Asparagine Amino Acids: Properties, Function, Benefits, and Sources

What is asparagine?

The non-essential amino acid asparagine (Asn) has a role in the metabolic regulation of cellular activities in brain and nerve tissue. Glycoprotein biosynthesis relies on this amino acid, which is essential for the production of many proteins due to its involvement in providing critical sites for N-linked glycosylation. When it comes to the metabolism of the poisonous ammonia, Asn is also a key player. The cells can easily manufacture l-asparagine because it is a nonessential amino acid. In the presence of a transaminase enzyme, oxaloacetate combines with l-glutamate to produce l-aspartate and α-ketoglutarate, which are the initial building blocks for aspartate in the biosynthesis process. The l-asparagine synthetase enzyme catalyzes an ATP-dependent process in which l-aspartate and l-glutamine are combined to produce l-asparagine.

In a cellular process that requires ATP, asparagine synthetase (ASNS) uses glutamine (Gln) and aspartate (Asp) as substrates to produce Asn. Metformin impedes the conversion of Asp from Gln and impacts de novo synthesis of Asn by inhibiting the mitochondrial electron transport chain (ETC), suggesting that the operation of the ETC is somewhat dependent on this intracellular production of Asn. Since KRAS, SOX12, and TP53 regulate ASNS expression, somatic mutation patterns inside cancer cells also impact ASNS-catalyzed Asn production.

Metabolism of asparagine (Asn). (Shen X., et al., 2022)

Cyclization of asparagine

A cyclic structure is formed when the amino acid asparagine passes through a chemical reaction, a process known as cyclization of asparagine. This usually happens when particular factors are present, including particular catalysts, heat, or other chemicals. One of the most prevalent types of cyclic derivatives that can be formed by the cyclization reaction is 5-membered ring structures, such as cyclic imides or lactams. Proteins can undergo post-translational modifications such as deamidation of Asn residues, which can happen in vivo and in vitro under physiological conditions without the need for enzymes. Asp and the biologically rare isoAsp residues are normally produced in a molar ratio of about 1:3 when this reaction goes through the cyclic reaction (cyclic succinimide intermediate). Protein structural stability and physiologically significant functions are both compromised by these deamidation-induced changes from amino to acidic residues. The ubiquitin-proteasome pathway is responsible for the destruction of some of the deamidated proteins. There is a theory that deamidation acts as a molecular clock that regulates biological processes including protein turnover. Deamidation is also thought to promote a number of age-related disorders due to the fact that it causes proteins to denaturate and aggregate. Crystallins extracted from the lens of patients with age-related nuclear cataracts often include deamidated residues. Even more so, autoimmune illnesses include isoAsp production in living organisms. The immune system attacks the native peptide with the original Asn residue when it encounters an isoAsp-containing peptide. Example: systemic lupus erythematosus may involve isoAsp production in histone H2B. Antibody medication quality control also relies on Asn deamidation. Remnant monoclonal antibodies and human endogenous IgG have been found to undergo deamidations in their complementarity-determining domains (CDRs) and Fc regions when in vivo. Asn deamidation in CDRs specifically decreases antigen-binding affinity. As a result, Asn deamidation is a crucial process in the fields of pathology and peptide/protein therapeutic development.

Cyclization of asparagine. (Kato K., et al., 2020)

Asparagine structure

Asparagine is a beta-amido derivative of aspartic acid that plays a significant role in the production of glycoproteins and other proteins. It serves as a non-toxic vehicle for the excretion of leftover ammonia from the body. Asparagines function as the transport and storage forms of nitrogen. The unique effects of enantiomers and their differing behaviors in biological systems are evident even at a macroscopic level. (R)-asparagine (d-Asn) elicits a sweet taste, but (S)-asparagine (l-Asn) induces a bitter taste in humans. The backbone comprises an amino group (-NH₂), a carboxyl group (-COOH), and a side chain including a carboxamide group (-CONH₂). The side chain (β-amino group) is an amide group, distinguishing asparagine from its analogous amino acid, aspartic acid, which contains a carboxyl group instead of the amide group.

Chemical structures of asparagine. (Bui C V., et al., 2021)

Asparagine pKa

Among the most crucial physicochemical characteristics of macromolecules and small molecules is the dissociation constant, which applies to weakly acidic or basic groups. The standard way to represent it is as the pK an of every group. When it comes to medication pharmacokinetics and protein-molecule interactions, this is a big deal.

The theoretically derived pKa values for asparagine and glycyl-asparagine (pK _a1=2.723527422 and 2.447560050) were comparable to the observed pK _a1 values (pK _a1= 2.01 and 2.942). The theoretically derived pKa2 values for asparagine and glycyl-asparagine (pK _a2 = 8.700170342 and 8.391184488) were relatively consistent with the empirically observed pK _a (pK _a2 = 8.8 and 8.44).

Values of pKa for the protonation of asparagine. (Kiani F., et al., 2015)

Asparagine solubility

Asparagine is highly soluble in water, which is characteristic of many polar amino acids. This high solubility is due to the presence of its carboxamide side chain (-CONH₂), which can form hydrogen bonds with water molecules, facilitating its dissolution. Asparagine is poorly soluble in non-polar organic solvents (e.g., chloroform, ether), due to its hydrophilic nature. It tends to prefer aqueous environments where it can form favorable interactions with water molecules.

Asparagine vs aspartic acid (aspartate)

Asparagine is neutral at physiological pH (~7) due to the deprotonation of the carboxyl group on the backbone ( -COO^-), the protonation of the amino group (-NH₃⁺), and the non-ionization of the carboxamide side chain (-CONH₂). Aspartic acid possesses a negative charge at physiological pH due to the deprotonation of the carboxyl group in its side chain ( -COO^-). The amino group is protonated (-NH₃⁺), categorizing aspartic acid as an acidic amino acid.

The presence of aspartic acid (Asp) and asparagine (Asn) residues is crucial for plenty of activities. As an example, the aspartate residue is very important in apoptosis since it is a caspase specific cleavage site. Regarding Asn, it is where N-glycosylation takes place, resulting in N-linked glycosaccharides. These many recognition activities rely on this surface response that proteins undergo. To make proteins more resistant to denaturation and proteolysis, N-glycosylation stabilizes their structure.

Aspartic acid (Asp) and asparagine (Asn). (Strzelczak G., et al., 2007)

Asparagine vs glutamine

Asparagine possesses a 2-carbon side chain, specifically a -CH₂ group preceding the carboxamide group. Glutamine possesses a 3-carbon side chain, specifically a -CH₂-CH₂ group preceding the carboxamide group. Asparagine and glutamine are derivatives of aspartic and glutamic acid, respectively, containing an amide functional group on the carbon furthest from the α carbon. In contrast to their acidic counterparts, the side chains of asparagine and glutamine possess no electric charge; they are polar. Both are engaged in the biosynthesis of various amino acids, such as glutamate, and function as precursors for the synthesis of nucleotides, proteins, and other nitrogenous substances. Asparagine can participate in the manufacture of aspartate by the activity of asparaginase, an enzyme that catalyzes the hydrolysis of asparagine to aspartic acid. Glutamine serves as a precursor to the neurotransmitter glutamate, which is essential for excitatory communication in the central nervous system.

Asparagine (N) and Glutamine (Q). (Blanco A., et al., 2017)

Asparaginase mechanism of action

Breaking down L-asparagine into L-aspartic acid and ammonia is catalyzed by L-asparaginase, a tetrameric amidohydrolase enzyme. It has been one of the main chemotherapeutic medicines for acute lymphoblastic leukemia since its development as an anticancer medicine. In addition to its application in biopharmaceuticals, it is employed to decrease the production of acrylamide, a carcinogenic compound, in meals that are fried, baked, or roasted. Plants, bacteria, fungus, and actinomycetes are among the numerous sources of L-asparaginase. The current clinical treatment for acute lymphoblastic leukemia involves the use of L-asparaginase preparations derived from Escherichia coli and Erwinia chrysanthemi. Protein and ribonucleic acid (RNA) production in normal and lukemic cells both require L-asparagine. It is produced in normal cells by an enzyme called asparagine synthetase. Conversely, lukemic cells lack the enzyme asparagine synthetase, which prevents them from producing L-asparagine. Therefore, the presence of L-asparagine in the surrounding cells and tissues is essential for these cells. This means that they will be deficient in L-asparagine after taking L-asparaginase. In the end, this causes leukemic cells to die by blocking their ability to synthesize RNA and proteins, which in turn causes cell cycle arrest and apoptosis.

Mechanism of action of L-asparaginase. (Parashiva J., et al., 2023)

Asparagine function

It was a stroke of luck in 1953 when John Kidd's group found that blood malignancies depend on asparagine. When Kidd et al. investigated the use of animal sera as a complement source for lymphoma treatment, they discovered that guinea pig serum significantly reduced the size of engrafted mice lymphomas, in contrast to serum from other species. Asparaginase, an enzyme that efficiently depletes serum asparagine, was determined to be the component of guinea pig serum responsible for tumor regression eight years later by JD Broome. While prior research had shown that these lymphomas needed l-asparagine to develop, the discovery of l-asparaginase provided the first definitive proof of a metabolic necessity for tumors. Isolation of an Escherichia coli l-asparaginase sped up the clinical usage of the technique, which had previously relied on guinea pig serum in an effort to take advantage of patients' dependence. Induction chemotherapy for ALL relies on l-asparaginase, a monotherapy that has demonstrated tumor regression in 20-60% of ALL patients.

While leukemias are unable to produce asparagine from aspartate, the majority of human cell types are able to do so via an ATP-dependent process facilitated by asparagine synthetase (ASNS). This is in line with the fact that normal cells may withstand asparagine deficiency by increasing ASNS transcription and synthesis of de novo asparagine. The integrated stress response (ISR) is a conserved transcriptional pathway that helps maintain homeostasis in response to various physiological stimuli. Nutrient scarcity triggers the insensitive signal transduction pathway (ISR) through the buildup of uncharged tRNAs, which in turn activate the kinase GCN2, which phosphorylates the eukaryotic initiation factor 2 alpha subunit (eIF2alpha) of the eIF2 complex. Activating transcription factor 4 (ATF4) upregulation is a key result of ISR. In response to nutrient scarcity, ATF4 stabilizes and organizes the overexpression of many transporters and enzymes that carry nutrients, including ASNS. Since ribosome profiling investigations have demonstrated that asparagine limitation stops translation at asparagine residues, it is likely that the only function of asparagine in leukemia cells is its proteogenic role in translation. Apoptosis in ALL cells is induced and global protein synthesis is potently restricted when serum asparagine is reduced by l-asparaginase. On the other hand, asparagine may perhaps coordinate the synthesis of both proteins and nucleotides as an amino acid exchange factor, which has only recently come to light.

It has been suggested that reduced or complete loss of ASNS expression may be the exact source of the asparagine dependency of leukemias, however this has not been completely confirmed. The response to l-asparaginase is highly correlated with ASNS protein levels. The activity of the ASNS enzyme was positively correlated with l-asparaginase resistance, according to a 1969 publication by Haskell et al. Similarly, sensitivity to l-asparaginase is inversely correlated with ASNS cDNA levels, and this link is much stronger among leukemic cell lines, according to a study of 60 human cancer cell lines from the National Cancer Institute. Cell lines with strong ASNS expression outcompeted those with low expression, according to a competitive growth assay for 554 barcoded cell lines. Methylation of several expression-associated histone marks regulates the ASNS promoter in ALLs. Hypermethylation of CpG islands within the ASNS gene promoter has been detected in cell lines with low baseline ASNS expression. Also, a new study linked improved results following asparaginase treatment to reduced ASNS levels in T-ALLs, which the researchers attribute to hypermethylation of the ASNS promoter. It is intriguing to note that the exact reason ALLs decrease or eliminate ASNS expression is not yet known. At several points in the hematopoiesis process, ASNS expression may be downregulated. On the other hand, some cancers may benefit from downregulating ASNS expression to keep aspartate levels high, since it is a growth-limiting molecule. When considered collectively, these results point to ASNS as the main cell autonomous factor determining l-asparaginase sensitivity in leukemia. Nevertheless, there have been contradictory conclusions reported when trying to link ASNS expression to l-asparaginase sensitivity in ALLs. One study that looked at clinical ALL samples neglected to find any link between the expression of ASNS at baseline and the in vitro response to l-asparaginase. Possible other biological sources of asparagine from catabolic protein degradation could help to explain this disparity. Asparaginase treatment may actually supply enough asparagine to leukemia cells via the proteasomal breakdown route. Raising asparaginase's therapeutic index is possible through activating WNT-dependent inhibition of proteasomal degradation. Leukemia cells treated with asparaginase may potentially receive asparagine from other bone marrow cell types. Mesenchymal cells originating from bone marrow, for instance, express ASNS at high levels, and this expression correlates with asparagine production and secretion. In addition, when co-cultured with bone marrow stromal cells, ALL cells release insulin-like growth factor (IGF)-binding protein 7 (IGFBP7), which stimulates the production of ASNS and the secretion of asparagine. In addition to counteracting the effects of l-asparaginase, adipocytes may supply leukemic cells with anaplerotic substrates. These findings point to several ways that l-asparaginase treatment could be resistant.

A lot of people are starting to think about using l-asparaginase to treat solid tumors after seeing how well it worked for blood malignancies. Patients with melanoma and lymphoma showed some improvement in early l-asparaginase clinical studies that included all neoplastic disorders. Another finding is that l-asparaginase is effective against hepatocellular carcinomas, gastric malignancies, and pancreatic tumors that have poor ASNS expression. Treatment with l-asparaginase decreases invasiveness, metastatic progression, and the epithelial-to-mesenchymal transition, demonstrating that asparagine availability tightly controls metastatic potential. It should be mentioned that l-asparaginase, when taken at greater doses, can also cause a substantial decrease in serum glutamine levels. On the other hand, l-asparaginase's asparaginase activity, rather than its glutaminase activity, is enough to cause cell death in cells expressing low amounts of ASNS. A potential solution to this problem is the engineering of L-asparaginases with very low levels of off-target glutaminase activity; this could lead to a decrease in the harmful side effects of treatment.

Asparagine availability is dictated by both synthesis and uptake. (Garcia-Bermudez J., et al., 2020)

Asparagine amino acids at Creative Peptides

Asparagine activity

For almost half a century, therapeutic medicines derived from bacterial L-asparaginases (ASNases) have been utilized to treat acute lymphoblastic leukemia (ALL) in children. These enzymes catalyze the hydrolysis of asparagine to aspartate. The fact that ALL cells are known to express very little ASNS suggests that ASNase and ASNS are closely connected. As a rule, kidney cells express low levels of ASNS compared to those in the pancreas, brain, testes, and thyroid. Fifty years ago, researchers found that ASNases had anti-tumor capabilities; they work especially well against ALL. Because leukemia and lymphoma cells typically express little to no ASNS or none at all, ASNase treatment aims to destroy these cells. Hypermethylation of CpG islands in the ASNS promoter is likely the culprit. Asparagine is an auxotroph for these low-ASNS cells since they are unable to produce the amount of Asn required for proliferation and basic activities. Leukemia cells treated with ASNase suppress the mTOR pathway, which in turn inhibits proteosynthesis and nucleic acid synthesis, causes cell cycle arrest in the G1 phase, activates autophagy, and ultimately leads to apoptosis. In addition to altering leukemia cells' metabolism, ASNase enhances maximum respiration and FAO while decreasing glucose absorption. Their glutaminase activity, in comparison to asparaginase activity, is approximately 5% in ASNases that are employed in clinical settings. Glutaminase activity is higher in some ASNases compared to asparaginase activity. The glutaminase activity of ASNase appears to be very useful in the treatment of tumors. For instance, oligodendroglioma, multiple myeloma, and ovarian cancer all exhibit low or nonexistent glutamine synthetase (GS) expression. Astoundingly, ASNase's glutaminase activity aids in the treatment of malignancies expressing GS. Additionally, ASNase's glutaminase activity may be crucial to its efficacy in treating ALL, according to some research.

Researchers engineered ASNases with varying glutaminase activities to learn more about the enzyme's function. Tumor cells lacking ASNS expression were nevertheless susceptible to enzymes devoid of glutaminase activity. A study conducted by Offman et al. demonstrated that ALL cells devoid of ASNS expression exhibited heightened sensitivity to ASNase with high glutaminase activity compared to ASNase with low glutaminase activity. Glutaminase activity a prerequisite for tumor cell proliferation if ASNS-negative cells do not utilize glutamine to generate asparagine but rather to augment anaplerosis. Extracellular glutamine is required for the synthesis of asparagine and other metabolites; however, cells expressing ASNS have their glutaminase activity reduced by ASNase. These cells die because there isn't enough glutamine outside their cells. Furthermore, in tumor cells that express ASNS, cytotoxicity and glutaminase activity of ASNase are positively correlated. Glutamate is an essential amino acid for the body, therefore running out of it could cause issues. An approach to replenish glutamine has been proposed due to concerns about metabolic harm caused by ASNase therapy-induced glutamine depletion. While some research suggests that glutamine supplements can help patients undergoing ALL treatment and chemotherapy, other research has found no such benefit.

Asparagine uses

(1) Establish the model of promoting tumor growth

Recent studies in a model of non-small-cell lung cancer (NSCLC) have shown that oncogenic KRAS signaling can influence the production of asparagine in order to promote the growth of tumors. Through the downstream PI3K/AKT pathway, KRAS promotes NRF2, a critical transcription factor for anti-oxidant defense. Consequently, NRF2 triggers the ASNS gene's transcription by increasing ATF4 transcription factor expression. The study highlights the fact that L-asparaginase and AKT inhibitors can work together to suppress tumor growth in mice. A different study indicated that NSCLC cells with a KEAP1 mutation were more reliant on asparagine and other NEAAs. Asparagine and other NEAAs rely on intracellular glutamate as an intermediary in their manufacture; however, this work showed that increased NRF2 activity causes cystine import at the cost of intracellular glutamate. This is because KEAP1 is a negative regulator of NRF2. Consequently, xenograft tumor models can be used to specifically limit the growth of KEAP1 mutant NSCLC cells through the use of L-asparaginase therapy or asparagine dietary restriction. Nevertheless, it remains unclear if NRF2 activation induces reliance on exogenous asparagine or its de novo manufacture; additional research is necessary to resolve this conflict.

Through the discharge of stroma cells, solid tumor cells can also obtain asparagine from their surroundings. Cancer associated fibroblasts (CAFs) have the ability to promote asparagine production and secretion in a prostate cancer model, which can aid in the proliferation of tumor cells. According to this scenario, p62, an autophagy adaptor protein, plays a crucial regulatory role in ubiquitin-mediated degradation of ATF4, hence suppressing its expression. This suggests that tumor cell adaptation to a glutamine-limiting environment may be one mechanism by which p62 deletion in the CAFs promotes prostate cancer growth in a mouse model via the ATF4-ASNS axis. Nevertheless, the production of asparagine cannot begin without glutamine. It is worth exploring further why CAFs, and not tumor cells directly, are able to synthesize asparagine in a glutamine-limited setting.

Depending on the stage of tumor growth, asparagine bioavailability can have an impact as well. A recent study in a mouse model of metastatic breast cancer found that inhibiting ASNS expression with shRNA preferentially stops tumors from spreading to the lungs without affecting tumor growth in the original sites. Reduced lung metastases was observed in mice treated with L-asparaginase or given an asparagine-free diet. Environmental asparagine may be limited in the lung or during lung metastasis, according to this study, which means that de novo production is crucial. Mechanistically, genes implicated in the epithelial-mesenchymal transition (EMT) are downregulated by asparagine restriction, which is a critical step in the initiation of metastasis. Since EMT occurs at the first locations of tumor growth, the relative amounts of asparagine required for primary tumor growth and EMT still necessitate additional exploration.

(2) As an exchange factor

A foundational study published the first mention of asparagine's role in controlling cancer cell signaling by showing that it can activate mTORC1 to accelerate protein and nucleotide production. One of the ways in which asparagine activates mTORC1 is by acting as an antiporter exchange factor, allowing additional amino acids to be imported. In a recent study, the same group discovered that asparagine can activate mTORC1, which helps tumor cells deal with stress caused by ETC inhibition. This stress is associated with ATF4 activation. The work raises the question of which limiting metabolite—asparagine or aspartate—drives the ETC inhibition-associated phenomena in tumor cells and offers an alternate explanation for the synergy between the two. Asparagine can activate mTORC1 directly through an ADP-ribosylation factor 1 (Arf1)-dependent but Rag GTPase-independent method, in addition to its role as an activation exchange factor.

By attaching directly to signaling molecules, asparagine can also change their activity. The inhibitory impact of LKB1 on AMP-activated protein kinase (AMPK) can be enhanced when asparagine binds to it, according to current research. Therefore, asparagine shortage inside cells activates AMPK, which in turn causes p53 phosphorylation, further suppressing ASNS transcription by recruiting promoters. It is yet unclear why, in the face of asparagine deficiency, tumor cells employ this feedforward loop to regulate ASNS expression. Because p53 is often defunct in malignancies, it will be intriguing to see if its status can be used to predict sensitivity to asparagine depletion in a wider range of tumors. It is worth noting that the same group has just published a research stating that asparagine can specifically improve TCR signaling, which in turn increases CD8+ T cell activation and its anti-tumor responses. The process by which asparagine improves TCR signaling involves its direct binding to lymphocyte-specific protein tyrosine kinase (LCK), which causes autophosphorylation of LCK on Tyr 394 and 505. Critically, a subcutaneous B16 melanoma model reveals that immunocompetent mice pre-fed with an asparagine-free diet have impaired anti-tumor responses and reduced CD8+ T cell activity. This study also emphasizes the possible difficulty of treating cancer patients with T cell-based treatment while also administering L-asparaginase.

Signaling pathways regulated by asparagine or its depletion. (Jiang J., et al., 2021)

(3) Regulate vessel formation

Despite the growing body of evidence linking endothelial cell (EC) metabolism to angiogenesis regulation, our understanding of glutamine metabolism's specific function in ECs remains limited. Damage to vascular sprouting, migration, and pathological ocular angiogenesis were all caused by EC glutamine deprivation or GLS1 inhibition. Energy distress was not caused by inhibiting glutamine metabolism in ECs, but macromolecule synthesis, redox homeostasis, and tricarboxylic acid (TCA) cycle anaplerosis were compromised. Restoring the metabolic abnormalities and proliferative deficiency induced by glutamine deprivation required a combination of replenishing the TCA cycle and supplementing with asparagine. In order to maintain cellular homeostasis, glutamine served as a nitrogen source for asparagine production. Even though ECs can absorb asparagine, inhibiting the activity of ASNS, which is responsible for converting glutamine-derived nitrogen and aspartate to asparagine, hindered the sprouting of ECs, regardless of whether glutamine and asparagine were involved. In addition, glutamine-deprived ECs relied on asparagine to reinstate protein synthesis, reduce endoplasmic reticulum stress (ER stress), and activate mTOR signaling. These results show that endothelial glutamine and asparagine metabolism in sprouting of vessels is not well understood.

Asparagine is required for vessel sprouting. (Huang H., et al., 2017)

FAQ

Is asparagine acidic or basic?

Asparagine is an amide derivative of aspartic acid with no acidic or basic qualities. It is polar and contributes to hydrogen bond formation, hence at physiological pH (about 7), it is regarded neutral in terms of total acidity or basicity.

What type of amino acid is asparagine?

Due to the presence of electronegative oxygen and nitrogen atoms, the side chain of asparagine contains a polar carboxamide group (-CONH₂). This enables asparagine to establish hydrogen bonds with polar molecules like water. The protonation of the amino group (-NH₂) on the α-carbon to -NH₃⁺ and the deprotonation of the carboxyl group (-COOH) to -COO^- occur at physiological pH, which is around 7. Asparagine does not have a charge at physiological pH since the side chain's carboxamide group is neutral. Protein folding and stability rely on asparagine's capacity to engage in hydrogen bonding, a process made possible by its polar nature. This means that asparagine is an uncharged, polar amino acid (nonessential amino acid).

References

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