Pool Of Asparagine Research Articles

Development of a Novel L-Asparaginase with Enhanced Properties Using Ancestral Sequence Reconstruction

The overall survival of children with acute lymphoblastic leukemia (ALL) now exceeds 90% due to the precise disease risk stratification and intensification of multi-agent chemotherapy regimens. L-asparaginase (L-ASNase) is an indispensable biotherapeutic enzyme used in treatment of pediatric ALL. Discontinuation of L-ASNase treatment due to immunological or adverse reactions leads to significantly inferior disease-free survival (73% ±7% vs 92% ± 2%; P <0.01, Gupta et al. JCO 2020). Clinical L-ASNases are bacterial in origin, derived from either Escherichia coli or Erwinia chrysanthemi and are therefore highly immunogenic. Additionally, emerging data suggests that the concurrent glutaminase activity seen in bacterial asparaginases exacerbates its liver and pancreatic toxicity, especially in adults thereby limiting its extensive use beyond children. Therefore, finding alternative sources with preferable biochemical features remains paramount. The inferior catalytic properties of human L-ASNase restricts its functionality in ALL treatment. However, characterization has shown that guinea pig (GP) L-ASNase demonstrates significant anti-tumor properties and improved enzyme kinetics despite a 69.8% amino acid sequence identity with human L-ASNase, as compared to 30% for bacterial L-ASNases. Additionally, guinea pig L-ASNase has no glutaminase activity. Thus, to overcome the major deficiencies of bacterial L-ASNase and develop a more humanized form of L-ASNase we performed ancestral sequence reconstruction (ASR). ASR is a protein drug discovery / optimization platform that predicts ancient DNA and protein sequences from extant sequences. This approach permits higher-resolution mapping potentially highlighting functional resides. The objective therefore is to exploit functional diversity refined by natural selection using ASR to identify therapeutic variants with superior properties. Recent work on guinea pig (GP) L-ASNase (Schalk et al. J Biol Chem 2014) identified a low micromolar (μM) Michaelis constant (Km) at 57.7 ± 6.4 μM essential for hydrolyzing the extra-cellular pool of asparagine ~50 μM in human blood; thus, equipping us with the ideal mammalian L-ASNase ortholog to serve as a template to resurrect ancient ASNase sequences. Ancestral L-ASNase sequences were generated utilizing 54 extant L- asparaginase sequences, aligned using MUSCLE and an evolutionary tree inferred using MrBayes. A total of 53 ancestral nodes were identified, and the ten ancestral L-ASNase variants spanning the ancient primate and guinea pig (GP) lineage were resurrected. The sequence identity of these ancestral L-ASNase variants ranged from 81 to 98% when compared to human L-ASNase. E. coli codon optimized complementary DNA (cDNA) sequences were synthesized and subcloned intoan expression vector. Correctly cloned plasmids were transformed into E. coli BL21 (DE3) cells for protein expression. An-ASNase candidates were isolated by Ni 2+ affinity chromatography and then further purified by size exclusion chromatography. Asparaginase activity of the An-ASNase candidates were determined via a modified Nessler's reagent assay by using a continuous spectroscopic enzyme-coupled assay. Three of the ancestral L-ASNase candidates, An-88, An-104 and An-107 demonstrated excellent asparaginase activity when tested at an enzyme concentration of 0.1 mg/mL and an excess substrate concentration, with activity within a comparable range to the clinically relevant E. coli and Erwinia asparaginases. An-88 had 81% similarity whereas both An-104 and An-107 ASNases had 88% identity to human L-ASNase, which is in stark contrast to the only 30% similarity seen with bacterial L-ASNases. Detailed enzymatic characterization of these three lead drug candidates is currently being performed. Preliminary cytotoxicity data testing the ancestral L-ASNase candidates against a T-cell ALL cell line CCRF-CEM showed an expected dose response, with An-107 showing the highest cytotoxicity. Our findings demonstrate that ASR is an efficient protein drug design/optimization platform that we have now extended beyond our previous work with recombinant coagulation factors (Zakas et al. Nat Biotech 2017, Knight et al. Blood Adv 2021) to therapeutic enzymes; thereby, enabling the creation of novel recombinant L-ASNases with translational implications due to improved enzymatic specificity and reduced toxicity.

BSH Guideline: management of thrombotic and haemostatic issues in paediatric malignancy.

BSH Guideline: management of thrombotic and haemostatic issues in paediatric malignancy.

Adverse Hepatobiliary Effects of Pegaspargase: Diagnostic Features and Potential Treatment

Introduction: Pegaspargase is frequently used to treat patients with B-cell acute lymphoblastic leukemia (B-ALL). Hepatotoxicity is a potential adverse effect. We describe 3 cases of suspected pegaspargase-associated hepatotoxicity and effects of treatment.Figure: Liver biopsy histology demonstrating features of cholestatic hepatitis, including cholestasis (arrow) and feathery degeneration of hepatocytes (arrowhead) in a background of bile ductular proliferation, acute cholangitis, and microvesicular and macrovesicular steatosis (H&E, 200X).Case Description: Case 1: A 45-year-old man with relapsing B-ALL was treated with chemotherapy including pegaspargase. ALT rose from 154 to 626 U/L and total bilirubin (TB) from 0.9 to 21.6 mg/dL, despite ursodiol. Right upper quadrant abdominal ultrasound showed interval hepatic steatosis, compared to CT abdomen 3 months prior. Liver biopsy showed marked steatosis, cholestasis, and hepatocellular degeneration without fibrosis. After 1 month of daily oral vitamin B complex, ALT improved to 73 with TB of 1.1 mg/dL. Case 2: A 41-year-old man with relapsing B-ALL and hepatic graft-versus-host-disease (managed with steroids), was treated with chemotherapy including pegaspargase and methotrexate. ALT rose from 52 to 163 U/L, and TB rose from 0.6 to 23.5 mg/dL despite ursodiol. After 6 days of intravenous (IV) levocarnitine and 1.5 months of oral vitamin B complex, ALT improved to 45 U/L and TB to 2.1 mg/dL. Case 3: A 24-year-old woman with pre-B-ALL who previously underwent stem cell transplants was evaluated for relapse. She was treated with chemotherapy including pegaspargase. Total parenteral nutrition (TPN) was provided for nutrition. After pegaspargase treatment, ALT rose from 26 to 108 U/L, and TB rose from 0.4 to 13.1 mg/dL despite ursodiol. Abdominal ultrasound demonstrated hepatomegaly. TB improved to 4.3 mg/dL after a week of IV levocarnitine and oral vitamin B complex. Persistent elevation in ALT was attributed to effects of other pharmacologic agents and possibly to TPN. Discussion: Pegaspargase is a form of L-asparaginase used in treatment of B-ALL. Although rarely fatal, pegaspargase may cause hepatic impairment. Via depletion of asparagine pools, it impairs mitochondrial oxidation of fatty acids. Microvesicular and macrovesicular steatosis may result. Exclusion of other hepatotoxic agents is important, as other pharmacologic agents may also contribute to abnormal liver enzymes. Through replenishing substrates important to mitochondrial metabolism, treatment with levocarnitine and vitamin B complex has demonstrated efficacy in case reports. No standard treatment regimen has been defined. Empiric treatment may be beneficial in complex cases if clinical suspicion for the diagnosis is high.

Fresh frozen plasma contains free asparagine and may replace the plasma asparagine pool during L-asparaginase therapy

Fresh frozen plasma contains free asparagine and may replace the plasma asparagine pool during L -asparaginase therapy

Gene expression associated with N‐induced shifts in resource allocation in poplar

ABSTRACTSurprisingly little is known about molecular mechanisms by which nitrogen (N) availability acts to modulate the growth of forest trees. To address this issue, differential display was used in conjunction with filter‐based arrays to identify 52 partial cDNA clones that were significantly regulated within days in response to limiting or luxuriant levels of NH4NO3 fertilization in Populus trichocarpa Torr. & Gray × deltoides Bartr. ex Marsh. A subset of these cDNAs also demonstrated shifts in expression patterns in stem‐girdled trees, a manipulative physiology technique that disrupts phloem transport. Stem girdling also induced changes in glutamine and asparagine pools which were correlated with the observed changes in expression profiles for these genes. The identity of these genes provides insight into biochemical processes that are altered by N availability in poplar. Carbon–nitrogen interactions appear to figure prominently in the N‐response. The gene expression data suggest that N availability modulates the partitioning of C and N resources into metabolic fates that have the potential to alter both wood quality and quantity, including synthesis of vegetative storage proteins, cell wall components, and terpenoids.

Glomus intraradices causes differential changes in amino acid and starch concentrations of in vitro strawberry subjected to water stress.

The effect of colonization of tissue-cultured strawberry (Fragaria×ananassa Duch. cv. Kent) plantlets in vitro by the arbuscular mycorrhizal fungus (AMF) Glomus intraradices on plantlet response to poly(ethylene glycol) (PEG)-8000-induced water stress was investigated. The plantlets were inoculated axenically and co-cultured with the AMF for 4 wk, then transferred to 15% PEG-8000 solutions for 4, 8 and 12 h. Relative water content, water potential, osmotic potential, leaf conductance for water vapour diffusion and photosynthetic efficiency as estimated by chlorophyll a fluorescence were all affected by the PEG treatment and its duration but not by the presence of the intraradical phase of the AMF. However, distinct differences in PEG-induced changes in amino acid content were observed between nonmycorrhizal and mycorrhizal plantlets. In the latter, the treatment with PEG caused a substantial decrease in asparagine levels in leaves that was accompanied by a marked increase in asparagine concentration in roots. The opposite was observed in nonmycorrhizal plantlets. Furthermore, concentrations of aspartic acid, serine, threonine, amino-N-butyric acid, alanine and starch increased in roots of mycorrhizal and decreased in nonmycorrhizal plantlets. Our results suggest the presence of a mobile pool of asparagine that can be translocated from leaves to roots or vice versa in response to PEG-induced water stress, depending on the mycorrhizal status of the plantlets. These opposite patterns suggest different strategies of mycorrhizal and nonmycorrhizal plantlets to water stress, which seem to involve different adjustments in nitrogen and carbon metabolism.

Regulation of ammonium and potassium uptake by glutamine and asparagine in Pisum arvense plants

Pisum arvense plants were subjected to 5 days of nitrogen deprivation. Then, in the conditions that increased or decreased the root glutamine and asparagine pools, the uptake rates of 0.5 mM NH4 + and 0.5 mM K+ were examined. The plants supplied with 1 mM glutamine or asparagine took up ammonium and potassium at rates lower than those for the control plants. The uptake rates of NH4 + and K+ were not affected by 1 mM glutamate. When the plants were pre-treated with 100 µM methionine sulphoximine, an inhibitor of glutamine synthesis, the efflux of NH4 + from roots to ambient solution was enhanced. On the other hand, exposure of plants to methionine sulphoximine led to an increase in potassium uptake rate. The addition of asparagine, glutamine or glutamate into the incubation medium caused a decline in the rate of NH4 + uptake by plasma membrane vesicles isolated from roots of Pisum arvense, whereas on addition of methionine sulphoximine increased ammonium uptake. The results indicate that both NH4 + and K+ uptake appear to be similarly affected by glutamine and asparagine status in root cells.

Structure and function of the ectomycorrhizal association between Paxillus involutus and Betula pendula. II. Metabolic changes during mycorrhiza formation

Seedlings of Betula pendula Roth. were grown in the presence of Paxillus involutus (Batsch) Fr., and metabolic changes during mycorrhiza formation were examined by measuring organic acid and amino acid pools and related enzyme activities, following sequential harvests. Glutamine, aspartate and asparagine pools were always lower in infected roots than in non‐infected roots, especially during Hartig net initiation and formation. Glutamate concentration was similar in both tissues. Citrate and malate were the two major organic acids detected and their concentrations were equal in infected and non‐infected roots. Aspartate aminotransferase, glutamine synthetase, NAD‐dependent malate dehydrogenase and glucose‐6‐phosphate dehydrogenase activities were higher in infected roots than non‐infected roots. For all enzymes revealed on polyacrylamide gels, both root and fungal isoforms were present in infected roots. Quantitative changes in enzyme capacities and metabolite pools indicated that mycorrhiza formation caused a re‐arrangement of the main metabolic pathways during the very early stages following contact, which might be related to the structural changes.

Rapid assimilation of recently fixed N2 in root nodules of Myrica gale

In Myrica gale L. plants the assimilation of ammonia released by symbiotic Frankia was observed by 15N2 labelling and subsequent analysis of the isotopic enrichment of nodule amino acids over time by single ion monitoring gas chromatography‐mass spectrometry. In detached nodules of Myrica, glutamine was the first amino acid labelled at 30 s and subsequently the amino acids glutamate, aspartate, alanine and γ‐amino butyric acid (GABA) became labelled. This pattern of labelling is consistent with the incorporation of ammonium via glutamine synthetase [GS; EC 6.3.1.2]. No evidence for the ammonium assimilation via glutamate dehydrogenase [GDH; EC 1.4.1.2] was observed as glutamate became labelled only after glutamine. Using attached nodules and pulse‐chase labelling, we observed synthesis of glutamine, glutamate, aspartate, alanine, GABA and asparagine, and followed the transport of fixed nitrogen in the xylem largely as glutamine and asparagine. Estimation of the cost of nitrogen fixation and asparagine synthesis in Myrica nodules suggests a minimum of one sucrose required per asparagine produced. Rapid translocation of recently fixed nitrogen was observed in Myrica gale nodules as 80% of the nitrogen fixed during a 1‐h period was translocated out of the nodules within 9 h. The large pool of asparagine that is present in nodules may buffer the transport of nitrogen and thus act to regulate nitrogen fixation via a feedback mechanism.

Changes in coagulation and fibrinolysis in childhood ALL: a two‐step dose reduction of one E. coli asparaginase preparation

The influence of two different E. coli asparaginase (ASP) preparations on fibrinolytic proteins in childhood ALL was recently reported, demonstrating a clearly significant association between ASP activity and haemostatic changes. Since the Bayer preparation is no longer available for treatment of large series of patients with ALL, the present study was designed to prospectively evaluate coagulation and fibrinolytic changes in leukaemic children receiving different doses of Medac ASP, which is now available for treatment of childhood ALL. Leukaemic children in whom ASP Medac was administered at 3 d intervals in a two-step dose reduction (5000 IU/m2, n = 10; 2500 IU/m2, n = 15) were compared with children who had received Bayer ASP 10,000 IU/m2 in the same time schedule in a former randomized trial; at the same venipuncture, blood samples for coagulation studies were obtained before each ASP administration together with serum samples for pharmacokinetic monitoring. Compared with Bayer ASP 10,000 IU/m2, patients receiving Medac ASP 5000 IU/m2 showed significantly decreased values of fibrinogen, plasminogen, and alpha 2-antiplasmin, along with significantly enhanced thrombin generation. Improvement occurred in children treated with 2500 IU/m2 Medac ASP; alpha 2-antiplasmin and D-dimer no longer differed from the Bayer group. Since both patient groups showed complete asparagine depletion during the course of ASP administration, the lower dosage of 2500 IU/m2 administered at 3 d intervals should guarantee the specific metabolic therapy for ALL, leading to depletion of the circulating pool of asparagine.

Analysis of aspartic acid and asparagine metabolism in Xenopus laevis oocytes using a simple and sensitive HPLC method

The amino acids in methanol-soluble extracts of Xenopus oocytes were measured using a method involving precolumn derivatization with phenylisothiocyanate and reverse phase HPLC of the derivatized amino acids. This technique allows the estimation of asparagine and glutamine pools in oocytes, estimated as 70 and 283 pmoles per oocyte, respectively. The pool sizes of the other amino acids were similar to previously reported results obtained using conventional ion exchange chromatography and postcolumn derivatization with ninhydrin. The advantages of the method developed here include picomolar sensitivity and the enhanced resolution of asparagine and glutamine from other amino acids. The kinetics of aspartic acid and asparagine utilization were monitored following microinjection of oocytes with [3H]aspartic acid and [14C]asparagine. The aspartic acid pool turned over rapidly with a half-time of < 30 min. The asparagine pool was metabolized much more slowly and appeared to be utilized almost completely for protein synthesis. The absolute rate of protein synthesis in oocytes was calculated from the incorporation data and chemical pool measurements as approximately 25 ng/hr-oocyte. The methodology developed here may be useful in experimental situations involving limited amounts of biological material.

Intracellular asparagine pool as a factor in control of ammonium uptake by Saccharomyces cerevisiae

During growth of Saccharomyces cerevisiae in defined media containing 1 m m ammonium and a single amino acid at 2 m m , asparagine, and to a lesser extent aspartic acid and glutamine, significantly delayed ammonium uptake. Addition of asparagine or glutamine to the medium during growth on ammonium caused an immediate cessation of ammonium uptake. Measurement of the pool sizes indicated that ammonium uptake responded to the internal concentration of asparagine rather than that of glutamine and, because of the speed of the response the most likely mechanism was inhibition of the ammonium permease system.

Amino Acid Metabolism of Lemna minor L.

A serious limitation to the use of N(O,S)-heptafluorobutyryl isobutyl amino acid derivatives in the analysis of (15)N-labeling kinetics of amino acids in plant tissues, is that the amides glutamine and asparagine undergo acid hydrolysis to glutamate and aspartate, respectively, during derivatization. This led us to consider an alternative procedure (G Fortier et al. [1986] J Chromatogr 361: 253-261) for derivatization of glutamine and asparagine with N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide in pyridine. Gas chromatography-mass spectrometry (electron ionization) yielded fragment ions (M-57) of mass 417 and 431 for the [(14)N]asparagine and [(14)N]glutamine derivatives, respectively, suitable for monitoring unlabeled, single-(15)N- and double-(15)N-labeled amide species from the ion clusters at mass to charge ratio (m/z) 415 to 423 for asparagine, and m/z 429 to 437 for glutamine. From separate analyses of the specific isotope abundance of the amino-N groups of asparagine and glutamine as their N-heptafluorobutyryl isobutyl derivatives, the specific amide-[(15)N] abundance of these amino acids was determined. We demonstrate that this approach to (15)N analysis of the amides can yield unique insights as to the compartmentation of asparagine and glutamine in vivo. The ratios of unlabeled:single-(15)N:double-(15)N-labeled species are highly diagnostic of the relative sizes and turnover of metabolically active and inactive pools of the amides and their precursors. Kinetic evidence is presented to indicate that a significant proportion (approximately 10%) of the free asparagine pool may be metabolically inactive (vacuolar). If the amide group of asparagine is derived exclusively from glutamine-amide, then asparagine must be synthesized in a compartment of the cell in which both glutamine-amide and aspartate are more heavily labeled with (15)N than the bulk pools of these amino acids. This compartment is presumably the chloroplast. The transaminase inhibitor aminooxyacetate is shown to markedly inhibit amino acid synthesis; several amino acid pools accumulated in the presence of aminooxyacetate and [(15)N]H(4) (+) are (14)N-enriched and must be derived primarily from protein turnover.

Characteristics of Asparagine Pool in Soybean Nodules in Comparison with Ureide Pool

The changes in the content of nitrogenous compound in intact soy bean nodules were investigated by inhibiting nitrogen fixation with acetylene application. The contents of ureides (allantoin and allantoic acid), asparagine, and glutamine decreased by the acetylene treatment. Incorporation of 15N into asparagine, ureides, and other nitrogenous compounds in intact soybean nodules was also investigated by the long term feeding of gaseous 15N2. Approximately 68% nitrogen in the ureide pool and 35% nitrogen in the asparagine pool were derived from gaseous 15N2 at 5.5 h after 15N2 exposure. The flow rate of newly fixed nitrogen via the asparagine pool was from 17 to 22% of that via the ureide pool, which was estimated based on the data of the acetylene inhibition experiment and 15N tracer experiment. A larger quantity of UN was incorporated into the amido-N of asparagine than into its amino-N, and 15N incorporation into ammonia was almost similar to that into asparagine at 5.5 h after 15N2 exposure. The charact...

Galactose, aspartate crossway and rhythm inhibition in Aspergillus niger

AbstractWhen the glucose/K+ balance in the medium is favourable to rhythm expression in Aspergillus niger Van Tieghem, galactose has an inhibitory effect on amplitude but not on period length of the rhythm. Galactose is active when its level reaches 1/10 of that of glucose.On liquid media a small quantity of glucose is necessary to start growth on galactose. Under these conditions U14C‐galactose is rather slowly metabolized. After 6 h of feeding on the labelled galactose medium, this sugar is converted into glucose, which is used both for the synthesis of compounds derived from C3 and C4 units and for synthesis of polysaccharides and perhaps small peptides. The labelling of the macromolecules always remains low. The insoluble carbohydrates of the mycelium are little affected by the type of sugar supplied to the fungus.The metabolism on galactose differs from the metabolism on glucose mainly in a decrease of the free asparagine pool and a simultaneous equivalent increase of the free aspartate pool; such an effect could not be correlated with an increase of the aspartate aminotransferase activity. Supply of aspartate but not of gluta‐mate into the agar medium inhibits the rhythm amplitude. So, the damping effect of galactose on the rhythm might be at least partly due to its effect on the regulation of the aspartate cross‐way.

Synthesis and activation of asparagine in asparagine auxotrophs of Saccharomyces cerevisiae.

L-Asparagine synthesis in Saccharomyces cerevisiae is performed by a glutamine-dependent asparagine synthetase of the type found in higher organisms. Auxotrophy for asparagine has been obtained in two classes of mutants. In class I, asparagine synthetase activity is cancelled. These mutants combine two mutations, asnA- and asnB-. Neither asnA- nor asnB- mutation alone leads to total auxotrophy. Partial auxotrophy as well as a strong decrease in enzyme activity result from asnA- mutation. No change is detectable in cells with the asnB- mutationalone. This, and Jones' report [J. Bacteriol. 134, 200-207 (1978)] of auxotrophy resulting from the combination of two mutations, are strong supports for asparagine synthesis being an unusual biosynthetic operation. In class II, auxotrophy results from a single mutation which leads to a modification of the efficiency of the asparaginyl-tRNA synthetase (asnRS- mutation). This auxotrophy is cancelled if asparaginase I activity (the only one present in sigma 1278b wild type) is cancelled by casnI- mutation. This latter mutation allows an increase in the asparagine pool which is able to compensate for the asparaginyl-tRNA synthetase partial defect of the asnRS- mutant.