Expression of a glycosylphosphatidylinositol-anchored Trypanosoma brucei transferrin-binding protein complex in insect cells.

Abstract

The expression site-associated gene ESAG 6 was previously implicated in transferrin binding in the protozoan parasite Trypanosoma brucei. ESAG 6 and the closely related ESAG 7 of T. brucei strain AnTatl.3 have now been expressed in insect cells using the baculovirus expression system. Expression of ESAG 6 alone in insect cells gives rise to a glycosylated protein of =52 kDa, which is cell surfaceassociated through a glycosylphosphatidylnoltol anchor at its C terminus. The ESAG 7 product of about 42 kDa is also glycosylated, but lacks the glycosylphosphatidylinositol modification, and is located intracellularly. No terrm-binding activity is observed when either ESAG is expressed independently. However, their coexpression results in a cell surface complex of ESAG 6 and 7 products that specifically binds trusferrin. This shows that both ESAG 6 and 7 products are necessary and s ent for binding to transferrin.Trypanosoma brucei is widespread in Africa and causes a wasting disease-Nagana-in cattle and sleeping sickness in humans. In the mammalian host the parasite exists extracellularly in the blood and tissues, where it evades the immune response by sequentially expressing a repertoire of antigenically distinct surface proteins, encoded by the variant surface glycoprotein (VSG) genes. Each transcriptionally active VSG gene is located in a so-called expression site, which contains at least eight other cotranscribed expression siteassociated genes (ESAGs) (1). Although highly immunogenic, the VSGs are unsuitable for immunization against the parasitic infection. Potential targets for protective immunization may be surface proteins that deliver essential nutrients to the parasite and that are invariant.Living extracellularly, the parasite has access to the host's serum components. It has been demonstrated that T. brucei requires the major serum iron-transport protein, transferrin (TF), for growth (2). In T. brucei, a specific TF-binding protein (TFBP) that could function as a receptor in transferrin uptake was sought using TF-Sepharose as an affinity matrix (3). N-terminal sequencing of a purified TFBP isolated in extremely small ajnounts from T. brucei strain MITat 1.4 (clone 117a) revealed it to be identical to the products of two highly similar ESAGs, ESAGs 6 and 7 (3, 4). The putative products of these genes share about 85% sequence identity. Both contain a potential signal peptide, suggesting they are secreted or located on the surface. The ESAG 6 but not ESAG 7 product has a potential recognition site for attachment of a glycosylphosphatidylinositol (GPI) anchor at the C-terminal end. ESAG 6 was originally reported as encoding the TFBP (3). Subsequent work, however, has suggested that the data were misinterpreted (M. Ligtenberg and P. Borst, personal communication; this work; refs. 5 and 6) and that ESAG 6 and 7 products are involved in TF binding (this work;refs. 6 and 7).In this paper we report the expression of both proteins individually and together in insect cells and unequivocally demonstrate that in...

Characterization of a Novel, Stage-specific, Invariant Surface Protein in Trypanosoma brucei Containing an Internal, Serine-rich, Repetitive Motif

“…The N-and C-terminal regions that flank the internal repeat are indicated by solid lines. receptor (5,7). The distribution of both of these proteins differed from the Ca 2ϩ -ATPase of the endoplasmic reticulum (TBA1) (32) and ISG 70 (19), which were localized predominantly in the endoplasmic reticulum and plasma membrane fractions, respectively.…”

Factors Affecting the Level and Localization of the Transferrin Receptor in Trypanosoma brucei

“…African trypanosomes cover their iron need by taking up host transferrin (Tf) (3)(4)(5). Uptake occurs in the flagellar pocket (3,6,7), an invagination of the plasma membrane where all endocytosis and traffic to the trypanosome surface takes place (8 -12), and is mediated by a transferrin receptor (Tf-R), a heterodimer consisting of subunits encoded by expression site-associated gene (ESAG) 6 and 7 (13)(14)(15)(16)(17), which are located in the telomeric VSG gene expression sites (for review, see Refs. 18 -21).…”

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

“…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 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]. Binding of one molecule of transferrin Tf) [2] requires association of both pESAG6 and pESAG7 as ~hown by coexpression in heterologous systems [3][4][5]. Despite :he profound difference in receptor structure, the apparent tissociation constant (Kd) for iron-loaded Tf (holo-Tf) is of ::he same order of magnitude for both the trypanosome TfR 3.6-108 nM [2]) and the mammalian TfR (2-110 nM [6,7]).…”