, which is transmitted by the bite of the tsetse fly, is the causative agent of sleeping sickness in humans and Nagana in livestock. Within the mammalian host, trypanosomes live U
Corresponding author. Tel.: q49-6221-567855; fax: q496221-564643. E-mail address: Dietmar ] [email protected] ŽD. Steverding.
and multiply extracellulary in the blood and tissue fluids. Like all living organisms, bloodstream forms of T. brucei require iron for growth w1,2x although this life-cycle stage lacks cytochromes. All iron that bloodstream-form trypanosomes need for propagation is delivered by transferrin of the host w1,2x. The parasite internalizes host transferrin by receptor-mediated endocytosis; this process is 200 times faster than the rate of pinocytosis w3x. The trypanosomal receptor for transferrin uptake is an unusual protein which bears no structural similarity with its mammalian
1383-5769r00r$ - see front matter Q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 1 3 8 3 - 5 7 6 9 Ž 9 9 . 0 0 0 1 8 - 5
192
D. Ste¨ erding r Parasitology International 48 (2000) 191]198
counterpart. In this review, the transferrin receptors of T. brucei and human cells are compared with regard to structure, biochemical properties and function.
2. Receptor identification In search of a receptor involved in transferrin uptake, a heterodimeric protein of very low abundance Žapprox. 3000 moleculesrcell. was isolated from bloodstream forms of T. brucei by affinity chromatography w4]9x. The receptor is encoded by two homologous expression site-associated genes, ESAG6 and ESAG7 w4]9x. These genes are located upstream of the variant surface glycoprotein ŽVSG. gene in a polycistronic telomeric expression site w10]13x. Like the VSG, ESAGs are expressed exclusively in the mammalian stage of the parasite w6x. In Table 1, the features of the trypanosomal and the human transferrin receptors are compared. The receptor of T. brucei differs in primary structure, subunit organization and mode of membrane anchorage from its human counterpart. The latter is a transmembrane glycoprotein composed of two identical disulphide-linked sub-
units of 90 kDa, which are phosphorylated and acylated w14]16x. The subunits of the T. brucei transferrin receptor ŽESAG6 and ESAG7. are heterogeneously glycosylated proteins of 50]60 kDa and 40]42 kDa, respectively w4]7x. These proteins are synthesized with N-terminal signal sequences which are absent from the mature products w13x. The amino acid sequences of the two subunits are almost identical over their Nterminal half but show differences in the C-terminal region w13x. The hydrophobic C-terminal stretch of the primary ESAG6 is replaced by a glycosylphosphatidylinositol ŽGPI. anchor which attaches the heterodimeric receptor to the plasma membrane w4x. However, a significant proportion of the receptor including the GPI anchor is also found in the soluble fraction of cell lysates w4x. When purified from a lysate of bloodstream forms, part of the receptor is free and part is loaded with transferrin w4x. The cellular uptake of transferrin can be inhibited with IgGs w7x and Fab fragments w8,9x of anti-ŽESAG6r7 heterodimer. antibodies. Conversely, transferrin prevents receptor-dependent binding and uptake of anti-ŽESAG6r7 heterodimer. antibodies w8x. According to these findings it is most likely that the ESAG6r7 heterodimer is indeed the transferrin receptor in
Table 1 Features of the transferrin receptors ŽTfR. of T. brucei and human cells Features
Trypanosome TfR
Human TfR
Subunit organization
Heterodimer of ESAG6 Ž50]60 kDa. and ESAG7 Ž40]42 kDa.
Homodimer of 90-kDa subunits
Post-translational modifications
ESAG6: 2]5 N-linked glycans ESAG7: 2]3 N-linked glycans
Per subunit: 3 N-linked glycans 1 Phosphorylation ŽSer 24. 1 Acylation ŽCys 62.
Membrane anchorage
GPI-anchor at the C-terminus of ESAG6
1 Transmembrane domain per subunit
Copy number per cell
3000
20 000]700 000
Ligandrreceptor stoichiometry
1 Transferrin molecule per heterodimer
1 Transferrin molecule per monomer
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bloodstream forms of T. brucei. This is corroborated by the fact that no other proteins operating in transferrin uptake have been found in bloodstream-form trypanosomes.
3. Receptor structure The three-dimensional structure of the human transferrin receptor remains to be established although a large fragment of the extracellular domain Žresidues 121]760. has been analyzed by preliminary X-ray crystallography w17x. In contrast, the putative structure of the trypanosomal transferrin receptor was deduced by homology modeling on the basis of known structure of VSG, the surface coat protein of the parasite w18,19x. Both VSG and transferrin receptor occur as dimers and are attached to the plasma membrane by GPI-anchors. Significant sequence similarity Ž60]76%. has been reported between the Nterminal domain of VSG molecules and both subunits of the transferrin receptor w19,20x. In addition, the patterns of heptad repeats and of four cysteine residues are conserved in different VSGs and in the transferrin receptor subunits w18,19,21,22x. Based on these findings it has been predicted that ESAG6 and ESAG7 are folded in a similar fashion as the VSG molecule w18,19x. As shown in Fig. 1, computer modeling revealed an elongated structure of ESAG6 and ESAG7 resembling that of the N-terminal domain of VSGs w23x.
4. Ligand binding Each monomer of the human receptor can bind a single transferrin molecule w26x. In contrast, association of both subunits of the trypanosomal receptor is required for binding of one ligand molecule ŽTable 1. w8x. This was shown by coexpression of ESAG6 and ESAG7 in insect cells w5x, in procyclic forms of T. brucei w6x, and in Xenopus oocytes w7x. The ligand-binding site of the human receptor has yet not been defined. However, functional analysis of chimeric humanr chicken transferrin receptors indicates that the
193
C-terminal 192 amino acids are critical for ligand binding w27x. In contrast, the ligand-binding site of the trypanosomal transferrin receptor was recently identified as two small sequence stretches comprising amino acids 205]215 and 223]238 of ESAG6 and ESAG7 w19x. These two stretches form surface-exposed loops ŽFig. 1. as it had previously been predicted from sequence homology between ESAG6r7 and the N-terminal domains of two different VSG molecules w19,23x.
5. Binding affinities Despite the profound difference in receptor structure, the transferrin receptors of the T. brucei strain 427 and human cells have similar high affinities for holotransferrin Ždiferric transferrin . at pH 7 and pH 5, the estimated apparent dissociation constants Ž K d . being in the nanomolar range Žsee Table 2.. However, higher K d-values Ž5.3]830 nM. have been reported for transferrin receptors from other T. brucei clones and for transferrins from a variety of mammals w7,8,28x. This can be explained by the large sequence diversity of mammalian transferrins w33x, and by variations in the amino acid sequences of ESAG6 and ESAG7 expressed from different expression sites w13,19x. There are up to 20 expression sites in the genome of T. brucei but only one is active at a given time w10,34x. Different expression sites encode similar but not identical transferrin receptors w35x. During natural transmission from one host species to another, trypanosomes have to deal with the diversity of mammalian transferrins. Expression of a receptor with low affinity for transferrin will lead to limited iron supply of the parasite. By switching between different expression sites, the parasite can express a transferrin receptor more suitable for that host. Thus, expression of different transferrin receptors allows T. brucei to utilize transferrin from various mammalian hosts and permits the parasite to grow in a large range of mammalian species as hosts w28x. The affinities of the transferrin receptors of the T. brucei strain 427 and human cells for apotransferrin Žiron-free transferrin . at pH 7 and pH 5 are
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Fig. 1.
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Table 2 K d-Values of ligand]receptor complexes for apo- and holotransferrin at pH 7 and pH 5 Kd-Value ŽnM.
Transferrin receptor
Transferrin
pH
T. brucei strain 427
Holo bovine
7 5 7 5
2.6]3.6 12 20 1100
7 5 7 5
1.9]7.7 13 ) 700 13]21
Apo bovine
Human cells
Holo human Apo human
diametrically opposite Žsee Table 2.. Whereas the trypanosomal receptor shows at pH 7 a high and at pH 5 a low affinity for apotransferrin, the mammalian receptor exhibits a low and a high affinity. On the basis of the different affinities between receptor and apotransferrin at pH 7 and pH 5, the different intracellular processing of the ligand in trypanosomes and mammalian cells can be explained Žsee Section 6 and Fig. 2..
6. Transferrin uptake The cellular uptake of transferrin in bloodstream forms of T. brucei occurs by a mechanism distinct from the well-established transferrin cell cycle in human cells w30,31x. Binding of holotransferrin to the receptor takes place at the cell surface in human cells w36x, whereas in T. brucei it occurs in the flagellar pocket ŽFig. 2. w4,6,8x. The flagellar pocket is an invagination of the plasma membrane which serves as the sole site for endoand exocytosis w37]39x. Transferrin bound to the GPI-anchored trypanosomal receptor is pre-
References w8,28x w29x w2x w29x w30]32x w31x w31x w30,31x
sumably translocated into the cell interior by bulk membrane flow while the human ligand]receptor complex is internalized by clathrin-coated vesicles w40x. In both trypanosomes and human cells, the ligand]receptor complex is delivered into endosomes where the acidic pH triggers the release of iron from transferrin w29,31,41x. Because of different affinities at pH 5 Žsee Table 2., the apotransferrin remains bound to the human receptor w30,31x but dissociates from the trypanosomal receptor ŽFig. 2. w29x. In human cells, apotransferrin is recycled back to the cell surface while iron is retained within the cell w31,41x. At the neutral pH of the extracellular fluid, apotransferrin has a low affinity for the human receptor and is released to mediate further cycles of iron uptake w30,31x. In contrast, within trypanosomes apotransferrin is delivered into lysosomes where it is proteolytically degraded ŽFig. 2. w8,42x. The resulting large peptide fragments are released from the trypanosomes while iron remains cell-associated w8x. The receptor is probably recycled to the membrane of the flagellar pocket ŽFig. 2.. This is suggested by the observation that internalisation
Fig. 1. Putative structure of T. brucei transferrin receptor subunits as deduced from homology with VSG MITat 1.2 using the Inside 2 program w24x. Ža. ESAG6; Žb. ESAG7. Shown are backbone models excluding uncertain loop regions. Open ends are indicated by the number of amino acids. The amino- and carboxy-termini are labeled with N and C, respectively. a-Helices are shown in green, b-strands in red, and loops in gray. The two stretches Žamino acids 205]215 and 223]238 w19x. of ESAG6 involved in ligand binding are shown in blue. The heterodimeric receptor is presumably formed by close association of the two long a-helices of each subunit into a four-helix bundle, similar to that previously determined for the VSG MITat 1.2 dimer w25x. In the models, the top is exposed to the extracellular environment while the bottom faces the trypanosomal plasma membrane.
196
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Fig. 2. Transferrin uptake in bloodstream forms of T. brucei. See text for details. E6rE7, heterodimeric transferrin receptor; ellipsoid with Fe, holotransferrin; empty ellipsoid, apotransferrin; FP, flagellar pocket; FL, flagellum; V, endo- and exocytotic vesicles; EN, endosome; LY, lysosome; CY, cytosol.
of the ligand exceeds the total receptor content w8x. It should be noted that the uptake of transferrin in bloodstream forms of T. brucei resembles the internalization of low-density lipoproteins w43x and asialoglycoproteins w44x by mammalian cells. The rate of transferrin uptake is approximately 4.5 moleculesrreceptorrh in trypanosomes w2,8x which is only four times less than in immature erythroid cells showing an uptake rate of 18 moleculesrreceptorrh w45x. This is surprising since uptake via a GPI-anchored receptor through bulk membrane flow is expected to be much less efficient than via a transmembrane receptor in clathrin coated vesicles. However, bloodstream forms of T. brucei compensate the low effectiveness of their GPI-anchored transferrin receptor by a very high turnover of the flagellar pocket membrane w3,46,47x.
would probably not. As the subunits of the transferrin receptor are approximately 25% smaller than a VSG molecule, the surface coat may shield the trypanosomal receptor from antibodies leaving only the ligand-binding site accessible w48x. Acknowledgements I would like to thank Prof. R. Heiner Schirmer, BZH, Ruprecht-Karls-Universitat ¨ Heidelberg, for reading the manuscript, and Dr Thomas Dandekar, EMBL, Heidelberg, for computer modeling of ESAG6 and ESAG7. This work was supported by the Deutsche Forschungsgemeinschaft ŽSTE 803r1-1 and 1-2. and by the Bundesministerium fur ¨ Forschung und Technologie, Schwerpunkt fur ¨ tropenmedizinische Forschung in Heidelberg Ž01 KA 9301r3.. References
7. Conclusion The transferrin receptor of T. brucei has to function within the surface of the parasite. It is reasonable to assume that the ESAG6r7 heterodimer with its VSG-like structure fits perfectly into the monolayer of densely packed VSG dimers while a protein similar to the human receptor
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