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The interplay between the sex-specific differences in tumor metabolome and colorectal cancer (CRC) prognosis has never been studied and represents an opportunity to improve patient outcomes. This study examines the link between tumor metabolome and prognosis by sex for CRC patients. Using untargeted metabolomics analysis, abundances of 91 metabolites were obtained from primary tumor tissues from 197 patients (N = 95 females, N = 102 males) after surgical colectomy for stage I-III CRC. Cox Proportional hazard (PH) regression models estimated the associations between tumor metabolome and 5-year overall survival (OS) and recurrence-free survival (RFS), and their interactions with sex. Eleven metabolites had significant sex differences in their associations with 5-year OS, and five metabolites for 5-year RFS. The metabolites asparagine and serine had sex interactions for both OS and RFS. Furthermore, in the asparagine synthetase (ASNS)-catalyzed asparagine synthesis pathway, asparagine was associated with substantially poorer OS (HR (95% CI): 6.39 (1.78–22.91)) and RFS (HR (95% CI): 4.36 (1.39–13.68)) for female patients only. Similar prognostic disadvantages in females were seen in lysophospholipid and polyamine synthesis. Unique metabolite profiles indicated that increased asparagine synthesis was associated with poorer prognosis for females only, providing insight into precision medicine for CRC treatment stratified by sex.

Asparagine biosynthesis and breakdown are tightly regulated in mammalian cells. Recent studies indicate that asparagine supply could be a limiting factor for the replication of some viruses such as vaccinia virus and human cytomegalovirus. In this Viewpoint, we highlight the importance of asparagine metabolism during virus replication and rationalize that asparagine metabolism could be a viable target for broad-spectrum antiviral development. To achieve this goal, more studies into asparagine metabolism during viral infections are demanded. These efforts would benefit beyond viral diseases, as asparagine supply is also a limiting factor in various stages of cancer development.

Free (soluble, non-protein) asparagine concentration can increase many-fold in wheat grain in response to sulphur deficiency. This exacerbates a major food safety and regulatory compliance problem for the food industry because free asparagine may be converted to the carcinogenic contaminant, acrylamide, during baking and processing. Here, we describe the predominant route for the conversion of asparagine to acrylamide in the Maillard reaction. The effect of sulphur deficiency and its interaction with nitrogen availability is reviewed, and we reiterate our advice that sulphur should be applied to wheat being grown for human consumption at a rate of 20 kg per hectare. We describe the genetic control of free asparagine accumulation, including genes that encode metabolic enzymes (asparagine synthetase, glutamine synthetase, glutamate synthetase, and asparaginase), regulatory protein kinases (sucrose nonfermenting-1 (SNF1)-related protein kinase-1 (SnRK1) and general control nonderepressible-2 (GCN2)), and basic leucine zipper (bZIP) transcription factors, and how this genetic control responds to sulphur, highlighting the importance of asparagine synthetase-2 (ASN2) expression in the embryo. We show that expression of glutamate-cysteine ligase is reduced in response to sulphur deficiency, probably compromising glutathione synthesis. Finally, we describe unexpected effects of sulphur deficiency on carbon metabolism in the endosperm, with large increases in expression of sucrose synthase-2 (SuSy2) and starch synthases.

Contemporary chemoenzymatic approaches can provide highly complex multi-antennary N-linked glycans. These procedures are, however, very demanding and typically involve as many as 100 chemical steps to prepare advanced intermediates that can be diversified by glycosyltransferases in a branch selective manner to give asymmetrical structures commonly found in Nature. Only highly specialized laboratories can perform such syntheses, which greatly hampers progress in glycoscience. Here we describe a biomimetic approach in which a readily available bi-antennary glycopeptide can be converted in 10 or fewer chemical and enzymatic steps into multi-antennary N-glycans that at each arm can be uniquely extended by glycosyltransferases to give access to highly complex asymmetrically branched N-glycans. A key feature of our approach is the installation of additional branching points using recombinant MGAT4 and MGAT5 in combination with unnatural sugar donors. At an appropriate point in the enzymatic synthesis, the unnatural monosaccharides can be converted into their natural counterpart allowing each arm to be elaborated into a unique appendage., Graphical Abstract

BackgroundThe loss-of-function mutation of fumarate hydratase (FH) is a driver of hereditary leiomyomatosis and renal cell carcinoma (HLRCC). Fumarate accumulation results in activation of stress-related mechanisms leading to upregulation of cell survival-related genes. To better understand how cells compensate for the loss of FH in HLRCC, we determined the amino acid nutrient requirements of the FH-deficient UOK262 cell line (UOK262) and its FH-repleted control (UOK262WT).MethodsWe determined growth rates and survival of cell lines in response to amino acid depletion and supplementation. RNAseq was used to determine the transcription changes contingent on Asn and Gln supplementation, which was further followed with stable isotope resolved metabolomics (SIRM) using both [U-13C,15N] Gln and Asn.ResultsWe found that Asn increased the growth rate of both cell lines in vitro. Gln, but not Asn, increased oxygen consumption rates and glycolytic reserve of both cell lines. Although Asn was taken up by the cells, there was little evidence of Asn-derived label in cellular metabolites, indicating that Asn was not catabolized. However, Asn strongly stimulated Gln labeling of uracil and precursors, uridine phosphates and hexosamine metabolites in the UOK262 cells and to a much lesser extent in the UOK262WT cells, indicating an activation of the hexosamine biosynthetic pathway (HBP) by Asn. Asn in combination with Gln, but not Asn or Gln alone, stimulated expression of genes associated with the endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in UOK262 to a greater extent than in FH-restored cells. The changes in expression of these genes were confirmed by RT-PCR, and the stimulation of the UPR was confirmed orthogonally by demonstration of an increase in spliced XBP1 (sXBP1) in UOK262 cells under these conditions. Asn exposure also increased both the RNA and protein expression of the HBP regulator GFPT2, which is a transcriptional target of sXBP1.ConclusionsAsn in the presence of Gln induces an ER stress response in FH-deficient UOK262 cells and stimulates increased synthesis of UDP-acetyl glycans indicative of HBP activity. These data demonstrate a novel effect of asparagine on cellular metabolism in FH-deficient cells that could be exploited therapeutically.

Acrylamide is a Class 2a carcinogen that was discovered in a variety of popular foods, including baked cereal products, in 2002. The predominant route for its formation is from free asparagine and reducing sugars in the Maillard reaction, with free asparagine concentration being the main determinant of acrylamide-forming potential in cereal products. The European Commission set “indicative” levels for acrylamide in food in 2011 and 2013, and is currently reviewing its options for further measures. Agronomic and genetic approaches to reducing the acrylamide-forming potential of wheat include the evaluation of existing varieties for low asparagine accumulation in the grain, ensuring adequate sulfur fertilization in relation to nitrogen supply, developing an understanding of the genetic control of asparagine metabolism, and identifying quantitative trait loci or molecular markers for low asparagine accumulation in the grain. Asparagine concentration in grain is affected by environmental factors (E), genetic factors (G), and interactions between the two (G × E). This paper reviews the continuing efforts being made to reduce the acrylamide-forming potential of wheat, and to increase awareness of the issue among wheat breeders, farmers, and the food industry.

L-Asparaginases (EC 3.5.1.1) are enzymes that catalyze the hydrolysis of L-asparagine to L-aspartic acid and found in a variety of organisms from microorganisms to mammals. However, they are mainly expressed and produced by microorganisms. Microbial L-asparaginases have received sustained attention due to their irreplaceable role in the therapy of acute lymphoblastic leukemia and for their inhibition of acrylamide formation during food processing. In this article, we review the application of microbial L-asparaginases in medical treatments and acrylamide mitigation. In addition, we describe in detail recent advances in the existing sources, purification, production, properties, molecular modification, and immobilization of L-asparaginase.

Cancer cells consume and utilize glucose at a higher rate than normal cells. However, some microenvironments limit the availability of nutrients and glucose. In 2018, researchers found that tumours depend on a variety of different nutrient sources, both locally and systemically, to overcome metabolic limitations and promote tumour progression and metastasis.

It has recently been suggested that the analytical methods that have been developed to date for the determination of acrylamide (AA) may underestimate the concentration of AA in certain foods, because significantly higher results were obtained upon extraction of the food matrix under alkaline conditions. The present study employs food (potato, rye) and chemical model systems to better understand the tentative release of AA under high pH extraction conditions. The experimental design is based on the generation of AA in an environment containing an AA-isotopomer, and by comparing the ratio of AA, respectively the AA-isotopomer, after extraction at pHs 7 and 12. The results show that the additional AA released is not due to improved extractability of AA from the food matrix, and should therefore be regarded as an extraction artefact. Strongly alkaline conditions seem to induce net formation of AA from water-soluble precursors formed during thermolysis.

Instability of peptides and proteins can be divided into two forms: chemical and physical instability. Chemical instability is due to modification/alteration of amino acid residues. There are several types of degradation reactions responsible for this instability. Most frequently described reactions are oxidation, reduction, deamidation, hydrolysis, arginine conversion, beta-elimination and racemisation. However, any study of the degradation of a chemical substance lacks reliability when the analytical methodology, that is used is not properly validated. Especially in the investigation, where degradation processes lead to their parent compounds, validation of the analysis is pivotal for the correct interpretation of the results. It is therefore appropriate and useful to assemble an overview of degradation processes in relation to the analytical methods to monitor them. An overview like this can help investigators to make the right choices in their analytical approach of stability problems. The degradation reactions involved in peptide/protein degradation as well as the methods to monitor them are summarized and discussed.

Acrylamide in potato chips made from tubers stored at 2 or 20 degrees C for two weeks after harvest was analyzed by GC-MS. The acrylamide level in the former chips was higher than ten times of that in the latter, which was highly correlated with both glucose and fructose levels in the tubers.

Acrylamide forms during cooking and processing predominately from the reaction of free asparagine and reducing sugars in the Maillard reaction. The identification of low free asparagine and reducing sugar varieties of crops is therefore an important target. In this study, nine varieties of potato (French fry varieties Maris Piper (from two suppliers), Pentland Dell, King Edward, Daisy, and Markies; and chipping varieties Lady Claire, Lady Rosetta, Saturna, and Hermes) grown in the United Kingdom in 2009 were analyzed at monthly intervals through storage from November 2009 to July 2010. Acrylamide formation was measured in heated flour and chips fried in oil. Analysis of variance revealed significant interactions between varieties nested within type (French fry and chipping) and storage time for most free amino acids, glucose, fructose, and acrylamide formation. Acrylamide formed in chips correlated significantly with acrylamide formed in flour and with chip color. There were significant correlations between glucose or total reducing sugar concentration and acrylamide formation in both variety types, but with fructose the correlation was much stronger for chipping than for French fry varieties. Conversely, there were significant correlations with acrylamide formation for both total free amino acid and free asparagine concentration in the French fry but not chipping varieties. The study showed the potential of variety selection for preventing unacceptable levels of acrylamide formation in potato products and the variety-dependent effect of long-term storage on acrylamide risk. It also highlighted the complex relationship between precursor concentration and acrylamide risk in potatoes.

The sites and levels of Asn deamidation in proteins are often determined by LC-MS analysis of peptides obtained from enzymatic digestion. However, deamidation that occurs during sample preparation steps results in overestimation of the original level of deamidation. The inherent deamidation and those introduced by sample preparation can be differentiated by preparing samples in (18)O water. When using H(2)(18)O, the formation of isoAsp and Asp by Asn deamidation during sample preparation results in a molecular weight increase of 3 Da due to the incorporation of the (18)O atom to the side chains of isoAsp or Asp; in contrast, inherent deamidation only results in a molecular weight increase of 1 Da. In addition, up to two (18)O atoms can also be incorporated into the peptide C-terminal carboxyl group during enzymatic digestion. Therefore, the 2 Da molecular weight difference at the deamidation sites can only be used to differentiate deamidation that occurs prior to or during sample preparation under conditions that a fixed number of (18)O atoms are incorporated into the peptide C-terminal carboxyl groups. Otherwise, it is challenging to apply this procedure because of the resulting complicated isotopic distributions. Here, a new procedure of using (18)O-water for sample preparation coupled with tandem mass spectrometry (MS/MS) was established to calculate the deamidation artifacts. In this method, b ions were used for the calculation of Asn deamidation that occurred prior to or during sample preparation, which eliminated the complicated factor of various number of (18)O-atoms to the peptide carboxyl groups. This procedure has the potential to be applied under the general peptide mapping conditions.

Depletion of L -asparagine supply and apoptosis of leukemia cells induced by human glycosylasparaginase

Glutaminase-free L-asparaginase is known to be an excellent anticancer agent. In the present study, statistically based experimental designs were applied to maximize the production of glutaminase-free L-asparaginase from Pectobacterium carotovorum MTCC 1428. Nine components of the medium were examined for their significance on the production of L-asparaginase using the Plackett-Burman experimental design. The medium components, viz., glucose, L-asparagine, KH2PO4, and MgSO(4).7H2O, were screened based on their high confidence levels (P0.04). The optimum levels of glucose, L-asparagine, KH2PO4, and MgSO(4).7H2O were found to be 2.076, 5.202, 1.773, and 0.373 g L(-1), respectively, using the central composite experimental design. The maximum specific activity of L-asparaginase in the optimized medium was 27.88 U mg(-1) of protein, resulting in an overall 8.3-fold increase in the production compared to the unoptimized medium.

A host–pathogen interaction whereby GAS exploits host asparagine metabolism for its own benefit.

Cellular signalling pathways depend on the proper activation and inactivation of mediators. New structural information reveals an unusual mechanism by which one such mediator, Rap1, is switched off.

In this review we have critically evaluated the application of the diagenetic racemization of amino acids to geochronology and geothermometry. Although there has been enthusiastic support given to this new method, it is our opinion that recent developments suggest a more cautious approach. We have discussed the pitfalls and inhereent complications, while outlining the advances which have been accomplished. We conclude that this is an innovative approach which will add valuable information to the scientific literature. However, since our fundamental understanding of diagnetic racemization is still limited, many of the age and paleotemperature estimates which have been assigned to fossil specimens may be unreliable.

When [14C]-asparagine was supplied to growing pea leaves aspartate and other compounds were formed, but after 4 hours more than half of the metabolised carbon skeleton was present as one compound, identified as 2-hydroxysuccinamate. This compound was also formed, with a high rate of conversion, when 2-ketosuccinamate (the product of transamination of asparagine) was supplied to the leaves. There was some synthesis of amino acids from 2-hydroxysuccinamate, and in the dark it was metabolised to release some carbon dioxide. Accumulation and metabolism of 2-hydroxysuccinamate has also been observed in soybean leaves. It is suggested that 2-hydroxysuccinamate is a major intermediate in the metabolism of the carbon skeleton of asparagine following transamination, an important route for asparagine ultilisation.

Candida albicans and six other medically important Candida species were cultured on glucose–salts–biotin media containing tryptophan as the major nitrogen source. Candida albicans, C. tropicalis, and C. parapsilosis produced a pink pigment; C. krusei, C. pseudotropicalis, and C. gulliermondii formed brown pigments. Pigmentation of C. stellatoidea was variable, some strains produced brown pigments and others a pink pigment. Production of pink pigment was catalyzed by light. Cultures incubated in the dark produced only trace amounts of the pink pigment. Iron enhanced pigment production. Intense autofluorescence was observed for strains producing the brown pigment and weak autofluorescence for strains producing the pink pigment.

Asparaginase and asparagine synthetase specific activity (enzyme units/mg protein) was determined in chicks and rats fed various protein diets. The specific activity of asparaginase was twofold higher in the kidney compared to the liver of chicks fed a 25% protein control diet, whereas the specific activity of asparaginase was eightfold greater in the liver compared to kidney tissue of rats fed the 25% protein control diet. The asparaginase specific activity in the chick liver, chick kidney, and rat liver were all significantly increased when animals were fed the 75% protein diet. Asparaginase specific activity increased significantly in rat and chick kidneys when the animals were fasted. Dietary supplementation of asparagine did not significantly increase asparaginase specific activity in either animal. The combined kidney and liver asparagine synthestase specific activity for chicks fed the control diets is over seven hundred times less than the corresponding combined asparaginase specific activity. Rats fed the control diet had a combined kidney and liver asparagine synthetase specific activity about 75 times less than the corresponding asparaginase specific activity. The asparagine synthetase specific activity was significantly increased in the rat and chick kidney when a 2% protein diet was fed.

(1979). Hb J Lome β59 (E3) Lys → Asn Associated with Hpfh in a Togolese Family. Hemoglobin: Vol. 3, No. 1, pp. 87-91.

1. 1. Radioactive asparagine has been isolated from lupine seedlings, which were permitted to carry on photosynthesis for 6 days in the presence of C14O2. It was found to contain 55% of its activity in the α-carboxyl. 2. 2. Radioactive asparagine was vacuum infiltrated into wheat and lupine seedlings, and its subsequent metabolism was observed. 3. 3. Utilization of asparagine for both CO2 production and the synthesis of cells was observed in all cases. This was true even in young lupine seedlings before they develop their photosynthetic mechanism. 4. 4. Radioactive glucose, infiltrated into lupine seedlings, was utilized 2 1 2 –5 times faster than asparagine. Although glucose was used about equally for respiration and cell synthesis, asparagine was used mainly for the synthesis of cells.

The utilization of N compounds by P. ultimum Trow (strain I and II), P. rostratum Butler, and P. irregulare Buisman was examined in a chemically denned medium under controlled conditions in surface culture. All species were able to metabolize nitrate, ammonium, and organic nitrogen, and the amount of growth varied with the nitrogen source. In general, yeast extract, peptone, glycine, serine, histidine, cysteine, asparagine, aspartic acid, and glutamic acid supported favorable growth, whereas γ-aminobutyric acid, threonine, and alanine supported poor growth of these fungi. The addition of succinic acid at 0.02 M concentration to ammonium compounds further increased growth of four isolates.Preferential utilization of amino acids from a given mixture was recorded using paper chromatographic techniques. All four isolates gave more vegetative growth on mixtures of amino acids than when they were supplied singly.

Asparagine, a major transport compound, is metabolized in Pisum sativum by two enzymes, asparaginase (EC 3.5.1.1) and asparagine-pyruvate aminotransferase. The relative amount of the two enzymes varies between tissues. In developing seeds, there are very high levels of asparaginase but only trace amounts of the aminotransferase. Asparaginase is high in young leaves but falls rapidly during leaf growth; the aminotransferase remains high throughout development. Inhibitor studies with aminooxyacetate and methionine sulfoximine confirm that the aminotransferase is the main enzyme involved in asparagine utilisation in the leaf. Root tissue has low levels of asparaginase and only trace amounts of the aminotransferase. The asparaginase is potassium dependent, but is also partially activated by ammonium ions. The leaf aminotransferase has a lower K m for asparagine (4.5 mM) than the leaf asparaginase (8 mM). The seed asparaginase has a lower K m for asparagine (3 mM) than the leaf asparaginase.

The effect of dietary asparagine and protein-equivalents from crystalline amino acid mixtures upon asparagine metabolism in chicks were studied. Liver and kidney asparaginase activities were significantly increased in chicks fed 44.6% protein-equivalents compared to chicks fed the Illinois chick standard amino acid mixture containing 14.8% protein-equivalents. The asparagine synthetase activity in chick liver and kidney was not significantly changed by protein-equivalents or dietary asparagine. Liver and kidney asparaginase activities in chicks fed 14.8% protein-equivalent standard diets were decreased with increasing levels of dietary asparagine (0,2 and 6%). Kidney asparaginase activities in chicks fed 44.6% protein-equivalents also were decreased with increasing levels of asparagine but liver asparaginase in these chicks was not changed with dietary asparagine. The plasma asparagine concentration was dependent on the amount of dietary asparagine and protein-equivalents. Dietary asparagine increased plasma asparagine in chicks fed 14.8 and 44.6% protein-equivalent diets but plasma asparagine in chicks fed the 14.8% protein-equivalent diet plus 6% asparagine was 3.5 times higher than plasma asparagine in chicks fed the diet containing 44.6% protein-equivalent plus 6% dietary asparagine. Plasma asparagine in chicks fed the 44.6% protein-equivalent diet with 6% asparagine was reduced due to increased asparaginase activity.

Legumes have been examined for asparaginase activity, taking into consideration their symbiotic association with bacteria in their nitrogen-fixing nodules. It has been shown that legumes grown without these bacterial asociations can hydrolyse asparagine and that asparaginase is located principally in the roots of the plants. However, in a normally nodulated plant the non-bacteroid portion of the nodule has a high concentration of asparagine, and asparaginase activity is much higher per g of tissue in this part of the nodule than in the remainder of the root section of the plant. Bacteroids from the nodule are also able to hydrolyse asparagine. The possible contribution of the nodule to the asparagine metabolism of the whole plant is discussed.

Mit Hilfe der Papierchromatographie wurden losliche und unlosliche Proteine in gesunden und durch «Carica-Curl»-Virus erkrankten Blattern von Carica-Papaya bestimmt. In kranken Blattern nehmen die unloslichen Proteine ab, der Gehalt an Asparagin,β-Alanin +α-Aminobuttersaure zu, was wohl auf eine Anderung des Eiweisstoffwechsels der infizierten Blatter unter dem Einfluss des Virus zuruckgefuhrt werden darf.

Summary The factors causing the production of large amounts of asparagine in seedlings, excised leaves, and in the transport system of legumes and certain trees are discussed. An attempt has been made to correlate the radioactive precursor studies with the known enzymology of asparagine synthesis. Although asparagine is considered to act as a nitrogen store, there is little evidence to demonstrate its breakdown. Three possible routes of asparagine degradation are investigated.

© 2018 The Authors. A detailed network describing asparagine metabolism in plants was constructed using published data from Arabidopsis (Arabidopsis thaliana) maize (Zea mays), wheat (Triticum aestivum), pea (Pisum sativum), soybean (Glycine max), lupin (Lupus albus), and other species, including animals. Asparagine synthesis and degradation is a major part of amino acid and nitrogen metabolism in plants. The complexity of its metabolism, including limiting and regulatory factors, was represented in a logical sequence in a pathway diagram built using yED graph editor software. The network was used with a Unique Network Identification Pipeline in the analysis of data from 18 publicly available transcriptomic data studies. This identified links between genes involved in asparagine metabolism in wheat roots under drought stress, wheat leaves under drought stress, and wheat leaves under conditions of sulfur and nitrogen deficiency. The network represents a powerful aid for interpreting the interactions not only between the genes in the pathway but also among enzymes, metabolites and smaller molecules. It provides a concise, clear understanding of the complexity of asparagine metabolism that could aid the interpretation of data relating to wider amino acid metabolism and other metabolic processes. Biotechnology and Biological Sciences Research Council. Grant Number: BB/I020918/1; Designing Future Wheat

Background Understanding the determinants of free asparagine concentration in wheat grain is necessary to reduce levels of the processing contaminant acrylamide in baked and toasted wheat products. Although crop management strategies can help reduce asparagine concentrations, breeders have limited options to select for genetic variation underlying this trait. Asparagine synthetase enzymes catalyse a critical step in asparagine biosynthesis in plants and, in wheat, are encoded by five homeologous gene triads that exhibit distinct expression profiles. Within this family, TaASN2 genes are highly expressed during grain development but TaASN-B2 is absent in some varieties. Results Natural genetic diversity in the asparagine synthetase gene family was assessed in different wheat varieties revealing instances of presence/absence variation and other polymorphisms, including some predicted to affect the function of the encoded protein. The presence and absence of TaASN-B2 was determined across a range of UK and global common wheat varieties and related species, showing that the deletion encompassing this gene was already present in some wild emmer wheat genotypes. Expression profiling confirmed that TaASN2 transcripts were only detectable in the grain, while TaASN3.1 genes were highly expressed during the early stages of grain development. TaASN-A2 was the most highly expressed TaASN2 homeologue in most assayed wheat varieties. TaASN-B2 and TaASN-D2 were expressed at similar, lower levels in varieties possessing TaASN-B2. Expression of TaASN-A2 and TaASN-D2 did not increase to compensate for the absence of TaASN-B2, so total TaASN2 expression was lower in varieties lacking TaASN-B2. Consequently, free asparagine concentrations in field-produced grain were, on average, lower in varieties lacking TaASN-B2, although the effect was lost when free asparagine accumulated to very high concentrations as a result of sulphur deficiency. Conclusions Selecting wheat genotypes lacking the TaASN-B2 gene may be a simple and rapid way for breeders to reduce free asparagine concentrations in commercial wheat grain.

THIS is a report of the formation of polyaspartic acid from asparagine1 in aqueous solution under conditions which satisfy suggested primitive terrestrial conditions2 better than thermal polycondensation3,4.