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Asparagine synthetase: Function, structure, and role in disease

  • Carrie L. Lomelino
    Footnotes
    Affiliations
    Department of Biochemistry and Molecular Biology, Shands Cancer Center, College of Medicine, University of Florida, Gainesville, Florida 32610
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  • Jacob T. Andring
    Footnotes
    Affiliations
    Department of Biochemistry and Molecular Biology, Shands Cancer Center, College of Medicine, University of Florida, Gainesville, Florida 32610
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  • Robert McKenna
    Footnotes
    Affiliations
    Department of Biochemistry and Molecular Biology, Shands Cancer Center, College of Medicine, University of Florida, Gainesville, Florida 32610
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  • Michael S. Kilberg
    Correspondence
    To whom correspondence should be addressed. Tel.: 352-294-8388
    Footnotes
    Affiliations
    Department of Biochemistry and Molecular Biology, Shands Cancer Center, College of Medicine, University of Florida, Gainesville, Florida 32610
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  • Author Footnotes
    5 Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site.
    6 S. J. Sacharow, E. E. Dudenhausen, C. L. Lomelino, L. Rodan, C. Moufawad El Achkar, H. E. Olson, C. A. Genetti, P. B. Agrawal, R. McKenna, and M. S. Kilberg, submitted for review.
    1 These authors should be considered as co-first authors.
    2 These authors should be considered as co-senior authors.
Open AccessPublished:October 30, 2017DOI:https://doi.org/10.1074/jbc.R117.819060
      Asparagine synthetase (ASNS) converts aspartate and glutamine to asparagine and glutamate in an ATP-dependent reaction. ASNS is present in most, if not all, mammalian organs, but varies widely in basal expression. Human ASNS activity is highly responsive to cellular stress, primarily by increased transcription from a single gene located on chromosome 7. Elevated ASNS protein expression is associated with resistance to asparaginase therapy in childhood acute lymphoblastic leukemia. There is evidence that ASNS expression levels may also be inversely correlated with asparaginase efficacy in certain solid tumors as well. Children with mutations in the ASNS gene exhibit developmental delays, intellectual disability, microcephaly, intractable seizures, and progressive brain atrophy. Thus far, 15 unique mutations in the ASNS gene have been clinically associated with asparagine synthetase deficiency (ASD). Molecular modeling using the Escherichia coli ASNS-B structure has revealed that most of the reported ASD substitutions are located near catalytic sites or within highly conserved regions of the protein. For some ASD patients, fibroblast cell culture studies have eliminated protein and mRNA synthesis or stability as the basis for decreased proliferation.

      ASNS function

      Asparagine synthetase (ASNS)
      The abbreviations used are: ASNS
      asparagine synthetase
      ASNase
      asparaginase
      AAR
      amino acid response
      ALL
      acute lymphoblastic leukemia
      ASD
      asparagine synthetase deficiency
      ATF
      activating transcription factor
      UPR
      unfolded protein response.
      catalyzes the synthesis of asparagine and glutamate from aspartate and glutamine in an ATP-dependent amidotransferase reaction (Fig. 1A) (
      • Richards N.G.
      • Kilberg M.S.
      Asparagine synthetase chemotherapy.
      ). The recent discovery of a neurologic disorder associated with asparagine synthetase deficiency (ASD) and its long recognized importance in the treatment of acute lymphoblastic leukemia (ALL) highlight the clinical relevance of ASNS as a topic of current interest.
      Figure thumbnail gr1
      Figure 1Mechanism and structural features of human asparagine synthetase. A, the reaction begins when the aspartate carboxyl is activated by an ATP-dependent process, forming a β-aspartyl-AMP intermediate. Glutamine deamidation releases ammonia, which performs a nucleophilic attack on the aspartyl intermediate to produce asparagine. Glutamate is the second product of the overall reaction. B, sequence of human ASNS isoform 1 with residues colored light and dark gray for the N- and C-terminal domains, respectively. α-Helical and β-sheet secondary structures are shown above the sequence. Residues associated with glutamine and ATP binding are colored purple and orange, respectively. Clinically identified ASD mutations are colored according to . C, the N- and C-terminal domains are represented within the surface structure and colored light and dark gray, respectively. The substrates glutamine (purple) and ATP (orange) are indicated by an arrow and shown as sticks. Insets show the substrate-binding pockets. Hydrogen bonds are shown as black dashes with distances labeled in angstroms.
      Many prokaryotes express two forms of ASNS that are characterized by their source of nitrogen donor, either ammonia (AS-A) or glutamine (AS-B (
      • Richards N.G.
      • Kilberg M.S.
      Asparagine synthetase chemotherapy.
      ). The highly conserved residue Cys2 is required for the nucleophilic deamidation of glutamine, and the mutation of this residue reverts AS-B to use ammonia exclusively as the nitrogen source (
      • Richards N.G.J.
      • Schuster S.M.
      Mechanistic issues in asparagine synthetase catalysis.
      ). In mammalian cells, a single form of the ASNS enzyme is expressed that utilizes glutamine as the nitrogen donor in a reaction corresponding to bacterial AS-B (
      • Richards N.G.J.
      • Schuster S.M.
      Mechanistic issues in asparagine synthetase catalysis.
      ). Structure–function studies have previously been performed on Escherichia coli AS-B and have revealed two distinct catalytic domains that are conserved in the human enzyme (
      • Larsen T.M.
      • Boehlein S.K.
      • Schuster S.M.
      • Richards N.G.
      • Thoden J.B.
      • Holden H.M.
      • Rayment I.
      Three-dimensional structure of Escherichia coli asparagine synthetase B: a short journey from substrate to product.
      ) (Fig. 1B). Enzymatic analyses have shown that the catalytic mechanism requires magnesium ions and ATP (
      • Patterson Jr., M.K.
      • Orr G.R.
      Asparagine biosynthesis by the Novikoff Hepatoma isolation, purification, property, and mechanism studies of the enzyme system.
      ). The reaction begins in the C-terminal domain with the activation of the aspartate carboxyl group. This ATP-dependent process forms a β-aspartyl-AMP intermediate that is stabilized by proximal residues of the active site. Within the N-terminal domain, the hydrolysis of glutamine yields glutamate and ammonia, the latter of which diffuses through an intramolecular tunnel to perform a nucleophilic attack on the electrophilic β-aspartyl-AMP intermediate, producing asparagine (Fig. 1A) (
      • Richards N.G.
      • Kilberg M.S.
      Asparagine synthetase chemotherapy.
      ,
      • Larsen T.M.
      • Boehlein S.K.
      • Schuster S.M.
      • Richards N.G.
      • Thoden J.B.
      • Holden H.M.
      • Rayment I.
      Three-dimensional structure of Escherichia coli asparagine synthetase B: a short journey from substrate to product.
      ,
      • Tesson A.R.
      • Soper T.S.
      • Ciustea M.
      • Richards N.G.
      Revisiting the steady state kinetic mechanism of glutamine-dependent asparagine synthetase from Escherichia coli.
      ). Human ASNS is categorized as a class II or N-terminal nucleophile glutamine amidotransferase because the hydrolysis of glutamine occurs in the N-terminal domain of the enzyme (
      • Richards N.G.
      • Kilberg M.S.
      Asparagine synthetase chemotherapy.
      ).

      ASNS protein structure

      The crystal structure of E. coli AS-B (Protein Data Bank (PDB) 1CT9) has been solved (
      • Larsen T.M.
      • Boehlein S.K.
      • Schuster S.M.
      • Richards N.G.
      • Thoden J.B.
      • Holden H.M.
      • Rayment I.
      Three-dimensional structure of Escherichia coli asparagine synthetase B: a short journey from substrate to product.
      ), but a structure of human ASNS has yet to be published or deposited into the PDB. Therefore, the structural features of the human enzyme are based on homology modeling using the E. coli AS-B structure as a template because they share ∼40% sequence identity. The canonical human ASNS enzyme consists of 561 amino acid residues with an approximate molecular mass of 65 kDa (Fig. 1B). However, the UniProt protein database contains two other putative isoforms of human ASNS that differ in the length of the N-terminal domain: isoform 2 (residues 22–561) and isoform 3 (residues 84–561). The expression and physiological importance of these putative isoforms have not been described. With regard to the two functional domains, the N-terminal domain (residues 1–208) consists of a two-layer, antiparallel β-sheet core surrounded by four α-helices (Fig. 1C). This domain contains the glutamine-binding pocket, consisting of residues Arg49, Asn75, Glu77, and Asp97. As observed in the E. coli structure, glutamine is predicted to bind in a manner so that the carboxamide group is oriented toward the interface of the two domains to allow the transfer of an ammonia group from glutamine to aspartate. The C-terminal domain (residues 209–561) is composed primarily of α-helices, but also encompasses a five-stranded, parallel β-sheet that contains the ATP-binding site: residues Leu256, Val288, Asp295, Ser363, Gly364, Glu365, and Asp401 (Fig. 1C). A distance of ∼20 Å separates the two active sites.

      Asparagine synthetase and cancer

      ASNS expression and asparagine metabolism have received considerable attention in transformed cells, beginning with the observation that childhood ALL is susceptible to treatment by the infusion of bacterial asparaginase (ASNase). Primary ALL cells and many ALL cell lines exhibit little or no detectable ASNS and are sensitive to asparagine depletion (
      • Broome J.D.
      Studies on the mechanism of tumor inhibition by l-asparaginase.
      ,
      • Asselin B.L.
      • Kurtzburg J.
      Asparaginase.
      • Rizzari C.
      Asparaginase treatment.
      ). Standard treatment of childhood ALL includes infusion of bacterial ASNase as a principle component of a combinatorial chemotherapy (
      • Pieters R.
      • Hunger S.P.
      • Boos J.
      • Rizzari C.
      • Silverman L.
      • Baruchel A.
      • Goekbuget N.
      • Schrappe M.
      • Pui C.H.
      l-Asparaginase treatment in acute lymphoblastic leukemia: a focus on Erwinia asparaginase.
      ). The circulating ASNase causes rapid depletion of plasma asparagine, followed by the efflux of intracellular asparagine, and thus, starving the leukemia cells to prevent further growth (
      • Aslanian A.M.
      • Kilberg M.S.
      Multiple adaptive mechanisms affect asparagine synthetase substrate availability in asparaginase-resistant MOLT-4 human leukaemia cells.
      ,
      • Avramis V.I.
      Asparaginases: biochemical pharmacology and modes of drug resistance.
      ). In contrast to the increase in ASNS expression in response to substrate deprivation in most normal cells within the body, there is little or no up-regulation of ASNS protein content in ALL cells, rendering them preferentially sensitive to ASNase (
      • Aslanian A.M.
      • Fletcher B.S.
      • Kilberg M.S.
      Asparagine synthetase expression alone is sufficient to induce l-asparaginase resistance in MOLT-4 human leukaemia cells.
      ,
      • Su N.
      • Pan Y.X.
      • Zhou M.
      • Harvey R.C.
      • Hunger S.P.
      • Kilberg M.S.
      Correlation between asparaginase sensitivity and asparagine synthetase protein content, but not mRNA, in acute lymphoblastic leukemia cell lines.
      ).
      The importance of ASNS expression in solid tumor growth and the sensitivity to ASNase have not been investigated as extensively. A screen of 98 human pancreatic ductal carcinomas established that ASNS protein was low or below detection in about 70% of the samples, suggesting that some pancreatic tumors may be susceptible to ASNase therapy (
      • Dufour E.
      • Gay F.
      • Aguera K.
      • Scoazec J.Y.
      • Horand F.
      • Lorenzi P.L.
      • Godfrin Y.
      Pancreatic tumor sensitivity to plasma l-asparagine starvation.
      ). Cui et al. (
      • Cui H.
      • Darmanin S.
      • Natsuisaka M.
      • Kondo T.
      • Asaka M.
      • Shindoh M.
      • Higashino F.
      • Hamuro J.
      • Okada F.
      • Kobayashi M.
      • Nakagawa K.
      • Koide H.
      • Kobayashi M.
      Enhanced expression of asparagine synthetase under glucose-deprived conditions protects pancreatic cancer cells from apoptosis induced by glucose deprivation and cisplatin.
      ) showed that pancreatic cancer cells overexpressing ASNS exhibited increased resistance to apoptosis induced by cisplatin. Similarly, using the NCI-60 human cancer cell panel, Lorenzi et al. (
      • Lorenzi P.L.
      • Reinhold W.C.
      • Rudelius M.
      • Gunsior M.
      • Shankavaram U.
      • Bussey K.J.
      • Scherf U.
      • Eichler G.S.
      • Martin S.E.
      • Chin K.
      • Gray J.W.
      • Kohn E.C.
      • Horak I.D.
      • Von Hoff D.D.
      • Raffeld M.
      • et al.
      Asparagine synthetase as a causal, predictive biomarker for l-asparaginase activity in ovarian cancer cells.
      ) noted a negative correlation between ASNS mRNA levels and susceptibility to ASNase in ovarian cells, as well as increased ASNase sensitivity after siRNA knockdown of ASNS expression. In a second study with a larger number of ovarian cell lines, an inverse correlation between ASNase efficacy and ASNS protein levels was observed (
      • Lorenzi P.L.
      • Llamas J.
      • Gunsior M.
      • Ozbun L.
      • Reinhold W.C.
      • Varma S.
      • Ji H.
      • Kim H.
      • Hutchinson A.A.
      • Kohn E.C.
      • Goldsmith P.K.
      • Birrer M.J.
      • Weinstein J.N.
      Asparagine synthetase is a predictive biomarker of l-asparaginase activity in ovarian cancer cell lines.
      ). These results were consistent with previous observations in human leukemia cells showing that increased ASNase sensitivity correlated with lower ASNS protein expression rather than mRNA content (
      • Su N.
      • Pan Y.X.
      • Zhou M.
      • Harvey R.C.
      • Hunger S.P.
      • Kilberg M.S.
      Correlation between asparaginase sensitivity and asparagine synthetase protein content, but not mRNA, in acute lymphoblastic leukemia cell lines.
      ).
      Protein limitation or an imbalanced dietary amino acid composition leads to intracellular amino acid depletion and activation of ASNS gene transcription through a signaling pathway called the amino acid response (AAR) (
      • Kilberg M.S.
      • Balasubramanian M.
      • Fu L.
      • Shan J.
      The transcription factor network associated with the amino acid response in mammalian cells.
      ). Likewise, endoplasmic reticulum stress also increases ASNS transcription via the unfolded protein response (UPR) (
      • Kilberg M.S.
      • Shan J.
      • Su N.
      ATF4-dependent transcription mediates signaling of amino acid limitation.
      ). These cell stress pathways result in activation of the eIF2 kinases GCN2 (general control nonderepressible) (AAR) and PERK (PKR-like endoplasmic reticulum kinase) (UPR). Phosphorylation of the α-subunit of eIF2 causes a transient, partial suppression of global protein synthesis, but a paradoxical increase in the translation for certain mRNAs, and among these is the transcription factor ATF4 (
      • Baird T.D.
      • Wek R.C.
      Eukaryotic initiation factor 2 phosphorylation and translational control in metabolism.
      ). ATF4 binds to an enhancer element within the proximal promoter of the ASNS gene and activates transcription (Fig. 2) (
      • Balasubramanian M.N.
      • Butterworth E.A.
      • Kilberg M.S.
      Asparagine synthetase: regulation by cell stress and involvement in tumor biology.
      ). Ye et al. (
      • Ye J.
      • Kumanova M.
      • Hart L.S.
      • Sloane K.
      • Zhang H.
      • DePanis D.N.
      • Bobrovnikova-Marjon E.
      • Diehl A.
      • Ron D.
      • Koumenis C.
      The GCN2-ATF4 pathway is critical for tumor cell survival and proliferation in response to nutrient deprivation.
      ) investigated the role of ATF4 in tumor cell survival and proliferation and observed that ATF4 knockdown caused reduced survival in HT1080 fibrosarcoma and DLD1 colorectal adenocarcinoma cells in the absence of nonessential amino acids. Reduced proliferative capacity and increased apoptosis correlated with lower ASNS expression in the ATF4-deficient cells. Supplementation of the tumor cells with asparagine, but not other amino acids, led to increased cell survival. Based on this and other experimental data, Ye et al. (
      • Ye J.
      • Kumanova M.
      • Hart L.S.
      • Sloane K.
      • Zhang H.
      • DePanis D.N.
      • Bobrovnikova-Marjon E.
      • Diehl A.
      • Ron D.
      • Koumenis C.
      The GCN2-ATF4 pathway is critical for tumor cell survival and proliferation in response to nutrient deprivation.
      ) concluded that induction of ATF4: 1) contributes to tumor cell proliferation during nutrient limitation, 2) is a component of starvation-induced autophagy in cancer cells, and 3) induces ASNS as a key factor in tumor initiation and growth under amino acid–limiting conditions. The exact role of ASNS activity in modulating tumor growth is unknown. One obvious possibility, an impact on protein synthesis, seems tenuous as other amino acid synthetic enzymes are not as highly regulated as ASNS.
      Figure thumbnail gr2
      Figure 2Regulation of ASNS expression. Asparagine depletion activates the AAR, whereas endoplasmic reticulum stress (ER Stress) activates the UPR. Each stress condition increases the activity of an eIF2 kinase. Phosphorylation of eIF2 slows global protein synthesis, but paradoxically increases translation of a subset of mRNAs, including that for the transcription factor ATF4. Binding of ATF4 to an enhancer element within the promoter of the ASNS gene induces expression of the enzyme.

      Asparagine synthetase deficiency

      ASD is a recently characterized neurological disorder having severe impacts on psychomotor development and mortality at an early age. Multiple patient studies have been conducted over the last few years due to the rising awareness of the disease. Symptoms include intellectual disability, microcephaly, severe developmental delay, intractable seizures, progressive brain atrophy, and more rarely, respiratory deficiency (
      • Ruzzo E.K.
      • Capo-Chichi J.M.
      • Ben-Zeev B.
      • Chitayat D.
      • Mao H.
      • Pappas A.L.
      • Hitomi Y.
      • Lu Y.F.
      • Yao X.
      • Hamdan F.F.
      • Pelak K.
      • Reznik-Wolf H.
      • Bar-Joseph I.
      • Oz-Levi D.
      • Lev D.
      • et al.
      Deficiency of asparagine synthetase causes congenital microcephaly and a progressive form of encephalopathy.
      • Alfadhel M.
      • Alrifai M.T.
      • Trujillano D.
      • Alshaalan H.
      • Al Othaim A.
      • Al Rasheed S.
      • Assiri H.
      • Alqahtani A.A.
      • Alaamery M.
      • Rolfs A.
      • Eyaid W.
      Asparagine synthetase deficiency: new inborn errors of metabolism.
      ,
      • Ben-Salem S.
      • Gleeson J.G.
      • Al-Shamsi A.M.
      • Islam B.
      • Hertecant J.
      • Ali B.R.
      • Al-Gazali L.
      Asparagine synthetase deficiency detected by whole exome sequencing causes congenital microcephaly, epileptic encephalopathy and psychomotor delay.
      ,
      • Palmer E.E.
      • Hayner J.
      • Sachdev R.
      • Cardamone M.
      • Kandula T.
      • Morris P.
      • Dias K.R.
      • Tao J.
      • Miller D.
      • Zhu Y.
      • Macintosh R.
      • Dinger M.E.
      • Cowley M.J.
      • Buckley M.F.
      • Roscioli T.
      • Bye A.
      • Kilberg M.S.
      • Kirk E.P.
      Asparagine Synthetase Deficiency causes reduced proliferation of cells under conditions of limited asparagine.
      ,
      • Seidahmed M.Z.
      • Salih M.A.
      • Abdulbasit O.B.
      • Samadi A.
      • Al Hussien K.
      • Miqdad A.M.
      • Biary M.S.
      • Alazami A.M.
      • Alorainy I.A.
      • Kabiraj M.M.
      • Shaheen R.
      • Alkuraya F.S.
      Hyperekplexia, microcephaly and simplified gyral pattern caused by novel ASNS mutations, case report.
      ,
      • Gataullina S.
      • Lauer-Zillhardt J.
      • Kaminska A.
      • Galmiche-Rolland L.
      • Bahi-Buisson N.
      • Pontoizeau C.
      • Ottolenghi C.
      • Dulac O.
      • Fallet-Bianco C.
      Epileptic phenotype of two siblings with asparagine synthesis deficiency mimics neonatal pyridoxine-dependent epilepsy.
      ,
      • Sun J.
      • McGillivray A.J.
      • Pinner J.
      • Yan Z.
      • Liu F.
      • Bratkovic D.
      • Thompson E.
      • Wei X.
      • Jiang H.
      • Asan
      • Chopra M.
      Diaphragmatic eventration in sisters with asparagine synthetase deficiency: a novel homozygous ASNS mutation and expanded phenotype.
      • Yamamoto T.
      • Endo W.
      • Ohnishi H.
      • Kubota K.
      • Kawamoto N.
      • Inui T.
      • Imamura A.
      • Takanashi J.I.
      • Shiina M.
      • Saitsu H.
      • Ogata K.
      • Matsumoto N.
      • Haginoya K.
      • Fukao T.
      The first report of Japanese patients with asparagine synthetase deficiency.
      ). The disease is associated with homozygous or compound heterozygous mutations within the ASNS gene on chromosome 7q2, but the exact mechanisms that cause the overt symptoms of the disease are not well understood (
      • Ruzzo E.K.
      • Capo-Chichi J.M.
      • Ben-Zeev B.
      • Chitayat D.
      • Mao H.
      • Pappas A.L.
      • Hitomi Y.
      • Lu Y.F.
      • Yao X.
      • Hamdan F.F.
      • Pelak K.
      • Reznik-Wolf H.
      • Bar-Joseph I.
      • Oz-Levi D.
      • Lev D.
      • et al.
      Deficiency of asparagine synthetase causes congenital microcephaly and a progressive form of encephalopathy.
      ,
      • Palmer E.E.
      • Hayner J.
      • Sachdev R.
      • Cardamone M.
      • Kandula T.
      • Morris P.
      • Dias K.R.
      • Tao J.
      • Miller D.
      • Zhu Y.
      • Macintosh R.
      • Dinger M.E.
      • Cowley M.J.
      • Buckley M.F.
      • Roscioli T.
      • Bye A.
      • Kilberg M.S.
      • Kirk E.P.
      Asparagine Synthetase Deficiency causes reduced proliferation of cells under conditions of limited asparagine.
      ,
      • Gupta N.
      • Tewari V.V.
      • Kumar M.
      • Langeh N.
      • Gupta A.
      • Mishra P.
      • Kaur P.
      • Ramprasad V.
      • Murugan S.
      • Kumar R.
      • Jana M.
      • Kabra M.
      Asparagine Synthetase deficiency: report of a novel mutation and review of literature.
      ). The fact that the children are born with epileptic-like seizures and microcephaly suggests that brain ASNS activity is critical for development of this organ. Currently, the disease can only be diagnosed through DNA sequencing. Some, but not all, affected individuals have a measurable decrease in the amount of asparagine in their serum or cerebrospinal fluid, which limits this analysis as a preliminary screen in suspected cases.
      Based on animal growth studies, asparagine is traditionally defined as a “nonessential” or “dispensable” amino acid. ASNS is expressed to varying degrees within human organs (http://www.proteinatlas.org/ENSG00000070669-ASNS/tissue),
      Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site.
      but is particularly high in pancreas (
      • Milman H.A.
      • Cooney D.A.
      The distribution of l-asparagine synthetase in the principal organs of several mammalian and avian species.
      ). However, when considering the requirement for asparagine at the level of individual cells, ASNS deficiency leads to extracellular asparagine dependence, as discussed above for ALL cells. Individuals with ASD express phenotypic impairments in the brain that are not readily apparent in other organs, suggesting a tissue-specific dependence on asparagine for neural development (
      • Ruzzo E.K.
      • Capo-Chichi J.M.
      • Ben-Zeev B.
      • Chitayat D.
      • Mao H.
      • Pappas A.L.
      • Hitomi Y.
      • Lu Y.F.
      • Yao X.
      • Hamdan F.F.
      • Pelak K.
      • Reznik-Wolf H.
      • Bar-Joseph I.
      • Oz-Levi D.
      • Lev D.
      • et al.
      Deficiency of asparagine synthetase causes congenital microcephaly and a progressive form of encephalopathy.
      ). Although asparagine can be obtained through the diet, it is transported by an equilibrating, bidirectional facilitated transporter (System n) across the luminal surface of the endothelial cells that make up the blood–brain barrier. Therefore, asparagine is not actively accumulated within the brain (
      • Hawkins R.A.
      • O’Kane R.L.
      • Simpson I.A.
      • Viña J.R.
      Structure of the blood-brain barrier and its role in the transport of amino acids.
      ). Indeed, Na+-dependent transport of asparagine across the abluminal membrane of the endothelial layer appears to be designed to remove the amino acid from the brain. Consistent with this hypothesis, the cerebrospinal fluid levels of asparagine, as with many amino acids, are only a fraction of those in the plasma (
      • Scholl-Bürgi S.
      • Haberlandt E.
      • Heinz-Erian P.
      • Deisenhammer F.
      • Albrecht U.
      • Sigl S.B.
      • Rauchenzauner M.
      • Ulmer H.
      • Karall D.
      Amino acid cerebrospinal fluid/plasma ratios in children: influence of age, gender, and antiepileptic medication.
      ,
      • Akiyama T.
      • Kobayashi K.
      • Higashikage A.
      • Sato J.
      • Yoshinaga H.
      CSF/plasma ratios of amino acids: reference data and transports in children.
      ). Consequently, a decrease in ASNS catalytic activity in the brain is presumed to cause the disease phenotype (
      • Ruzzo E.K.
      • Capo-Chichi J.M.
      • Ben-Zeev B.
      • Chitayat D.
      • Mao H.
      • Pappas A.L.
      • Hitomi Y.
      • Lu Y.F.
      • Yao X.
      • Hamdan F.F.
      • Pelak K.
      • Reznik-Wolf H.
      • Bar-Joseph I.
      • Oz-Levi D.
      • Lev D.
      • et al.
      Deficiency of asparagine synthetase causes congenital microcephaly and a progressive form of encephalopathy.
      ,
      • Alfadhel M.
      • Alrifai M.T.
      • Trujillano D.
      • Alshaalan H.
      • Al Othaim A.
      • Al Rasheed S.
      • Assiri H.
      • Alqahtani A.A.
      • Alaamery M.
      • Rolfs A.
      • Eyaid W.
      Asparagine synthetase deficiency: new inborn errors of metabolism.
      ,
      • Palmer E.E.
      • Hayner J.
      • Sachdev R.
      • Cardamone M.
      • Kandula T.
      • Morris P.
      • Dias K.R.
      • Tao J.
      • Miller D.
      • Zhu Y.
      • Macintosh R.
      • Dinger M.E.
      • Cowley M.J.
      • Buckley M.F.
      • Roscioli T.
      • Bye A.
      • Kilberg M.S.
      • Kirk E.P.
      Asparagine Synthetase Deficiency causes reduced proliferation of cells under conditions of limited asparagine.
      ). Plasma asparagine levels were reduced in 8 out of 16 ASD patients for whom plasma analysis was reported (reviewed in Ref.
      • Gupta N.
      • Tewari V.V.
      • Kumar M.
      • Langeh N.
      • Gupta A.
      • Mishra P.
      • Kaur P.
      • Ramprasad V.
      • Murugan S.
      • Kumar R.
      • Jana M.
      • Kabra M.
      Asparagine Synthetase deficiency: report of a novel mutation and review of literature.
      ). However, cerebrospinal fluid asparagine content has been analyzed in only four ASD patients, and it was undetectable in two of the four (
      • Alfadhel M.
      • Alrifai M.T.
      • Trujillano D.
      • Alshaalan H.
      • Al Othaim A.
      • Al Rasheed S.
      • Assiri H.
      • Alqahtani A.A.
      • Alaamery M.
      • Rolfs A.
      • Eyaid W.
      Asparagine synthetase deficiency: new inborn errors of metabolism.
      ,
      • Seidahmed M.Z.
      • Salih M.A.
      • Abdulbasit O.B.
      • Samadi A.
      • Al Hussien K.
      • Miqdad A.M.
      • Biary M.S.
      • Alazami A.M.
      • Alorainy I.A.
      • Kabiraj M.M.
      • Shaheen R.
      • Alkuraya F.S.
      Hyperekplexia, microcephaly and simplified gyral pattern caused by novel ASNS mutations, case report.
      ,
      • Yamamoto T.
      • Endo W.
      • Ohnishi H.
      • Kubota K.
      • Kawamoto N.
      • Inui T.
      • Imamura A.
      • Takanashi J.I.
      • Shiina M.
      • Saitsu H.
      • Ogata K.
      • Matsumoto N.
      • Haginoya K.
      • Fukao T.
      The first report of Japanese patients with asparagine synthetase deficiency.
      ). Recent studies with ASD patients’ fibroblasts have revealed that lowering the asparagine concentration in the extracellular milieu results in an inability to proliferate (
      • Palmer E.E.
      • Hayner J.
      • Sachdev R.
      • Cardamone M.
      • Kandula T.
      • Morris P.
      • Dias K.R.
      • Tao J.
      • Miller D.
      • Zhu Y.
      • Macintosh R.
      • Dinger M.E.
      • Cowley M.J.
      • Buckley M.F.
      • Roscioli T.
      • Bye A.
      • Kilberg M.S.
      • Kirk E.P.
      Asparagine Synthetase Deficiency causes reduced proliferation of cells under conditions of limited asparagine.
      ). These cell culture studies also showed that fibroblasts from asymptomatic heterozygote parents exhibit no ill effects in the presence of sufficient extracellular asparagine, but decline in proliferative capacity when placed in an asparagine-free medium. Collectively, investigation of ASD patients indicates that ASNS activity in the brain is crucial for organ development. Whether this dependence is the direct result of perturbations in asparagine metabolism, or one of the other ASNS reactants, must be established by further analysis and experimentation.

      Structural examination of ASD mutations

      To date, 15 unique mutations in the ASNS gene have been clinically identified in association with ASD (
      • Ruzzo E.K.
      • Capo-Chichi J.M.
      • Ben-Zeev B.
      • Chitayat D.
      • Mao H.
      • Pappas A.L.
      • Hitomi Y.
      • Lu Y.F.
      • Yao X.
      • Hamdan F.F.
      • Pelak K.
      • Reznik-Wolf H.
      • Bar-Joseph I.
      • Oz-Levi D.
      • Lev D.
      • et al.
      Deficiency of asparagine synthetase causes congenital microcephaly and a progressive form of encephalopathy.
      • Alfadhel M.
      • Alrifai M.T.
      • Trujillano D.
      • Alshaalan H.
      • Al Othaim A.
      • Al Rasheed S.
      • Assiri H.
      • Alqahtani A.A.
      • Alaamery M.
      • Rolfs A.
      • Eyaid W.
      Asparagine synthetase deficiency: new inborn errors of metabolism.
      ,
      • Ben-Salem S.
      • Gleeson J.G.
      • Al-Shamsi A.M.
      • Islam B.
      • Hertecant J.
      • Ali B.R.
      • Al-Gazali L.
      Asparagine synthetase deficiency detected by whole exome sequencing causes congenital microcephaly, epileptic encephalopathy and psychomotor delay.
      ,
      • Palmer E.E.
      • Hayner J.
      • Sachdev R.
      • Cardamone M.
      • Kandula T.
      • Morris P.
      • Dias K.R.
      • Tao J.
      • Miller D.
      • Zhu Y.
      • Macintosh R.
      • Dinger M.E.
      • Cowley M.J.
      • Buckley M.F.
      • Roscioli T.
      • Bye A.
      • Kilberg M.S.
      • Kirk E.P.
      Asparagine Synthetase Deficiency causes reduced proliferation of cells under conditions of limited asparagine.
      ,
      • Seidahmed M.Z.
      • Salih M.A.
      • Abdulbasit O.B.
      • Samadi A.
      • Al Hussien K.
      • Miqdad A.M.
      • Biary M.S.
      • Alazami A.M.
      • Alorainy I.A.
      • Kabiraj M.M.
      • Shaheen R.
      • Alkuraya F.S.
      Hyperekplexia, microcephaly and simplified gyral pattern caused by novel ASNS mutations, case report.
      ,
      • Gataullina S.
      • Lauer-Zillhardt J.
      • Kaminska A.
      • Galmiche-Rolland L.
      • Bahi-Buisson N.
      • Pontoizeau C.
      • Ottolenghi C.
      • Dulac O.
      • Fallet-Bianco C.
      Epileptic phenotype of two siblings with asparagine synthesis deficiency mimics neonatal pyridoxine-dependent epilepsy.
      ,
      • Sun J.
      • McGillivray A.J.
      • Pinner J.
      • Yan Z.
      • Liu F.
      • Bratkovic D.
      • Thompson E.
      • Wei X.
      • Jiang H.
      • Asan
      • Chopra M.
      Diaphragmatic eventration in sisters with asparagine synthetase deficiency: a novel homozygous ASNS mutation and expanded phenotype.
      ,
      • Yamamoto T.
      • Endo W.
      • Ohnishi H.
      • Kubota K.
      • Kawamoto N.
      • Inui T.
      • Imamura A.
      • Takanashi J.I.
      • Shiina M.
      • Saitsu H.
      • Ogata K.
      • Matsumoto N.
      • Haginoya K.
      • Fukao T.
      The first report of Japanese patients with asparagine synthetase deficiency.
      • Gupta N.
      • Tewari V.V.
      • Kumar M.
      • Langeh N.
      • Gupta A.
      • Mishra P.
      • Kaur P.
      • Ramprasad V.
      • Murugan S.
      • Kumar R.
      • Jana M.
      • Kabra M.
      Asparagine Synthetase deficiency: report of a novel mutation and review of literature.
      ). ASD is characterized as a pan-ethnic disorder because it has been identified within a variety of ethnic groups. Several patients have mutations localized at or near the ATP-binding site, whereas thus far, only one patient has been identified with a mutation in the glutamine-binding site (Table 1). The remaining mutations are located outside the active sites, but in highly conserved regions, which may reduce protein stability without necessarily inhibiting enzyme activity (
      • Ruzzo E.K.
      • Capo-Chichi J.M.
      • Ben-Zeev B.
      • Chitayat D.
      • Mao H.
      • Pappas A.L.
      • Hitomi Y.
      • Lu Y.F.
      • Yao X.
      • Hamdan F.F.
      • Pelak K.
      • Reznik-Wolf H.
      • Bar-Joseph I.
      • Oz-Levi D.
      • Lev D.
      • et al.
      Deficiency of asparagine synthetase causes congenital microcephaly and a progressive form of encephalopathy.
      ). Alignment of the three isoforms of human ASNS and renumbering of the mutations to correlate to the isoform 1 sequence highlights that previously reported mutations Y315C (
      • Seidahmed M.Z.
      • Salih M.A.
      • Abdulbasit O.B.
      • Samadi A.
      • Al Hussien K.
      • Miqdad A.M.
      • Biary M.S.
      • Alazami A.M.
      • Alorainy I.A.
      • Kabiraj M.M.
      • Shaheen R.
      • Alkuraya F.S.
      Hyperekplexia, microcephaly and simplified gyral pattern caused by novel ASNS mutations, case report.
      ) and Y377C (
      • Alfadhel M.
      • Alrifai M.T.
      • Trujillano D.
      • Alshaalan H.
      • Al Othaim A.
      • Al Rasheed S.
      • Assiri H.
      • Alqahtani A.A.
      • Alaamery M.
      • Rolfs A.
      • Eyaid W.
      Asparagine synthetase deficiency: new inborn errors of metabolism.
      ) are equivalent to Y398C (
      • Ben-Salem S.
      • Gleeson J.G.
      • Al-Shamsi A.M.
      • Islam B.
      • Hertecant J.
      • Ali B.R.
      • Al-Gazali L.
      Asparagine synthetase deficiency detected by whole exome sequencing causes congenital microcephaly, epileptic encephalopathy and psychomotor delay.
      ), whereas Arg324* would be renumbered to Arg407* (
      • Seidahmed M.Z.
      • Salih M.A.
      • Abdulbasit O.B.
      • Samadi A.
      • Al Hussien K.
      • Miqdad A.M.
      • Biary M.S.
      • Alazami A.M.
      • Alorainy I.A.
      • Kabiraj M.M.
      • Shaheen R.
      • Alkuraya F.S.
      Hyperekplexia, microcephaly and simplified gyral pattern caused by novel ASNS mutations, case report.
      ) (Fig. 1B).
      Table 1Reported mutations in ASD patients
      MutationTypeStructural consequenceReference
      A6ECHCharged amino acid in hydrophobic region, steric clash with Phe8
      • Ruzzo E.K.
      • Capo-Chichi J.M.
      • Ben-Zeev B.
      • Chitayat D.
      • Mao H.
      • Pappas A.L.
      • Hitomi Y.
      • Lu Y.F.
      • Yao X.
      • Hamdan F.F.
      • Pelak K.
      • Reznik-Wolf H.
      • Bar-Joseph I.
      • Oz-Levi D.
      • Lev D.
      • et al.
      Deficiency of asparagine synthetase causes congenital microcephaly and a progressive form of encephalopathy.
      R550CDecrease in side chain length likely to result in loss of interactions
      R49QHGlutamine-binding site, loss of hydrogen bondingSee Footnote 6
      L145SCHPolar side chain in hydrophobic region
      • Yamamoto T.
      • Endo W.
      • Ohnishi H.
      • Kubota K.
      • Kawamoto N.
      • Inui T.
      • Imamura A.
      • Takanashi J.I.
      • Shiina M.
      • Saitsu H.
      • Ogata K.
      • Matsumoto N.
      • Haginoya K.
      • Fukao T.
      The first report of Japanese patients with asparagine synthetase deficiency.
      L247WDecrease in van der Waals interactions
      G289ACHProximal to ATP-binding site, steric hindrance with Ser293
      • Palmer E.E.
      • Hayner J.
      • Sachdev R.
      • Cardamone M.
      • Kandula T.
      • Morris P.
      • Dias K.R.
      • Tao J.
      • Miller D.
      • Zhu Y.
      • Macintosh R.
      • Dinger M.E.
      • Cowley M.J.
      • Buckley M.F.
      • Roscioli T.
      • Bye A.
      • Kilberg M.S.
      • Kirk E.P.
      Asparagine Synthetase Deficiency causes reduced proliferation of cells under conditions of limited asparagine.
      T337IProximal to ATP-binding site, hydrophobic patch on protein that may decrease solubility
      R340HHLoss of hydrogen bonds, steric clash with Phe482
      • Sun J.
      • McGillivray A.J.
      • Pinner J.
      • Yan Z.
      • Liu F.
      • Bratkovic D.
      • Thompson E.
      • Wei X.
      • Jiang H.
      • Asan
      • Chopra M.
      Diaphragmatic eventration in sisters with asparagine synthetase deficiency: a novel homozygous ASNS mutation and expanded phenotype.
      F362VHDecrease in van der Waals interactions
      • Ruzzo E.K.
      • Capo-Chichi J.M.
      • Ben-Zeev B.
      • Chitayat D.
      • Mao H.
      • Pappas A.L.
      • Hitomi Y.
      • Lu Y.F.
      • Yao X.
      • Hamdan F.F.
      • Pelak K.
      • Reznik-Wolf H.
      • Bar-Joseph I.
      • Oz-Levi D.
      • Lev D.
      • et al.
      Deficiency of asparagine synthetase causes congenital microcephaly and a progressive form of encephalopathy.
      A380SHPolar residue in hydrophobic region
      • Gupta N.
      • Tewari V.V.
      • Kumar M.
      • Langeh N.
      • Gupta A.
      • Mishra P.
      • Kaur P.
      • Ramprasad V.
      • Murugan S.
      • Kumar R.
      • Jana M.
      • Kabra M.
      Asparagine Synthetase deficiency: report of a novel mutation and review of literature.
      Y398CHDecrease in van der Waals interactions, solvent-accessible thiol group
      • Alfadhel M.
      • Alrifai M.T.
      • Trujillano D.
      • Alshaalan H.
      • Al Othaim A.
      • Al Rasheed S.
      • Assiri H.
      • Alqahtani A.A.
      • Alaamery M.
      • Rolfs A.
      • Eyaid W.
      Asparagine synthetase deficiency: new inborn errors of metabolism.
      ,
      • Ben-Salem S.
      • Gleeson J.G.
      • Al-Shamsi A.M.
      • Islam B.
      • Hertecant J.
      • Ali B.R.
      • Al-Gazali L.
      Asparagine synthetase deficiency detected by whole exome sequencing causes congenital microcephaly, epileptic encephalopathy and psychomotor delay.
      ,
      • Seidahmed M.Z.
      • Salih M.A.
      • Abdulbasit O.B.
      • Samadi A.
      • Al Hussien K.
      • Miqdad A.M.
      • Biary M.S.
      • Alazami A.M.
      • Alorainy I.A.
      • Kabiraj M.M.
      • Shaheen R.
      • Alkuraya F.S.
      Hyperekplexia, microcephaly and simplified gyral pattern caused by novel ASNS mutations, case report.
      R407*HProtein truncation
      • Seidahmed M.Z.
      • Salih M.A.
      • Abdulbasit O.B.
      • Samadi A.
      • Al Hussien K.
      • Miqdad A.M.
      • Biary M.S.
      • Alazami A.M.
      • Alorainy I.A.
      • Kabiraj M.M.
      • Shaheen R.
      • Alkuraya F.S.
      Hyperekplexia, microcephaly and simplified gyral pattern caused by novel ASNS mutations, case report.
      S480FCHNonpolar residue on protein surface that may decrease solubility
      • Gataullina S.
      • Lauer-Zillhardt J.
      • Kaminska A.
      • Galmiche-Rolland L.
      • Bahi-Buisson N.
      • Pontoizeau C.
      • Ottolenghi C.
      • Dulac O.
      • Fallet-Bianco C.
      Epileptic phenotype of two siblings with asparagine synthesis deficiency mimics neonatal pyridoxine-dependent epilepsy.
      R550CDecrease in side chain length likely to result in loss of interactions
      V489DCHCharged amino acid in hydrophobic region
      • Yamamoto T.
      • Endo W.
      • Ohnishi H.
      • Kubota K.
      • Kawamoto N.
      • Inui T.
      • Imamura A.
      • Takanashi J.I.
      • Shiina M.
      • Saitsu H.
      • Ogata K.
      • Matsumoto N.
      • Haginoya K.
      • Fukao T.
      The first report of Japanese patients with asparagine synthetase deficiency.
      W541Cfs*5Protein truncation
      R550CHDecrease in side chain length likely to result in loss of interactions
      • Ruzzo E.K.
      • Capo-Chichi J.M.
      • Ben-Zeev B.
      • Chitayat D.
      • Mao H.
      • Pappas A.L.
      • Hitomi Y.
      • Lu Y.F.
      • Yao X.
      • Hamdan F.F.
      • Pelak K.
      • Reznik-Wolf H.
      • Bar-Joseph I.
      • Oz-Levi D.
      • Lev D.
      • et al.
      Deficiency of asparagine synthetase causes congenital microcephaly and a progressive form of encephalopathy.
      Cell culture analyses have shown that several of the aforementioned mutations do not affect mRNA stability (
      • Ruzzo E.K.
      • Capo-Chichi J.M.
      • Ben-Zeev B.
      • Chitayat D.
      • Mao H.
      • Pappas A.L.
      • Hitomi Y.
      • Lu Y.F.
      • Yao X.
      • Hamdan F.F.
      • Pelak K.
      • Reznik-Wolf H.
      • Bar-Joseph I.
      • Oz-Levi D.
      • Lev D.
      • et al.
      Deficiency of asparagine synthetase causes congenital microcephaly and a progressive form of encephalopathy.
      ,
      • Palmer E.E.
      • Hayner J.
      • Sachdev R.
      • Cardamone M.
      • Kandula T.
      • Morris P.
      • Dias K.R.
      • Tao J.
      • Miller D.
      • Zhu Y.
      • Macintosh R.
      • Dinger M.E.
      • Cowley M.J.
      • Buckley M.F.
      • Roscioli T.
      • Bye A.
      • Kilberg M.S.
      • Kirk E.P.
      Asparagine Synthetase Deficiency causes reduced proliferation of cells under conditions of limited asparagine.
      ). Rather, many of the disease-associated mutations are expected to decrease enzyme activity and/or protein stability. Analysis of the human ASNS structural model incorporating the observed amino acid mutations illustrates an overall theme of protein destabilization. The mutations can be classified into four groups based upon their putative effect on the protein structure: change in the amino acid type (hydrophobic to hydrophilic or vice versa), loss of hydrogen bonding or van der Waals interactions, truncation of the protein, and predicted decrease in substrate binding. The introduction of charged or polar residues into hydrophobic regions of the enzyme is expected to destabilize the structure as predicted for the A6E, L145S, A380S, and V489D mutations (
      • Ruzzo E.K.
      • Capo-Chichi J.M.
      • Ben-Zeev B.
      • Chitayat D.
      • Mao H.
      • Pappas A.L.
      • Hitomi Y.
      • Lu Y.F.
      • Yao X.
      • Hamdan F.F.
      • Pelak K.
      • Reznik-Wolf H.
      • Bar-Joseph I.
      • Oz-Levi D.
      • Lev D.
      • et al.
      Deficiency of asparagine synthetase causes congenital microcephaly and a progressive form of encephalopathy.
      ,
      • Yamamoto T.
      • Endo W.
      • Ohnishi H.
      • Kubota K.
      • Kawamoto N.
      • Inui T.
      • Imamura A.
      • Takanashi J.I.
      • Shiina M.
      • Saitsu H.
      • Ogata K.
      • Matsumoto N.
      • Haginoya K.
      • Fukao T.
      The first report of Japanese patients with asparagine synthetase deficiency.
      ). In addition, the increase in side-chain length of the A6E mutation results in steric hindrance with proximal residue Phe8. Conversely, the S480F mutation presents a nonpolar amino acid on the surface of protein that may decrease solubility (Fig. 3) (
      • Gataullina S.
      • Lauer-Zillhardt J.
      • Kaminska A.
      • Galmiche-Rolland L.
      • Bahi-Buisson N.
      • Pontoizeau C.
      • Ottolenghi C.
      • Dulac O.
      • Fallet-Bianco C.
      Epileptic phenotype of two siblings with asparagine synthesis deficiency mimics neonatal pyridoxine-dependent epilepsy.
      ). Several mutations result in the loss of stabilizing interactions. L247W causes a reorientation of the larger aromatic side chain out of a hydrophobic pocket to avoid steric clash, resulting in the loss of van der Waals interactions (
      • Yamamoto T.
      • Endo W.
      • Ohnishi H.
      • Kubota K.
      • Kawamoto N.
      • Inui T.
      • Imamura A.
      • Takanashi J.I.
      • Shiina M.
      • Saitsu H.
      • Ogata K.
      • Matsumoto N.
      • Haginoya K.
      • Fukao T.
      The first report of Japanese patients with asparagine synthetase deficiency.
      ). Similarly, the reduction in side-chain size is likely to cause a decrease in van der Waals interactions in F362V (
      • Ruzzo E.K.
      • Capo-Chichi J.M.
      • Ben-Zeev B.
      • Chitayat D.
      • Mao H.
      • Pappas A.L.
      • Hitomi Y.
      • Lu Y.F.
      • Yao X.
      • Hamdan F.F.
      • Pelak K.
      • Reznik-Wolf H.
      • Bar-Joseph I.
      • Oz-Levi D.
      • Lev D.
      • et al.
      Deficiency of asparagine synthetase causes congenital microcephaly and a progressive form of encephalopathy.
      ) and a loss of hydrogen bonding in the R340H mutant (
      • Sun J.
      • McGillivray A.J.
      • Pinner J.
      • Yan Z.
      • Liu F.
      • Bratkovic D.
      • Thompson E.
      • Wei X.
      • Jiang H.
      • Asan
      • Chopra M.
      Diaphragmatic eventration in sisters with asparagine synthetase deficiency: a novel homozygous ASNS mutation and expanded phenotype.
      ), which is further destabilized by steric hindrance with Phe482. In addition to the introduction of a reactive, solvent-accessible thiol group, the loss of van der Waals interactions in Y398C is predicted to disrupt contacts between the N and C domains (
      • Alfadhel M.
      • Alrifai M.T.
      • Trujillano D.
      • Alshaalan H.
      • Al Othaim A.
      • Al Rasheed S.
      • Assiri H.
      • Alqahtani A.A.
      • Alaamery M.
      • Rolfs A.
      • Eyaid W.
      Asparagine synthetase deficiency: new inborn errors of metabolism.
      ,
      • Ben-Salem S.
      • Gleeson J.G.
      • Al-Shamsi A.M.
      • Islam B.
      • Hertecant J.
      • Ali B.R.
      • Al-Gazali L.
      Asparagine synthetase deficiency detected by whole exome sequencing causes congenital microcephaly, epileptic encephalopathy and psychomotor delay.
      ,
      • Seidahmed M.Z.
      • Salih M.A.
      • Abdulbasit O.B.
      • Samadi A.
      • Al Hussien K.
      • Miqdad A.M.
      • Biary M.S.
      • Alazami A.M.
      • Alorainy I.A.
      • Kabiraj M.M.
      • Shaheen R.
      • Alkuraya F.S.
      Hyperekplexia, microcephaly and simplified gyral pattern caused by novel ASNS mutations, case report.
      ). Although the R550C mutation is not included in the human ASNS model due to the lack of inclusion in the E. coli AS-B structure, it is expected to result in destabilization of the C-terminal domain (Fig. 3).
      Figure thumbnail gr3
      Figure 3ASD associated mutations in the human ASNS enzyme. Mutations are represented as sticks within the predicted ASNS structure. Top panel, A6E (red), L145S (salmon), T337I (light green), R340H (dark green), A380S (light blue), and Y398C (dark blue). Bottom panel (180° rotation around y axis), R49Q (raspberry), L247W (light orange), G289A (yellow), F362V (teal), S480F (light pink), and V489D (dark pink). Interacting residues are also shown as sticks, and hydrogen bonds are represented as black dashes.
      Two observed mutations are predicted to result in the truncation of the human ASNS protein. The nonsense mutation Arg407* introduces a premature stop codon (
      • Seidahmed M.Z.
      • Salih M.A.
      • Abdulbasit O.B.
      • Samadi A.
      • Al Hussien K.
      • Miqdad A.M.
      • Biary M.S.
      • Alazami A.M.
      • Alorainy I.A.
      • Kabiraj M.M.
      • Shaheen R.
      • Alkuraya F.S.
      Hyperekplexia, microcephaly and simplified gyral pattern caused by novel ASNS mutations, case report.
      ), whereas the frameshift mutation W541Cfs*5 truncates the enzyme, containing only five codons after the mutated residue 541 (
      • Yamamoto T.
      • Endo W.
      • Ohnishi H.
      • Kubota K.
      • Kawamoto N.
      • Inui T.
      • Imamura A.
      • Takanashi J.I.
      • Shiina M.
      • Saitsu H.
      • Ogata K.
      • Matsumoto N.
      • Haginoya K.
      • Fukao T.
      The first report of Japanese patients with asparagine synthetase deficiency.
      ). The remaining reported mutations are not only predicted to destabilize the structure of the protein, but also potentially decrease the binding of substrate and possibly decrease the catalytic efficiency of the ASNS enzyme. R49Q is the first mutation reported to be in the glutamine-binding pocket of the N-terminal domain.
      S. J. Sacharow, E. E. Dudenhausen, C. L. Lomelino, L. Rodan, C. Moufawad El Achkar, H. E. Olson, C. A. Genetti, P. B. Agrawal, R. McKenna, and M. S. Kilberg, submitted for review.
      This mutation not only results in a loss of hydrogen bonds with the second β-sheet, but also a loss in hydrogen bonds with the glutamine substrate. The C-terminal domain mutations G289A and T337I are associated with an afflicted child who is a compound heterozygote. Both mutations are located proximal to the ATP-binding pocket (
      • Palmer E.E.
      • Hayner J.
      • Sachdev R.
      • Cardamone M.
      • Kandula T.
      • Morris P.
      • Dias K.R.
      • Tao J.
      • Miller D.
      • Zhu Y.
      • Macintosh R.
      • Dinger M.E.
      • Cowley M.J.
      • Buckley M.F.
      • Roscioli T.
      • Bye A.
      • Kilberg M.S.
      • Kirk E.P.
      Asparagine Synthetase Deficiency causes reduced proliferation of cells under conditions of limited asparagine.
      ). The G289A mutation results in a steric clash with Ser293, whereas the T337I mutation introduces a hydrophobic patch on the protein surface that may decrease solubility. Although these two residues are not predicted to interact directly with the ATP molecule, the mutations are expected to destabilize the region of the ATP-binding site, and therefore, potentially decrease the affinity of ASNS for this substrate (Fig. 3).

      Remaining questions

      Although many aspects of ASNS enzymology and regulation have been investigated over the last four decades, major gaps remain in our knowledge. The exact contribution of ASNS activity in maintaining the cellular and whole body homeostasis of its four amino acid reactants remains largely unanswered. Although the enzyme name leads one to focus on asparagine synthesis, given the overall reaction, the activity does consume glutamine and aspartate, and produce glutamate. Whether or not ASNS actually impacts the cellular homeostasis of one or more of the other reactants remains speculative. Given the much higher levels of glutamine in most cells, the chance of changes in ASNS activity contributing to the cellular aspartate and glutamate abundance would be more likely. However, the in vivo impact during embryonic development or physiological status during periods of up-regulation in response to cellular stresses that activate the AAR or UPR pathways would be valuable studies in tissue-specific ASNS knock-out animals. Furthermore, although limited, the results showing the importance of increased ASNS expression in solid tumor proliferation and the development of resistance to ASNase chemotherapy in childhood ALL illustrate the need for a better comprehension of ASNS regulation in transformed cells.
      The recent awareness of the neurological disease ASD highlights the need for a better understanding of the enzymatic consequences of ASNS mutations. The pioneering mouse model studies of Ruzzo et al. (
      • Ruzzo E.K.
      • Capo-Chichi J.M.
      • Ben-Zeev B.
      • Chitayat D.
      • Mao H.
      • Pappas A.L.
      • Hitomi Y.
      • Lu Y.F.
      • Yao X.
      • Hamdan F.F.
      • Pelak K.
      • Reznik-Wolf H.
      • Bar-Joseph I.
      • Oz-Levi D.
      • Lev D.
      • et al.
      Deficiency of asparagine synthetase causes congenital microcephaly and a progressive form of encephalopathy.
      ) should be expanded to generate brain-specific transgenic and knock-out animals to investigate the developmental effects of altered ASNS activity. Given that fibroblasts are available from only a few ASD children, the physiologic consequences for some mutations will require expression in ASNS null cells. At the protein level, modeling of the human ASNS protein has provided a means to tentatively map the structural consequences of ASD-associated mutations. However, the X-ray crystal structure of the human enzyme would be useful to confirm the structural effects proposed for ASD and allow for potential in silico drug screening approaches for future anti-tumor use. Furthermore, the current enzymatic assays for ASNS are cumbersome and variable in reproducibility when performed at the relatively low tissue levels of the enzyme. Overexpression of wild-type and mutant recombinant human ASNS protein in quantities suitable for new attempts at analysis would allow for activity comparisons of each mutation. With regard to ASD, the fact that asparagine uptake across the blood–brain barrier is not concentrative may limit dietary amino acid supplement therapy because artificially elevating blood asparagine levels too high may inhibit uptake of other amino acids due to competition for shared transporters (
      • Hawkins R.A.
      • O’Kane R.L.
      • Simpson I.A.
      • Viña J.R.
      Structure of the blood-brain barrier and its role in the transport of amino acids.
      ). In fact, an initial attempt at dietary asparagine therapy resulted in negative consequences (
      • Alrifai M.T.
      • Alfadhel M.
      Worsening of Seizures after asparagine supplementation in a child with asparagine synthetase deficiency.
      ). Affected ASD children are born with morphological and neurologic defects, indicating significant brain damage during embryonic development, and ASNS has been proven essential during brain development in a mouse model (
      • Ruzzo E.K.
      • Capo-Chichi J.M.
      • Ben-Zeev B.
      • Chitayat D.
      • Mao H.
      • Pappas A.L.
      • Hitomi Y.
      • Lu Y.F.
      • Yao X.
      • Hamdan F.F.
      • Pelak K.
      • Reznik-Wolf H.
      • Bar-Joseph I.
      • Oz-Levi D.
      • Lev D.
      • et al.
      Deficiency of asparagine synthetase causes congenital microcephaly and a progressive form of encephalopathy.
      ). These observations suggest that currently available therapeutic approaches are likely to prove ineffective, as significant and possibly irreversible tissue damage appears to occur during the earliest stages of development.

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