Substrate Affinity Is Not Crucial for Therapeutic L-Asparaginases: Antileukemic Activity of Novel Bacterial Enzymes

Abstract

L-asparaginases are used in the treatment of acute lymphoblastic leukemia. The aim of this work was to compare the antiproliferative potential and proapoptotic properties of novel L-asparaginases from different structural classes, viz. EcAIII and KpAIII (class 2), as well as ReAIV and ReAV (class 3). The EcAII (class 1) enzyme served as a reference. The proapoptotic and antiproliferative effects were tested using four human leukemia cell models: MOLT-4, RAJI, THP-1, and HL-60. The antiproliferative assay with the MOLT-4 cell line indicated the inhibitory properties of all tested L-asparaginases. The results from the THP-1 cell models showed a similar antiproliferative effect in the presence of EcAII, EcAIII, and KpAIII. In the case of HL-60 cells, the inhibition of proliferation was observed in the presence of EcAII and KpAIII, whereas the proliferation of RAJI cells was inhibited only by EcAII. The results of the proapoptotic assays showed individual effects of the enzymes toward specific cell lines, suggesting a selective (time-dependent and dose-dependent) action of the tested L-asparaginases. We have, thus, demonstrated that novel L-asparaginases, with a lower substrate affinity than EcAII, also exhibit significant antileukemic properties in vitro, which makes them interesting new drug candidates for the treatment of hematological malignancies. For all enzymes, the kinetic parameters (K_m and k_cat) and thermal stability (T_m) were determined. Structural and catalytic properties of L-asparaginases from different classes are also summarized.

Keywords: L-asparaginase; cell apoptosis; cell proliferation; leukemia; substrate affinity.

Figure 1

Catalytic properties and structures of L-asparaginases. Reaction catalyzed by enzymes with (A) L-asparaginase activity, (B) L-glutaminase (co-)activity, and (C) β-aspartyl aminopeptidase (co-)activity (NH₂-R—amino acid residue). (D) Symbols of different activities presented in panels (A–C). (E) Structure of EcAII from E. coli (PDB: 6v23). (F) Structure of EcAIII (PDB: 2zal) from E. coli. (G) AlphaFold2 model of KpAIII (K. pneumoniae) precursor (linker marked in red). (H) Structure of thermostable ReAIV (PDB: 8osw) and (I) thermolabile enzyme ReAV (PDB: 7os5) from R. etli. In all panels, protein subunits are shown in different colors. Red circles mark active sites with L-Asp in space-filling representation (panels (E,F)). Violet spheres (panels (H,I)) mark Zn²⁺ coordinated in the active sites of ReAIV and ReAV.

Figure 2

The effect of the tested enzymes on leukemic cell proliferation. Cells from the (A) MOLT-4, (B) RAJI, (C) THP-1, and (D) HL-60 lines were stained with PKH67 dye according to the manufacturer’s instructions. The maximum fluorescence was measured on day 0, and cells were then stimulated with the tested L-asparaginases for 24 and 48 h and analyzed using a flow cytometer (unstimulated cells and cells stimulated with the Control-P protein were used as controls). The presented results are from three independent experiments and are expressed as percentages of the mean (colored stripes) ± standard deviation (thin bar) of the maximum fluorescence value at day 0. * Marks a p < 0.05 probability according to Tukey’s post hoc test.

Figure 3

Influence of different L-asparaginases on the apoptosis of leukemia cell lines: (A) MOLT-4, (B) RAJI, (C) THP-1, and (D) HL-60 cells were cultured in the presence of selected enzymes in a full growth medium for 48 h, stained with a PE Annexin V Apoptosis Detection Kit I, and analyzed via flow cytometry. The L-asparaginases and their concentrations used in this experiment were based on the results from proliferation evaluation. The presented results are from three independent experiments and are expressed in Figure 2, i.e., as a percentage of the mean ± standard deviation of the dead cells (apoptotic and/or necrotic), *** marks a p < 0.005, and **** marks a p < 0.0001 probability according to Tukey’s post hoc test; ns: not significant. Gating strategies used in flow cytometry are in Figure S4.