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GPI anchor transamidase of Trypanosoma brucei: in vitro assay of the recombinant protein and VSG anchor exchange

Xuedong Kang^*, Alexander Szallies, Marc Rawer, Hartmut Echner and Michael Duszenko

Physiologisch-chemisches Institut, University of Tübingen, 72076 Tübingen, Germany
^* Present address: Howard Hughes Medical Institute, Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, CA 90095-1662, USA

View larger version (58K): [in a new window]   Fig. 1. Sequence alignment of TbGpi8 with Gpi8 sequences of other species and hydrophobicity plot of TbGpi8. (a) TbGpi8 protein sequence of Trypanosoma brucei aligned with homologues of Leishmania mexicana, Plasmodium falciparum, Saccharomyces cerevisiae, Schizosaccharomyes pombe and Homo sapiens. Amino acid identities and similarities are highlighted. TbGpi8 shows one potential N-glycosylation site at position 25 (NKT, overlined), which is not conserved in other Gpi8 molecules. In addition, TbGpi8 has a putative hydrophobic N-terminal ER-directing signal sequence (underlined), but shows no hydrophobic membrane-spanning region at its C-terminus when compared with other Gpi8 molecules. (b) Hydrophobicity plot of TbGpi8 using a window of 6 amino acids: note that there is no putative transmembrane domain on the C-terminus of the protein.

View larger version (47K): [in a new window]   Fig. 2. Induction and purification of MBP-TbGpi8. Cells were induced using 0.3 mM IPTG and lysed by sonification. MBP-TbGpi8 was eluted with elution buffer containing 10 mM maltose and analysed by SDS-PAGE. Lane 1, molecular weight markers; lane 2, lysate from non-induced cells; lane 3, cells induced with 0.3 mM IPTG; lane 4, MBP-TbGpi8 after purification using an amylose column.

View larger version (81K): [in a new window]   Fig. 3. Enterokinase treatment of MBP-TbGpi8. Enterokinase cleavage was carried out using an enzyme:MBP-TbGpi8 ratio of 1:50 (w/w) in 50 mM Tris/HCl buffer at pH 8.0 and room temperature for 8 hours. The cleavage mixture was run through an amylose column to remove MBP or MBP-linked proteins. Samples were analysed by SDS/PAGE. Lane 1, purified MBP-TbGpi8 after the first run of amylose column (Coomassie staining); lane 2, flow-through of the amylose column after enterokinase treatment of the fusion protein; lane 3, proteins eluted from the amylose column using 10 mM maltose; lanes 2 and 3, silver staining.

View larger version (21K): [in a new window]   Fig. 4. Enzyme activity in trypanosomal lysates to cleave Acetyl-SVLN-AMC. 1 mM substrate was incubated with trypanosomal cell lysates (10% in citrate buffer, pH 5.5) at 30°C. After incubation, the shifts of emission maxima were measured. Lysate and buffer shifts were also measured at mock condition. For determination of the pH optimum (b), samples were incubated in 50 mM citrate buffer ranging from pH 2.5 to 6.5, and 50 mM Hepes buffer ranging from pH 5.5 to 9.5. The emission at 440 nm was measured on a spectrofluorimeter as described in the Materials and Methods. (a) Addition of trypanosomal lysate led to the release of AMC from the peptide substrate (), while substrate alone (), lysate alone ([UNK]) or buffer alone () did not. (b) Determination of the pH optimum. (c) Effect of hydrazine addition on enzyme activity: liberation of AMC increased significantly after addition of 10 mM hydrazine () as compared with experiments without hydrazine ([UNK]). Again, substrate alone was stable. (d) Effect of pCMPSA addition on enzyme activity: trypanosome lysates and substrate were incubated in the presence ([UNK]) or absence () of 1 mM pCMPSA as described in the Materials and Methods.

View larger version (16K): [in a new window]   Fig. 5. Transamidase activity of TbGpi8 and MBP-TbGpi8. (a) Trypanosomal lysate and substrate were incubated at 30°C in the presence () or absence ([UNK]) of recombinant TbGpi8. (b) Recombinant TbGpi8 () or MBP-TbGpi8 ([UNK]) and substrate were incubated at 30°C. (c) Trypanosomal lysates were preincubated with Gpi8-specific antibodies for 1 hour at room temperature before the enzyme assay was performed. () control assay without antibodies; () 60 pg/ml antibody; () 120 µg/ml antibody, () 240 pg/ml antibody, ([UNK]) substrate alone.

View larger version (39K): [in a new window]   Fig. 6. Western blot detecting TbGpi8 with specific IgY antibodies. Trypanosomal lysate was separated by SDS-PAGE and analysed for TbGpi8 using affinity-purified chicken anti-TbGpi8 antibodies (lanes 2 and 3) or unspecific IgY antibodies isolated from non-immunized eggs (lanes 4 and 5). Lane 1, molecular weight standards, lanes 2 and 4, lysates equivalent to 1x107 cells per lane; lane 3 and 5, lysates equivalent to 5x106 cells per lane.

View larger version (59K): [in a new window]   Fig. 7. Intracellular localization of TbGpi8 in bloodstream-form trypanosomes. Affinity-purified chicken anti-TbGpi8 antibodies have been used to localize TbGpi8 in intracellular compartments. This labelling was localized specifically around the nucleus and in tubular and vesicular structures throughout the cell. (a-d), trypanosomes were sequentially stained with anti-Gpi8 antibodies and FITC-labelled anti-chicken IgG (a), anti-BiP antibodies and TRITC-labelled anti-rabbit IgG (b) and bisbenzamide (c). (d) shows an overlay of all images. (e) overlay of Gpi8- and BiP-stained cells. Bars, 5 µm.

View larger version (26K): [in a new window]   Fig. 8. Effect of tunicamycin and hydrazine or hydrazine-biotin on VSG formation. Trypanosomes were cultivated in the presence of 10 µg ml-1 tunicamycin (lanes 2, 3 and 4) and 10 mM hydrazine (lane 4) or 10 mM hydrazine-biotin (lane 3) for 4 hours at 37°C in a CO2 incubator. Following cultivation, cells were analysed by SDS-PAGE and western blotting using anti-VSG MITat 1.2 specific antibodies (a), anti-CRD specific antibodies (b) or streptavidin-HRP (c). Lane 1 shows the control sample incubated in the absence of both tunicamycin and hydrazine.

View larger version (82K): [in a new window]   Fig. 9. Intracellular localization of VSG-hydrazine-biotin. Bloodstream-form trypanosomes were grown for 44 hours in the presence (b) or absence (a) of 0.5 mM hydrazine. Parasites were fixed in HBS buffer containing 1% formaldehyde. VSG was immunolocalized using anti-VSG MITat 1.2 specific antibodies. Bars, 5 µm; arrows and arrowheads denote the nucleus and kinetoplast DNA respectively, which were stained by bisbenzimide.