Fig 1.
Tomato lectin blotting and fluorescence microscopy analyses.
(A) TL blotting on total protein extracts of three developmental forms of T. cruzi. Similar amounts of proteins (around 50 μg) from three T. cruzi stages were loaded (see Material and Methods). The same membrane blot was revealed with ponceau red as loading control. The lectin blot analyses indicate that TL-binding glycoproteins are significantly present in epimastigote forms. E: epimastigote, T: trypomastigote, A: amastigote. (B) Fluorescence microscopy of three developmental forms of T. cruzi probed with biotinylated tomato lectin. Arrows indicate the position of nucleus (N) and kinetoplast (K) stained in blue by DAPI. E: epimastigote; M: metacyclic, T: trypomastigote, A: amastigote. Bars scales represent 2μm. (C) TL blotting on total extract of T. brucei bloodstream forms (106 cells) vs T. cruzi epimastigote forms (5 x106 cells). (D) TL blots of T. cruzi CHAPS- and Triton-soluble (CHAPS+Triton X-114) cell lysate fractions. Fractions were enriched by TL chromatography and then treated (+) or not (-) with PNGase F and T represents the total cell lysate. Blots were either probed with TL (upper panel) or anti-TcrCATL (lower panel). The TL blot indicates the presence of N-glycan modification in both soluble and membrane fractions. Treatment of the fractions with PNGase F abolished the reactivity of TL confirming N-glycoprotein type modification. The lower panel shows the presence of TcrCATL, a poly-LacNAc-modified glycoprotein, in both fractions. PNGase F treatment results in the appearance of a lower band corresponding to the loss of the N-glycosylation. Apparent molecular weights are indicated in kDa on the left.
Fig 2.
Localization of TL and GSLII binding sites in T. cruzi.
Endocytosis kinetics of fluorescent Alexa Fluor 594 conjugated Tf was performed in order to follow T. cruzi endocytic pathway from the flagellar pocket/cytostome to the reservosomes. Parasites were fixed at different time points and probed with biotinylated TL (A), biotinylated ricin (B) or Alexa 488 conjugated GSLII (C). The addition of chitin hydrolysate clearly shows inhibition of TL and GSLII staining. (A) Co-localization of biotinylated-TL (green) and Tf (red). (B) Co-localization of biotinylated-ricin (green) and Tf (red). Addition of 200 mM galactose abolished the ricin staining. (C) Co-localization of Alexa 488 conjugated GSLII (green) and Tf (red). (D) Co-localization of Alexa 488 conjugated GSLII (green) and TcJ6 (red). (E) Co-localization of Alexa 488 conjugated GSLII (green) and anti-BiP (red). (F) GSLII blotting of cell extracts enriched by GSLII chromatography. GSLII blots of T. cruzi CHAPS- and Triton-soluble (CHAPS+Triton X-114) cell lysate fractions were enriched by GSLII chromatography and then treated (+) or not (-) with PNGase F. Blots were probed with biotinylated-GSLII. The GSLII blot indicates the presence of N-acetylglucosamine modification in both soluble and membrane fractions. Treatment of the fractions with PNGase F decreased the reactivity of GSLII confirming N-glycoprotein type modification.
Fig 3.
Subcellular localization of TL-binding sites in T cruzi by transmission electron microscopy (TEM).
Parasites were incubated for 5 min in PSG medium in presence (F) or absence (A-E) of BSA-gold as endocytic tracer (10 nm). Cells were fixed and processed for ultrathin frozen sectioning (Tokayasu method, [42]). Cryosections were sequentially probed with biotinylated TL, rabbit anti-biotin antibodies, protein A-gold (5 nm) and finally mounted in methyl cellulose-uranyl acetate films. Representative images are shown. K: kinetoplast, M: mitochondrion, R: reservosome, N: nucleus, FP: flagellar pocket, F: flagellum, G: golgi, Cy: cytostome. Arrows and arrowhead, point to gold particles that mark the presence of TL binding sites and BSA-gold particles, respectively. Asterisk show TL-binding matrix near the opening of the cytostome. Bars = 200 nm.
Fig 4.
Enrichment of glycoproteins from T. cruzi epimastigote using TL and GSLII affinity chromatography.
T.cruzi epimastigote proteins were fractionated by detergent extraction into CHAPS and CHAPS + Triton X-114 fractions. These fractions were loaded either onto agarose-coupled TL or GSLII beads columns and left overnight at 4°C on a rotating device. Whole cell extracts, columns flow-through and eluates were then separated on NuPAGE gels (4–12%) and proteins were revealed by SafeStain blue staining.
Fig 5.
Pie charts (A) displaying protein families function distribution and Venn diagram (B) showing the repartition of the identified proteins in the different lectin-binding fractions.
(A) Functional classification of proteins in TL- and GSLII-enriched fractions. The chart shows the different metabolic pathways to which the identified proteins with known or hypothetical function were assigned. The percentages within each group are indicated. (B) Numbers of identified proteins (with the exception of the proteins grouped under others and hypothetical) in TL and GSLII fractions are represented by a 4-tiered Venn diagram indicating the level of protein overlap between the different lectin-binding fractions. Notable regions include protein groups specific to only one lectin-type: blue (TL-CHAPS + Triton) and yellow (TL-CHAPS), violet (GSLII-CHAPS + Triton) and pink (GSLII-CHAPS) as well as groups identified across lectins and fractions (mixed color regions).
Fig 6.
Comparisons of the identified protein families in three independent proteomic studies [53, 54].
The percentages of different protein families identified in three different studies are compared. The stacked bar chart represents the cumulative distribution of the different fractions shown for each protein family. Functional classification of T. cruzi proteins was performed according to Atwood et al. [53]. Proteins grouped under others and hypothetical were discarded from this comparison.
Fig 7.
Inhibition of uptake of Tf by TL in epimastigote forms of T. cruzi.
Trypanosomes preincubated with biotinylated TL in the presence of 20 μM FMK-024 (25 μg/ml) and in the absence (A, left panel) or presence of competing chitin hydrolysate (A, right panel), were then incubated with Tf Alexa-594 for 5 or 30 min at 27°C. Cells were then fixed and treated for fluorescence microscopy. Similar incubations wherein TL was substituted by GSLII (B) were performed to assess the specificity of the TL labeling. Furthermore, live parasites preincubated with DyLight 488-TL and 20 μM protease inhibitor (FMK-024) for 5 min and then incubated for 60 min in the presence of Alexa Fluor 594 conjugated Tf showed a lectin labeling in the cytostome/cytopharynx (arrowhead), while no Tf labeling (red signal) was observed in these conditions (C, upper panel). In presence of a molar excess of chitin hydrolysate an intense labeling of Tf exclusively concentrate into reservosomes (arrow) while no green signal corresponding to TL was observed anymore (C, lower panel). Inhibition of trypanosomes Tf uptake with TL was furthermore quantified by flow cytometry (D). The TL signal was dropping from 913 to 273 of mfi in the absence or presence of chitin hydrolysate, respectively (D, left histogram). Conversely, Tf signal was increasing from 597 to 3793 of mfi in the absence or presence of chitin hydrolysate, respectively (D, right histogram).
Fig 8.
Uptake of Dextran in the presence of TL in epimastigote forms of T. cruzi.
Flow cytometry profiles of uptake of Dextran Alexa-647 by trypanosomes in the presence or absence of biotinylated TL. Trypanosomes preincubated (A) or not (B) with biotinylated TL in the presence of 20 μM FMK-024 (25 μg/ml) and in absence (A, left histogram) or presence of competing chitin hydrolysate (A, right histogram), were then incubated with Dextran Alexa-647 for 30 min at 27°C.
Fig 9.
TL blotting on Tf and glycophorin.
Different amounts of proteins (up to 5 μg) were loaded. The lectin blot analysis indicates that TL does not recognize Tf but reacts with the sialoglycoprotein glycophorin.