Figure 1.
SDS-PAGE analysis of purified T. brucei TfR and endoglycosidase digestions.
(A) An aliquot of T. brucei TfR, affinity purified on transferrin-Sepharose, was analyzed by SDS-PAGE and silver staining. (B) Aliquots of purified TfR were incubated with Endo H (lane 1), PNGase F (lane 3) or mock treated (lane 2) and analyzed by SDS-PAGE and Western blot with an antibody that reacts with the ESAG6 and ESAG7 subunits of TfR.
Figure 2.
Aliquots of purified T. brucei TfR (lanes 1, 2, 3, 6, 7, 10 and 11) and of bovine ribonuclease B, a positive control for ConA blotting (lanes 4 and 5), and bovine asialotransferrin, a positive control for ErCr lectin and ricin blotting (lanes 8, 9, 12 and 13), were separated by SDS-PAGE, transferred to nitrocellulose and subjected to blotting with anti-TfR antibody (lane 1), ConA (lanes 2–5), ErCr lectin (lanes 6–9) or ricin (lanes 10–13) in the absence (−) or presence (+) of the competing sugars α-methyl-mannose (lanes 3 and 5), lactose (lanes 7 and 9) or galactose and lactose (lanes 11 and 13). The positions of molecular weight markers are indicated for each group of blots.
Figure 3.
Fluorographs of HPTLC analyses of released and radiolabeled N-glycans from purified TfR.
(A) Total N-glycan fraction of TfR released by PNGase F and radiolabeled by reduction with NaB[3H]4. The positions of oligomannose N-linked glycan standards reduced with NaB[3H]4 are shown on the left. The proposed structures of the principal TfR glycans are shown on the right. The top three biantennary structures (Man3GlcNAc2 to Man5GlcNAc2) are of the paucimannose series and the bottom three triantennary structures (Man5GlcNAc2 to Man7GlcNAc2) are of the oligomannose series. (B) The three major components of the radiolabeled TfR N-glycan fraction isolated by Dionex HPAEC (peaks a, b and c; lanes 1, 2 and 3, respectively). The positions of NaB[3H]4 reduced oligomannose N-linked glycan and dextran oligomer standards are shown on the left and right, respectively. (C) The paucimannose Man4GlcNAc2 structure (Dionex peak a) before (lane 1) and after (lane 2) digestion with Manα1-2Man specific α-mannosidase (ASαM) and the triantennary oligomannose Man5GlcNAc2structure Dionex peak b before (lane 3) and after (lane 4) digestion with ASαM. The positions of NaB[3H]4-reduced dextran oligomers are shown on right.
Figure 4.
Glycosylation patterns of ESAG6 and ESAG7.
Maps of the major N-glycan types at each N-glycosylation site along the polypeptide backbones of ESAG6 and ESAG7, based on the data in Figure 3 and Table 1, and the GPI glycan structure of ESAG6, based on [31].
Table 1.
Experimental and theoretical occupancy of TfR N-glycosylation sites.
Figure 5.
TfR does not bind directly to tomato lectin but binds to other glycoproteins.
A ricin-binding glycoprotein fraction ([27] was purified from T. brucei, separated by SDS-PAGE and transferred to nitrocellulose. Identical lanes were incubated without (lane 1) or with (lane 2) purified TfR, followed by anti-TfR antibody, or with tomato lectin (TL) in the absence (lane 3) or presence (lane 4) of competing chitin hydrolysate. The positions of molecular weight markers are shown on the left.
Figure 6.
Molecular modeling of TfR in the VSG coat of T. brucei.
(A) A single glycosylated ESAG6/ESAG7 TfR heterodimer is shown flanked by two glycosylated VSG homodimers. The molecular models suggest that TfR is likely to be recessed into the VSG surface coat. (B) Top and side views of a glycosylated TfR molecule surrounded by VSG molecules. (C) The same views as (B) but with a glycosylated transferrin molecule approaching the TfR. TfR: peptide chains – green; N-glycans – yellow; GPI anchor – orange. VSG: peptide chains – blue; N-glycans – yellow; GPI anchor – orange. Transferrin: peptide chains – red; N- and O-glycans – purple.