Fig 1.
SDS Page of purified GST-fusion proteins.
Line 1: marker, line 2: GST, line 3: GST-ASPG, line 4: GST-ASPG (T19A), line 5–7: 50, 100 and 250 ng of bovine serum albumin respectively.
Fig 2.
Characterization of L-asparaginase activity of GST-ASPG.
L-Asparaginase activity of GST-ASPG (●) and its catalytically inactive mutant GST-ASPG (T19A) (▲) was evaluated by Nessler’s method in a time dependent manner (A), as a function of enzyme concentration (B) and substrate concentration (C). A hill slope of 6.9 and an S0.5 value of ≈13 mM were estimated. Ammonia release of GST-ASPG was measured using 15 mM of L-asparagine alone or in presence of 15 mM of D-asparagine; No detectable enzymatic activity was found using 15 mM of D-asparagine or 15 mM of L-glutamine as substrates (D). Steady state kinetic of recombinant GST-ASPG was calculated in presence of 0 mM (●), 10 mM (■) and 30 mM (▲) of D-asparagine and a Ki value of 71 mM was estimated (E). All the experiments were performed at 37°C as reported in Materials and Methods using 1.5 μg of each protein; data points are represented as means ± SD of triplicate sample measurements. *** (p < 0.001).
Table 1.
Effect of different compounds on the activity of GST-ASPG.
Fig 3.
(A) The PAF-AH activity of recombinant GST-ASPG (●) and its point mutant GST-ASPG (T19A) (▲) was measured as function of enzyme concentration using Abcam's PAF acetylhydrolase Assay Kit as reported in Materials and Methods. Data points are represented as means ± SD of triplicate sample measurements and were fitted with a second order polynomial equation in GraphPad Prism software. (B) The residual PAF-acetylhydrolase activity of GST-ASPG was measured after pre-incubation for 10 min of 1.5 μg of GST-ASPG with 40 mM and 200 mM of D-asparagine using Abcam's PAF-AH assay. Data are shown as means ± SD of triplicate measurements. ** (p < 0.01); *** (p < 0.001).
Fig 4.
Cell growth inhibition by GST-ASPG.
(A) K562 cells after 24h of incubation with 1000 ng (115 nM) of GST-ASPG and its inactive mutant GST-ASPG (T19A). (B) Time course of K562 cells treatment with 115 nM of GST-ASPG (T19A) (▲), 115 nM of GST ASPG (●) and after further addition of 115 nM of GST-ASPG at 12h (ο).Viability of K562 (C) and NALM-6 (D) cells after 24h of treatment with increasing concentrations of GST-ASPG (●) and its inactive catalytically mutant GST-ASPG (T19A) (▲). The results (the average and the standard deviation of three independent experiments) were evaluated by Trypan Blue exclusion assay and fitted using GraphPad Prism software. * (p < 0.05); ** (p < 0.01); *** (p < 0.001).
Fig 5.
Cytotoxicity of GST-ASPG on human leukemia cell lines and normal cells.
The percentage of cell survival was evaluated by CCK8 assay in K562 (A), NALM-6 (B) and MOLT-4 (C) cell lines after 24h of treatment with GST-ASPG (●) and its inactive catalytically mutant GST-ASPG (T19A) (▲). HDFA (□) and PBMCs (◊) were used to analyze the cytotoxicity of GST-ASPG in normal cells (D). The results were fitted using GraphPad Prism software and represent the average and the standard deviation of three independent experiments. * (p < 0.05); *** (p < 0.001).
Fig 6.
Cytotoxicity of GST-ASPG on K562 cell line in presence of D-asparagine, L-asparagine and PAF.
Data show the effects of 24h of treatment with 100 ng (A) or 1000 ng (B) of GST-ASPG in the presence of 200 mM of D-asparagine, 50 mM of L-asparagine and 10 μM of PAF (C and D) in the culture medium. The percentage of cell survival was evaluated by CCK8 assay and data represent means ± SD of triplicate measurements. ** (p < 0.01) *** (p < 0.001).
Fig 7.
(A) Representative Guava caspase assay graphs of K562 cells lines treated with 0.7 μg of GST, GST-ASPG or GST-ASPG (T19A) for 6, 12 and 24h. (B). Bar graphs representing the percentage of caspase and/or 7AAD positive cells after treatment with 0.7 μg of GST, GST-ASPG or GST-ASPG (T19A) for 6, 12 and 24h. (C) Table representing the statistical significances between groups. *(p < 0.05); **(p< 0.01); ***(p< 0.001).
Fig 8.
Schematic representation of ASPG action on the growth and survival of leukemia cells.
ASPG with its L-asparaginase activity could deprive leukemia cells of L-asparagine, which is an essential metabolite for its malignant growth, inducing apoptosis and blocking cell proliferation. ASPG could also inducing the arrest of cell proliferation converting the active PAF in the inactive form Lyso-PAF and down-regulating the expression of the Epithelial Sodium Channel [3, 26–27].