ABSTRACT
l-asparaginases play a crucial role in the treatment of acute lymphoblastic leukemia (ALL), a type of cancer that mostly affects children and teenagers. However, it is common for these molecules to cause adverse reactions during treatment. These downsides ignite the search for novel asparaginases to mitigate these problems. Thus, this work aimed to produce and characterize a recombinant asparaginase from Phaseolus vulgaris (Asp-P). In this study, Asp-P was expressed in Escherichia coli with high yields and optimum activity at 40 °C, pH 9.0. The enzyme Km and Vmax values were 7.05 mM and 1027 U/mg, respectively. Asp-P is specific for l-asparagine, showing no activity against l-glutamine and other amino acids. The enzyme showed a higher cytotoxic effect against Raji than K562 cell lines, but only at high concentrations. In silico analysis indicated that Asp-P has lower immunogenicity than a commercial enzyme. Asp-P induced biofilm formation by Candida sp. due to sublethal dose, showing an underexplored potential of asparaginases. The absence of glutaminase activity, lower immunogenicity and optimal activity similar to physiological temperature conditions are characteristics that indicate Asp-P as a potential new commercial enzyme in the treatment of ALL and its underexplored application in the treatment of other diseases.
Subject(s)
Asparaginase , Phaseolus , Recombinant Proteins , Asparaginase/chemistry , Asparaginase/pharmacology , Asparaginase/genetics , Asparaginase/immunology , Phaseolus/chemistry , Humans , Kinetics , Recombinant Proteins/pharmacology , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Cell Line, Tumor , Leukemia/drug therapy , K562 Cells , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Biofilms/drug effects , Hydrogen-Ion Concentration , TemperatureABSTRACT
Amino acid deprivation therapy (AADT) is a novel anticancer therapy, considered nontoxic and selective. Thermophilic L-asparaginase enzymes display high stability and activity at elevated temperatures. However, they are of limited use in clinical applications because of their low substrate affinity and reduced activity under physiological conditions, which may necessitate an improved dosage, leading to side effects and greater costs. Thus, in an attempt to improve the activity of L-Asn at 37 °C, with the use of a semi-rational design, eight active-site mutants of Thermococcus litoralis DSM 5473 L-asparaginase Tli10209 were developed. T70A exhibited a 5.11-fold increase compared with the wild enzyme in physiological conditions. Double-mutant enzymes were created by combining mutants with higher hydrolysis activity. T70A/F36Y, T70A/K48L, and T70A/D50G were enhanced by 5.59-, 6.38-, and 5.58-fold. The immobilized enzyme applied in MCF-7 breast cancer cells only required one-seventh of the dose of the free enzyme to achieve the same inhibition rate under near-infrared irradiation. This provides a proof of concept that it is possible to reduce the consumption of L-Asn by improving its activity, thus providing a method to manage side effects.
Subject(s)
Antineoplastic Agents , Asparaginase , Mutagenesis, Site-Directed , Asparaginase/genetics , Asparaginase/chemistry , Asparaginase/pharmacology , Asparaginase/metabolism , Humans , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , MCF-7 Cells , Thermococcus/enzymology , Thermococcus/genetics , Catalytic DomainABSTRACT
The Protein Data Bank (PDB) includes a carefully curated treasury of experimentally derived structural data on biological macromolecules and their various complexes. Such information is fundamental for a multitude of projects that involve large-scale data mining and/or detailed evaluation of individual structures of importance to chemistry, biology and, most of all, to medicine, where it provides the foundation for structure-based drug discovery. However, despite extensive validation mechanisms, it is almost inevitable that among the â¼215â 000 entries there will occasionally be suboptimal or incorrect structure models. It is thus vital to apply careful verification procedures to those segments of the PDB that are of direct medicinal interest. Here, such an analysis was carried out for crystallographic models of L-asparaginases, enzymes that include approved drugs for the treatment of certain types of leukemia. The focus was on the adherence of the atomic coordinates to the rules of stereochemistry and their agreement with the experimental electron-density maps. Whereas the current clinical application of L-asparaginases is limited to two bacterial proteins and their chemical modifications, the field of investigations of such enzymes has expanded tremendously in recent years with the discovery of three entirely different structural classes and with numerous reports, not always quite reliable, of the anticancer properties of L-asparaginases of different origins.
Subject(s)
Asparaginase , Databases, Protein , Asparaginase/chemistry , Humans , Models, Molecular , Crystallography, X-Ray/methods , Protein ConformationABSTRACT
We succeeded in homogeneously expressing and purifying L-asparaginase from Latilactobacillus sakei LK-145 (Ls-Asn1) and its mutated enzymes C196S, C264S, C290S, C196S/C264S, C196S/C290S, C264S/C290S, and C196S/C264S/C290S-Ls-Asn1. Enzymological studies using purified enzymes revealed that all cysteine residues of Ls-Asn1 were found to affect the catalytic activity of Ls-Asn1 to varying degrees. The mutation of Cys196 did not affect the specific activity, but the mutation of Cys264, even a single mutation, significantly decreased the specific activity. Furthermore, C264S/C290S- and C196S/C264S/C290S-Ls-Asn1 almost completely lost their activity, suggesting that C290 cooperates with C264 to influence the catalytic activity of Ls-Asn1. The detailed enzymatic properties of three single-mutated enzymes (C196S, C264S, and C290S-Ls-Asn1) were investigated for comparison with Ls-Asn1. We found that only C196S-Ls-Asn1 has almost the same enzymatic properties as that of Ls-Asn1 except for its increased stability for thermal, pH, and the metals NaCl, KCl, CaCl2, and FeCl2. We measured the growth inhibitory effect of Ls-Asn1 and C196S-Ls-Asn1 on Jurkat cells, a human T-cell acute lymphoblastic leukemia cell line, using L-asparaginase from Escherichia coli K-12 as a reference. Only C196S-Ls-Asn1 effectively and selectively inhibited the growth of Jurkat T-cell leukemia, which suggested that it exhibited antileukemic activity. Furthermore, based on alignment, phylogenetic tree analysis, and structural modeling, we also proposed that Ls-Asn1 is a so-called "Type IIb" novel type of asparaginase that is distinct from previously reported type I or type II asparaginases. Based on the above results, Ls-Asn1 is expected to be useful as a new leukemia therapeutic agent.
Subject(s)
Asparaginase , Asparaginase/genetics , Asparaginase/metabolism , Asparaginase/chemistry , Asparaginase/isolation & purification , Asparaginase/pharmacology , Humans , Bacillaceae/enzymology , Bacillaceae/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry , Hydrogen-Ion Concentration , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Jurkat Cells , Mutation , Amino Acid Sequence , KineticsABSTRACT
L-asparaginase is an essential drug used to treat acute lymphoid leukemia (ALL), a cancer of high prevalence in children. Several adverse reactions associated with L-asparaginase have been observed, mainly caused by immunogenicity and allergenicity. Some strategies have been adopted, such as searching for new microorganisms that produce the enzyme and applying protein engineering. Therefore, this work aimed to elucidate the molecular structure and predict the immunogenic profile of L-asparaginase from Penicillium cerradense, recently revealed as a new fungus of the genus Penicillium and producer of the enzyme, as a motivation to search for alternatives to bacterial L-asparaginase. In the evolutionary relationship, L-asparaginase from P. cerradense closely matches Aspergillus species. Using in silico tools, we characterized the enzyme as a protein fragment of 378 amino acids (39 kDa), including a signal peptide containing 17 amino acids, and the isoelectric point at 5.13. The oligomeric state was predicted to be a homotetramer. Also, this L-asparaginase presented a similar immunogenicity response (T- and B-cell epitopes) compared to Escherichia coli and Dickeya chrysanthemi enzymes. These results suggest a potentially useful L-asparaginase, with insights that can drive strategies to improve enzyme production.
Subject(s)
Asparaginase , Computer Simulation , Penicillium , Asparaginase/chemistry , Asparaginase/immunology , Asparaginase/metabolism , Penicillium/immunology , Penicillium/enzymology , Amino Acid Sequence , Fungal Proteins/chemistry , Fungal Proteins/immunology , Fungal Proteins/metabolism , Epitopes, B-Lymphocyte/immunology , Epitopes, B-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/immunology , Epitopes, T-Lymphocyte/chemistry , Humans , Aspergillus/immunology , Aspergillus/enzymology , Escherichia coli/genetics , Dickeya chrysanthemi/enzymology , Dickeya chrysanthemi/immunology , Models, MolecularABSTRACT
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 (Km and kcat) and thermal stability (Tm) were determined. Structural and catalytic properties of L-asparaginases from different classes are also summarized.
Subject(s)
Antineoplastic Agents , Apoptosis , Asparaginase , Cell Proliferation , Humans , Asparaginase/pharmacology , Asparaginase/chemistry , Asparaginase/metabolism , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Cell Proliferation/drug effects , Cell Line, Tumor , Substrate Specificity , HL-60 Cells , Leukemia/drug therapy , Leukemia/enzymologyABSTRACT
This study investigated the effect of polycationic and uncharged polymers (and oligomers) on the catalytic parameters and thermostability of L-asparaginase from Thermococcus sibiricus (TsA). This enzyme has potential applications in the food industry to decrease the formation of carcinogenic acrylamide during the processing of carbohydrate-containing products. Conjugation with the polyamines polyethylenimine and spermine (PEI and Spm) or polyethylene glycol (PEG) did not significantly affect the secondary structure of the enzyme. PEG contributes to the stabilization of the dimeric form of TsA, as shown by HPLC. Furthermore, neither polyamines nor PEG significantly affected the binding of the L-Asn substrate to TsA. The conjugates showed greater maximum activity at pH 7.5 and 85 °C, 10-50% more than for native TsA. The pH optima for both TsA-PEI and TsA-Spm conjugates were shifted to lower pH ranges from pH 10 (for the native enzyme) to pH 8.0. Additionally, the TsA-Spm conjugate exhibited the highest activity at pH 6.5-9.0 among all the samples. Furthermore, the temperature optimum for activity at pH 7.5 shifted from 90-95 °C to 80-85 °C for the conjugates. The thermal inactivation mechanism of TsA-PEG appeared to change, and no aggregation was observed in contrast to that of the native enzyme. This was visually confirmed and supported by the analysis of the CD spectra, which remained almost unchanged after heating the conjugate solution. These results suggest that TsA-PEG may be a more stable form of TsA, making it a potentially more suitable option for industrial use.
Subject(s)
Asparaginase , Biocatalysis , Enzyme Stability , Thermococcus , Asparaginase/chemistry , Asparaginase/metabolism , Thermococcus/enzymology , Hydrogen-Ion Concentration , Polyethylene Glycols/chemistry , Temperature , Archaeal Proteins/chemistry , Archaeal Proteins/metabolismABSTRACT
L-Asparaginases (ASNases) catalyze the hydrolysis of L-Asn to L-Asp and ammonia. Members of the ASNase family are used as drugs in the treatment of leukemia, as well as in the food industry. The protomers of bacterial ASNases typically contain 300-400 amino acids (typical class 1 ASNases). In contrast, the chain of ASNase from Rhodospirillum rubrum, reported here and referred to as RrA, consists of only 172 amino acid residues. RrA is homologous to the N-terminal domain of typical bacterial class 1 ASNases and exhibits millimolar affinity for L-Asn. In this study, we demonstrate that RrA belongs to a unique family of cytoplasmic, short-chain ASNases (scASNases). These proteins occupy a distinct region in the sequence space, separate from the regions typically assigned to class 1 ASNases. The scASNases are present in approximately 7% of eubacterial species, spanning diverse bacterial lineages. They seem to be significantly enriched in species that encode for more than one class 1 ASNase. Here, we report biochemical, biophysical, and structural properties of RrA, a member of scASNases family. Crystal structures of the wild-type RrA, both with and without bound L-Asp, as well as structures of several RrA mutants, reveal topologically unique tetramers. Moreover, the active site of one protomer is complemented by two residues (Tyr21 and Asn26) from another protomer. Upon closer inspection, these findings clearly outline scASNases as a stand-alone subfamily of ASNases that can catalyze the hydrolysis of L-Asn to L-Asp despite the lack of the C-terminal domain that is present in all ASNases described structurally to date.
Subject(s)
Asparaginase , Rhodospirillum rubrum , Asparaginase/chemistry , Rhodospirillum rubrum/genetics , Rhodospirillum rubrum/metabolism , Protein Subunits , Aspartic Acid , Catalytic DomainABSTRACT
A thermostable L-asparaginase was produced from Bacillus licheniformis UDS-5 (GenBank accession number, OP117154). The production conditions were optimized by the Plackett Burman method, followed by the Box Behnken method, where the enzyme production was enhanced up to fourfold. It secreted L-asparaginase optimally in the medium, pH 7, containing 0.5% (w/v) peptone, 1% (w/v) sodium chloride, 0.15% (w/v) beef extract, 0.15% (w/v) yeast extract, 3% (w/v) L-asparagine at 50 °C for 96 h. The enzyme, with a molecular weight of 85 kDa, was purified by ion exchange chromatography and size exclusion chromatography with better purification fold and percent yield. It displayed optimal catalysis at 70 °C in 20 mM Tris-Cl buffer, pH 8. The purified enzyme also exhibited significant salt tolerance too, making it a suitable candidate for the food application. The L-asparaginase was employed at different doses to evaluate its ability to mitigate acrylamide, while preparing French fries without any prior treatment. The salient attributes of B. licheniformis UDS-5 L-asparaginase, such as greater thermal stability, salt stability and acrylamide reduction in starchy foods, highlights its possible application in the food industry.
Subject(s)
Acrylamide , Asparaginase , Asparaginase/chemistry , Acrylamide/analysis , Acrylamide/chemistry , Asparagine , Food IndustryABSTRACT
L-Asparaginase is a key component in the treatment of leukemias and lymphomas. However, the glutamine affinity of this therapeutic enzyme is an off-target activity that causes several side effects. The modeling and molecular docking study of Yarrowia lipolytica L-asparaginase (YL-ASNase) to reduce its l-glutamine affinity and increase its stability was the aim of this study. Protein-ligand interactions of wild-type and different mutants of YL-ASNase against L-asparagine compared to l-glutamine were assessed using AutoDock Vina tools because the crystal structure of YL-ASNase does not exist in the protein data banks. The results showed that three mutants, T171S, T171S-N60A, and T171A-T223A, caused a considerable increase in L-asparagine affinity and a decrease in l-glutamine affinity as compared to the wild-type and other mutants. Then, molecular dynamics simulation and MM/GBSA free energy were applied to assess the stability of protein structure and its interaction with ligands. The three mutated proteins, especially T171S-N60A, had higher stability and interactions with L-asparagine than l-glutamine in comparison with the wild-type. The YL-ASNase mutants could be introduced as appropriate therapeutic candidates that might cause lower side effects. However, the functional properties of these mutated enzymes need to be confirmed by genetic manipulation and in vitro and in vivo studies.
Subject(s)
Antineoplastic Agents , Yarrowia , Asparaginase/chemistry , Glutamine/chemistry , Molecular Docking Simulation , Asparagine/metabolism , Yarrowia/genetics , Yarrowia/metabolism , Molecular Dynamics Simulation , Antineoplastic Agents/chemistryABSTRACT
Genome sequence of Geobacillus thermopakistaniensis contains an open reading frame annotated as a type II L-asparaginase (ASNaseGt). Critical structural analysis disclosed that ASNaseGt might be a type I L-asparaginase. In order to determine whether it is a type I or type II L-asparaginase, we have performed the structural-functional characterization of the recombinant protein as well as analyzed the localization of ASNaseGt in G. thermopakistaniensis. ASNaseGt exhibited optimal activity at 52 °C and pH 9.5. There was a > 3-fold increase in activity in the presence of ß-mercaptoethanol. Apparent Vmax and Km values were 2735 U/mg and 0.35 mM, respectively. ASNaseGt displayed high thermostability with >80 % residual activity even after 6 h of incubation at 55 °C. Recombinant ASNaseGt existed in oligomeric form. Addition of ß-mercaptoethanol lowered the degree of oligomerization and displayed that tetrameric form was the most active, with a specific activity of 4300 U/mg. Under physiological conditions, ASNaseGt displayed >50 % of the optimal activity. Localization studies in G. thermopakistaniensis revealed that ASNaseGt is a cytosolic protein. Structural and functional characterization, and localization in G. thermopakistaniensis displayed that ASNaseGt is not a type II but a type I L-asparaginase.
Subject(s)
Asparaginase , Geobacillus , Asparaginase/chemistry , Geobacillus/genetics , Geobacillus/metabolism , Mercaptoethanol , Recombinant Proteins/genetics , Enzyme StabilityABSTRACT
L-asparaginase from Escherichia coli (EcA) has been used for the treatment of acute lymphoid leukemia (ALL) since the 1970s. Nevertheless, the enzyme has a second specificity that results in glutaminase breakdown, resulting in depletion from the patient's body, causing severe adverse effects. Despite the huge interest in the use of this enzyme, the exact process of glutamine depletion is still unknown and there is no consensus regarding L-asparagine hydrolysis. Here, we investigate the role of T12, Y25, and T89 in asparaginase and glutaminase activities. We obtained individual clones containing mutations in the T12, Y25 or T89 residues. After the recombinant production of wild-type and mutated EcA, The purified samples were subjected to structural analysis using Nano Differential Scanning Fluorimetry, which revealed that all samples contained thermostable molecules in their active structural conformation, the homotetramer conformation. The quaternary conformation was confirmed by DLS and SEC. The activity enzymatic assay combined with molecular dynamics simulation identified the contribution of T12, Y25, and T89 residues in EcA glutaminase and asparaginase activities. Our results mapped the enzymatic behavior paving the way for the designing of improved EcA enzymes, which is important in the treatment of ALL.
Subject(s)
Asparaginase , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Humans , Asparaginase/genetics , Asparaginase/therapeutic use , Asparaginase/chemistry , Glutaminase/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Asparagine/chemistry , Molecular Dynamics Simulation , Escherichia coli/metabolismABSTRACT
L-asparaginase having low glutaminase activity is important in clinical and food applications. Herein, glutaminase-free L-asparaginase (type I) coding genes from Pseudomonas sp. PCH182 (Ps-ASNase I) and Rahnella sp. PCH162 (Rs-ASNase I) was amplified using gene-specific primers, cloned into a pET-47b(+) vector, and plasmids were transformed into Escherichia coli (E. coli). Further, affinity chromatography purified recombinant proteins to homogeneity with monomer sizes of ~37.0 kDa. Purified Ps-ASNase I and Rs-ASNase I were active at wide pHs and temperatures with optimum activity at 50 °C (492 ± 5 U/mg) and 37 °C (308 ± 4 U/mg), respectively. Kinetic constant Km and Vmax for L-asparagine (Asn) were 2.7 ± 0.06 mM and 526.31 ± 4.0 U/mg for Ps-ASNase I, and 4.43 ± 1.06 mM and 434.78 ± 4.0 U/mg for Rs-ASNase I. Circular dichroism study revealed 29.3 % and 24.12 % α-helix structures in Ps-ASNase I and Rs-ASNase I, respectively. Upon their evaluation to mitigate acrylamide formation, 43 % and 34 % acrylamide (AA) reduction were achieved after pre-treatment of raw potato slices, consistent with 65 % and 59 % Asn reduction for Ps-ASNase I and Rs-ASNase I, respectively. Current findings suggested the potential of less explored intracellular L-asparaginase in AA mitigation for food safety.
Subject(s)
Antineoplastic Agents , Rahnella , Asparaginase/chemistry , Rahnella/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Pseudomonas/genetics , Pseudomonas/metabolism , Glutaminase/genetics , Acrylamide , Asparagine/metabolismABSTRACT
A novel ternary mixture of inexpensive and nutrient-rich agro-substrates comprising groundnut de-oiled cake, corn gluten meal, and soybean meal has been explored to enhance the L-asparaginase production in solid-state fermentation. To achieve the aim, a hybrid strategy was implemented by utilizing a combination of a mixture design and artificial neural networks. The study initiated with the judicious selection of the agro-substrates based on their low C/N content in comparison to the control using the CHNS elemental analysis. The mixture composition of soybean meal (49.0%), groundnut de-oiled cake (31.5%), and corn gluten meal (19.5%) were found optimum using the simplex lattice mixture design. The agro-industrial substrates mix revealed synergistic effects on the L-asparaginase production than either of the substrates alone. The maximum L-asparaginase activity of 141.45 ± 5.24 IU/gds was observed under the physical process conditions of 70% moisture content, autoclaving period of 30 min and 6.0 pH by adopting the machine learning-derived artificial neural network (ANN) methodology. The ANN modeling showed excellent prediction ability with a low mean squared error of 0.7, a low root mean squared error of 0.84, and a high value of 0.99 for regression coefficient. Moisture content (%) was assessed to be the most sensitive process parameter in the global sensitivity analysis. The net outcome from the two sequential optimization designs is the selection of the ideal mixture composition followed by the optimum physical process parameters. The application of the enzyme demonstrated significant cytotoxicity against leukemia cell line and therefore exhibited an anti-cancer effect. The present study reports a novel mixture combination and methodology that can be used to lower the cost and enhance the production of L-asparaginase using an agro-industrial substrate mixture.
Subject(s)
Asparaginase , Industrial Waste , Asparaginase/chemistry , Asparaginase/metabolism , Fermentation , Neural Networks, Computer , Glutens/metabolismABSTRACT
Asparaginase holds significant commercial value as an enzyme in the food and pharmaceutical industries. This study examined the optimum and practical use of the l-asparaginase derived from Pseudomonas aeruginosa HR03. Specifically, the study focused on the effectiveness of the stabilized enzyme when applied to chitosan nanoparticles. The structure, size, and morphology of chitosan nanoparticles were evaluated in relation to the immobilization procedure. This assessment involved the use of several analytical techniques, including FT-IR, DLS, SEM, TEM, and EDS analysis. Subsequently, the durability of the enzyme that has been stabilized was assessed by evaluating its effectiveness under extreme temperatures of 60 and 70 °C, as well as at pH values of 3 and 12. The findings indicate that incorporating chitosan nanoparticles led to enhanced immobilization of the l-asparaginase enzyme. This improvement was observed in terms of long-term stability, stability under crucial temperature and pH conditions, as well as thermal stability. In addition, the optimum temperature increased from 40 to 50 °C, and the optimum pH increased from 8 to 9. Enzyme immobilization led to an increase in Km and a decrease in kcat compared to its free counterpart. Because of its enhanced long-term stability, l-asparaginase immobilization on chitosan nanoparticles may be a potential choice for use in industries that rely on l-asparaginase enzymes, particularly the pharmaceutical and food industries.
Subject(s)
Chitosan , Nanoparticles , Asparaginase/chemistry , Enzyme Stability , Chitosan/chemistry , Spectroscopy, Fourier Transform Infrared , Hydrogen-Ion Concentration , Enzymes, Immobilized/chemistry , Nanoparticles/chemistry , TemperatureABSTRACT
BACKGROUND: L-asparaginase (ASNase) has played a key role in the management of acute lymphoblastic leukaemia (ALL). As an amidohydrolase, it catalyzes the hydrolysis of L-asparagine, a crucial step in the treatment of ALL. Various ASNase variants have evolved from diverse sources since it was first used in paediatric patients in the 1960s. This review describes the available ASNase and approaches being used to develop ASNase as a biobetter candidate. SCOPE OF REVIEW: The review discusses the Glycosylation and PEGylation techniques, which are frequently used to develop biobetter versions of the majority of the therapeutic proteins. Further, it explores current ASNase biobetters in therapeutic use and discusses the protein engineering and chemical modification approaches that were employed to reduce immunogenicity, extend protein half-life, and enhance protease stability of ASNase. Emerging strategies like immobilization and encapsulation are also highlighted as potential pathways for improving ASNase properties. MAJOR CONCLUSIONS: The purpose of the development of ASNase biobetter is to achieve a novel therapeutic candidate that could improve catalytic efficiency, in vivo stability with minimum glutaminase (GLNase) activity and toxicity. Modification of ASNase by immobilization and encapsulation or by fusion technologies like Albumin fusion, Fc fusion, ELP fusion, XTEN fusion, etc. can be exploited to develop a novel biobetter candidate suitable for therapeutic approaches. GENERAL SIGNIFICANCE: This review emphasizes the importance of biobetter development for therapeutic proteins like ASNase. Improved ASNase molecules have the potential to significantly advance the treatment of ALL and have broader implications in the pharmaceutical industry.
Subject(s)
Antineoplastic Agents , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Humans , Child , Asparaginase/genetics , Asparaginase/therapeutic use , Asparaginase/chemistry , Antineoplastic Agents/chemistry , Asparagine , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Glutamine/metabolismABSTRACT
l-asparaginase (ASNase), an enzyme that catalyzes the hydrolysis of l-asparagine into l-aspartic acid, is frequently used as a medication for acute lymphoblastic leukemia (ALL). However, when derived from bacterial sources, this enzyme can elicit side effects, including allergic or hypersensitivity reactions, owing to immune responses. Here, we describe the synthesis of polyoxazoline-conjugated ASNase (POx-ASNase) and investigate its enzyme activity, anticancer efficacy, immunogenicity, and retention in the bloodstream. The water-soluble POx was coupled with surface lysine residues of ASNase using a bifunctional cross-linker. The average number of polymers bound to each enzyme was determined as 10. Although the enzymatic activity of POx-ASNase decreased to 56% of that of native ASNase, its temperature and pH dependencies remained unaltered. Remarkably, the lyophilized powder form of POx-ASNase retained its catalytic ability for 24 months. POx-ASNase demonstrated nearly identical anticancer efficacy compared to naked ASNase against leukemia and lymphoma cells (MOLT-4, CLBL-1, and K562) while displaying no cytotoxicity toward normal cells. Animal experiments conducted using rats revealed that the POx decoration suppressed the generation of anti-ASNase IgM and IgG antibodies with no detection of anti-POx antibodies. The half-life within the bloodstream extended to 34 h, representing a 17-fold increase compared to unmodified ASNase. These findings suggest that POx-ASNase serves as an anticancer therapeutic agent, characterized by the absence of antibody production and notably extended circulation persistence.
Subject(s)
Antineoplastic Agents , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Animals , Rats , Asparaginase/therapeutic use , Asparaginase/chemistry , Antibody Formation , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Antineoplastic Agents/therapeutic use , Asparagine/metabolism , Asparagine/therapeutic useABSTRACT
<b>Background and Objective:</b> Type 2 L-asparaginase enzyme can be used as a cancer therapy agent and prevent acrylamide formation in food products. Enzymes produced by thermohalophilic bacteria can provide high activity at high temperatures so they are needed on an industrial scale. Hence, this study aims to determine the characteristics of the gene encoding type 2 L-asparaginase enzyme in the thermohalophilic bacterial isolate CAT3.4. <b>Materials and Methods:</b> This research is a type of exploratory research. The characteristics of the gene encoding type 2 L-asparaginase were determined using the PCR technique using the primer pairs AsnBac2-F2 (5'-CTCACGGGAATCTCCATAACTC-3') and AsnBac2-R2 (5'CAGCGATGTAACAGACAGCATC-3'). The characterization process was carried out in stages: Isolation of genomic DNA using a modified alkali-lysis method, nucleotide and protein similarity analysis using BLASTn analysis on the NCBI website, construction of a phylogenetic tree using the MEGAX program, restriction enzyme mapping and amino acid analysis using the Bioedit program. <b>Results:</b> The characterization results showed that the PCR product has a size of 1594 bp with a CDS of 1128 bp, has a similarity value of 100% with <i>Bacillus subtilis</i>, has seven restriction enzymes as molecular markers for the type 2 L-asparaginase gene at the species level: <i>Bsr</i>GI, <i>Dra</i>I, <i>Eco</i>RV, <i>Hind</i>III, <i>Hpy</i>CH4IV , <i>Ssp</i>I and <i>Tai</i>I, have dominant hydrophilic regions and are in the same subclass as <i>Bacillus subtilis</i> strain GOT9. <b>Conclusion:</b> The target gene was similar to the gene encoding type 2 L-asparaginase from <i>Bacillus subtilis</i> with a max identity of 98.85%, query coverage value of 100% and E-value of 0.
Subject(s)
Asparaginase , Hot Springs , Asparaginase/genetics , Asparaginase/chemistry , Asparaginase/metabolism , Indonesia , Phylogeny , Bacillus subtilis/geneticsABSTRACT
The present study reports the structural and functional characterization of a new glutaminase-free recombinant L-asparaginase (PrASNase) from Pseudomonas resinovorans IGS-131. PrASNase showed substrate specificity to L-asparagine, and its kinetic parameters, Km, Vmax, and kcat were 9.49 × 10-3 M, 25.13 IUmL-1 min-1, and 3.01 × 103 s-1, respectively. The CD spectra showed that PrASNase consisted of 18.5 % helix, 21.5 % antiparallel sheets, 4.2 % parallel sheets, 14 % turns, and rest other structures. FTIR was used for the functional characterization, and molecular docking predicted that the substrate interacts with serine, alanine, and glutamine in the binding pocket of PrASNase. Differing from known asparaginases, structural characterization by small-angle X-ray scattering (SAXS) and analytical ultracentrifugation (AUC) unambiguously revealed PrASNase to exist as a monomer in solution at low temperatures and oligomerized to a higher state with temperature rise. Through SAXS studies and enzyme assay, PrASNase was found to be mostly monomer and catalytically active at 37 °C. Furthermore, this glutaminase-free PrASNase showed killing effects against WIL2-S and TF-1.28 cells with IC50 of 7.4 µg.mL-1 and 5.6 µg.mL-1, respectively. This is probably the first report with significant findings of fully active L-asparaginase in monomeric form using SAXS and AUC and demonstrated the potential of PrASNase in inhibiting cancerous cells, making it a potential therapeutic candidate.
Subject(s)
Asparaginase , Asparagine , Asparaginase/chemistry , Molecular Docking Simulation , Scattering, Small Angle , X-Ray Diffraction , Asparagine/chemistryABSTRACT
The genome of Rhizobium etli, a nitrogen-fixing bacterial symbiont of legume plants, encodes two L-asparaginases, ReAIV and ReAV, that have no similarity to the well characterized enzymes of class 1 (bacterial type) and class 2 (plant type). It has been hypothesized that ReAIV and ReAV might belong to the same structural class 3 despite their low level of sequence identity. When the crystal structure of the inducible and thermolabile protein ReAV was solved, this hypothesis gained a stronger footing because the key residues of ReAV are also present in the sequence of the constitutive and thermostable ReAIV protein. High-resolution crystal structures of ReAIV now confirm that it is a class 3 L-asparaginase that is structurally similar to ReAV but with important differences. The most striking differences concern the peculiar hydration patterns of the two proteins, the presence of three internal cavities in ReAIV and the behavior of the zinc-binding site. ReAIV has a high pH optimum (9-11) and a substrate affinity of â¼1.3â mM at pH 9.0. These parameters are not suitable for the direct application of ReAIV as an antileukemic drug, although its thermal stability and lack of glutaminase activity would be of considerable advantage. The five crystal structures of ReAIV presented in this work allow a possible enzymatic scenario to be postulated in which the zinc ion coordinated in the active site is a dispensable element. The catalytic nucleophile seems to be Ser47, which is part of two Ser-Lys tandems in the active site. The structures of ReAIV presented here may provide a basis for future enzyme-engineering experiments to improve the kinetic parameters for medicinal applications.