Figure 1.
Structure of kalata B1 and cyclotide mutants.
The structure of kalata B1 is shown in cartoon form (A). The six conserved cysteines are labeled with Roman numerals and the cystine knot disulfide connectivity (CI-CIV, CII-CV and CIII-CVI) is indicated. The amino acid sequence and the loops of the kalata B1 backbone are shown in C. The positions of the mutations are indicated by red arrows in the cartoon and are highlighted with red letters in the sequence. The α-H chemical shift comparison of kalata B1 (full circles), [V10K] kalata B1 (open triangles) and [T20K] kalata B1 (open squares) is shown for all residues starting from residue G1 (B). The amino acid sequence (with mutations highlighted in red) and a surface representation of kalata B1 (PDB code: 1NB1) is shown in (C). The structure is using a hydrophobicity scale according to Eisenberg et al. [53] to illustrate the amphiphilic nature of these peptides. Typical hydrophilic regions of cyclotides are in loops 2 and 3, whereas the hydrophobic patch is found on the opposite site in loops 5 and 6. Mutated amino acids, e.g., in [V10K] and [T20K] kalata B1 were modeled using the PyMol mutation wizard and have been shown in red color in the enlarged window. All Figure representations were prepared using PyMol.
Figure 2.
Effects of cyclotide mutants on cell proliferation of primary activated human lymphocytes.
The influence of medium (ctrl), CsA (0.8 µM) or different concentrations of the kalata B1 cyclotide mutants [T8K], [V10A], [V10K], [G18K], [T20K] and [N29K] (1.8-14 μM) on proliferation of activated primary lymphocytes was measured by cell division analysis using CFSE-based flow cytometry on day three post stimulation. Data are presented as mean ± SD of four (three for [T20K] kalata B1) independent donors and experiments. Corresponding IC50 values have been presented in Table 1.
Figure 3.
Effects of cyclotide mutants on IL-2 biology of primary activated human lymphocytes and purified T-cells.
For IL-2 receptor expression studies, lymphocytes were treated with CsA or cyclotides (4 µM each) and were cultivated in the presence of media (Ø) or activation stimuli alone (ctrl). At 24 h (A) or 36 h (B) after cultivation, cells were surface-stained with anti-human CD25 mAbs and were analyzed by flow cytometry. For IL-2 secretion analysis, lymphocytes (C) or purified T-cells (D) were restimulated with PMA (50 ng/mL) and ionomycin (500 ng/mL) for 6 h after 24 h of cultivation. Afterwards, the amount of IL-2 was individually measured in the supernatant by using ELISA-based techniques. The il-2 gene expression of lymphocytes was analyzed by quantitative RT-PCR (E). The data were normalized to the cycle threshold value of the internal housekeeping gene 18s rRNA and the relative mRNA level in the untreated stimulated group was used as calibrator. Data are expressed as mean ± SD of independent donors and experiments as indicated. For IL-2 receptor analysis representative data were additional depicted as dot plots. The asterisks represent significant differences (*P <0.05, **P <0.01, ***P <0.001) of treated cells in comparison to ctrl (PHA-L stimulated cells alone).
Figure 4.
Effects of exogenous IL-2 on proliferation capacity of cyclotide-treated lymphocytes.
CFSE-labeled primary human lymphocytes were pretreated with CsA or cyclotides (4 µM each) and were activated using PHA-L (10 µg/mL), followed by washing off the cyclotides and activation stimuli. Furthermore, the cells were cultured without (A and B) or in the presence of exogenous recombinant human IL-2 (C and D). The proliferation capacity of the cells was analyzed at day three by flow cytometry. Representative data are presented in dot plots (A and B) and summary of three independent experiments were presented as mean ± SD (B and D). The values in dot plots represents the amount of proliferated lymphocytes on which the quantification to control is based. The asterisks represent significant differences of treated cells in comparison to ctrl (PHA-L stimulated cells alone) (*P <0.05, **P <0.01, ***P <0.001).
Figure 5.
Influence of cyclotide mutants on effector function of primary activated human lymphocytes.
Purified lymphocytes were preincubated with CsA or cyclotides (4 µM each), then stimulated with PHA-L (10 µg/mL) and exposed to a re-stimulation impulse with PMA and ionomycin for 6 h before analysis of effector functions, expressed as amounts of IFN-γ (24 h: A; 36 h: B), TNF-α (24 h: C; 36 h: D) or degranulation capacity (E). The amount of IFN-γ and TNF-α was measured in the supernatant of cultured cells using an ELISA-based method for early time points, or intracellular cytokine detection flow cytometric analysis for late time points. The degranulation capacity was detected by using classical CD107a assay. Data of three independent experiments were presented as mean ± SD. The asterisks represent significant differences of treated cells in comparison to ctrl or PHA-L stimulated cells alone (*P <0.05, **P <0.01, ***P <0.001).