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Mutational analysis of the variant surface glycoprotein GPI-anchor signal sequence in Trypanosoma brucei

Laboratory of Molecular Parasitology, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA

View larger version (19K): [in a new window]   Fig. 1. Procedure for introducing mutations into the VSG 117 C-terminal GPI signal sequence. In the first PCR reaction, the mutation was introduced into VSG 117 with the wild-type and mutant primers P1 and P2. The second PCR reaction used the first PCR product (template 2) and a BspM I fragment (template 3) as templates. The forward primer in this reaction was P1 and the reverse primer P3. Because the final PCR product is a mixture of unmutated and mutated sequence, several plasmids were subjected to sequencing to identify the desired mutant derivative.

View larger version (32K): [in a new window]   Fig. 2. (A) Western blot of T7-VSG 117 (wild-type VSG 117 expressed in the same context as the mutants) compared with mutants S505G, S505A, S505T, K510R and K510L. The cells were induced with 100 ng/ml doxycycline for 24 hours, and the equivalent of 2x104 cells were loaded per lane. (B,C) Western blots of cell line T7-VSG 117 after endogenous GPIPLC activation. Aliquots representing 2x104 cells were loaded per lane. Cells coexpressing VSGs 117 and 221 were osmotically lysed in ice-cold water and centrifuged. The cell ghosts (P1) were resuspended and incubated in 10 mM sodium phosphate buffer, pH 8.0, at 37°C for 15 minutes and centrifuged to generate pellet (P2) and supernatant (S) fractions. Lanes C, P1, P2 and S contain, respectively, whole cells, pellet after lysis and pellet and supernatant after GPIPLC activation. (B) VSG 117 release in the absence or presence of 5 mM pCMPS during PIPLC cleavage, probed with antibodies to VSG 117. (C) Analysis of the same samples, in the absence of pCMPS, with anti-VSG 221 antibodies. (D) Analysis with anti-BiP antibodies. (E) The same protocol was applied to a GPIPLC null-mutant cell line expressing VSG 221 (Leal et al., 2001) and the blot was probed with VSG 221 antibodies.

View larger version (67K): [in a new window]   Fig. 3. Immunofluorescence of (A) T7-VSG117, (B) 515-520, (C) S505A and (D) D503C. Fixed cells were treated with rabbit anti-VSG 117 plus fluorescein-conjugated secondary antibody and DAPI (4',6-diamidino-2-phenylindole), and the images were digitally merged.

View larger version (31K): [in a new window]   Fig. 4. Western blots showing VSG release for mutants D503C (A), D503E (B), 506-508T (C), 504-526 (D), 504-511 (E), 512-526 (F) and 519-526 (G). Cells were induced with 100 ng/ml doxycline for 24 hours in HMI9 medium. Aliquots representing 2x104 cells were loaded per lane. Lanes C, P1, P2 and S contain, respectively, whole cells, pellet after lysis, and pellet and supernatant after GPIPLC activation. In (A) and (B) the amount of mutated VSG is compared with unmutated VSG 117 (T7-VSG117). The exposure was 10 seconds for all panels except (A), which was exposed for 1 minute.

View larger version (45K): [in a new window]   Fig. 5. Immunofluorescence of 504-526 (A-D) and 512-526 (E-H). Fixed and permeabilized cells were treated with rabbit anti-VSG 117 and fluorescein-conjugated secondary antibody (A,E), with mouse anti-BiP and rhodamine-conjugated secondary antibody (B,F), with DAPI (C,G), and the three images were digitally merged (D, H), which results in a yellow pseudo-color in regions of VSG-BiP colocalization.

View larger version (44K): [in a new window]   Fig. 6. Dimerization analysis of mutation 519-526. 2x108 cells of cell line 519-526 were permeabilized with 500 µl 20 mM HEPES, 0.15 M NaCl, pH 7.6 containing 1% (v/v) Nonidet P40. Insoluble material was removed by centrifugation (16 000 g for 15 minutes), and the supernatant was loaded onto a Sephacryl S-200 column. Fractions were TCA-precipitated and analysed by SDS-PAGE. Western blot with anti-VSG 117 (A) or anti-VSG 221 (B) antibodies. As determined by running standards (see Materials and Methods), dimerized VSG should peak in fraction 19. The lower bands on the gel correspond to VSG degradation products, which are exaggerated in this long exposure.

View larger version (39K): [in a new window]   Fig. 7. Effects of elongation mutations on GPI anchoring and expression level. A western blot with rabbit anti-VSG 117, showing the release of VSG117 511FK (A) and VSG117Ty (B). Aliquots representing 2x104 cells were loaded per lane. Lanes C, P1, P2 and S contain, respectively, whole cells, pellet after lysis, and pellet and supernatant after GPIPLC activation. (C) Comparison of VSG expression in four independent clones of VSG117TM, which is not GPI anchored. Aliquots representing 2x104 cells and 2x106 cells were loaded for T7-VSG117 and VSG 117TM, respectively.

View larger version (74K): [in a new window]   Fig. 8. Immunolocalization of VSG117TM. Fixed and permeabilized VSG117TM cells were treated with rabbit anti-VSG 117, mouse anti-p67 (a lysosomal marker) and DAPI, and the images were merged.

View larger version (36K): [in a new window]   Fig. 9. Effects of selected mutations on RNA and protein stability. (A) Degradation kinetics of unmutated VSG 117 and 519-526 RNA. Cell lines were cultured for 8 hours in medium containing 10 µg/ml actinomycin D to prevent transcription. Samples were taken at 4 hour intervals and a northern blot was probed with VSG 117. (B) Degradation kinetics of unmutated VSG 117, 504-526, 512-526, 519-526, D503C and VSG117TM. Cell lines were cultured for 8 hours in medium containing 50 µg/ml cycloheximide to prevent translation. Samples were taken at different time points and analyzed on a western blot with anti-VSG 117 antibodies. (C) Degradation kinetics of wild-type VSG 117, 504-526, 512-526 and 519-526 in the presence of lactacystin. Cell lines were cultured for 8 hours in medium containing 50 µg/ml cycloheximide and 1 µM lactacystin. Samples were taken at different time points and analyzed on a western blot with anti-VSG 117 antibodies. Aliquots representing 2x104 cells were loaded per lane with the exception of VSG117TM where 2x106 cells were loaded per lane.