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International Journal of Molecular... Oct 2022L-Asparagine (Asn) has been regarded as one of the most economical molecules for nitrogen (N) storage and transport in plants due to its relatively high N-to-carbon (C)...
L-Asparagine (Asn) has been regarded as one of the most economical molecules for nitrogen (N) storage and transport in plants due to its relatively high N-to-carbon (C) ratio (2:4) and stability. Although its internal function has been addressed, the biological role of exogenous Asn in plants remains elusive. In this study, different concentrations (0.5, 1, 2, or 5 mM) of Asn were added to the N-deficient hydroponic solution for poplar 'Nanlin895'. Morphometric analyses showed that poplar height, biomass, and photosynthesis activities were significantly promoted by Asn treatment compared with the N-free control. Moreover, the amino acid content, total N and C content, and nitrate and ammonia content were dramatically altered by Asn treatment. Moreover, exogenous Asn elicited root growth inhibition, accompanied by complex changes in the transcriptional pattern of genes and activities of enzymes associated with N and C metabolism. Combined with the plant phenotype and the physiological and biochemical indexes, our data suggest that poplar is competent to take up and utilize exogenous Asn dose-dependently. It provides valuable information and insight on how different forms of N and concentrations of Asn influence poplar root and shoot growth and function, and roles of Asn engaged in protein homeostasis regulation.
Topics: Asparagine; Biomass; Populus; Nitrogen; Plants
PubMed: 36361911
DOI: 10.3390/ijms232113126 -
Oncogene Oct 2022Amino acid restriction has recently emerged as a compelling strategy to inhibit tumor growth. Recent work suggests that amino acids can regulate cellular signaling in...
Amino acid restriction has recently emerged as a compelling strategy to inhibit tumor growth. Recent work suggests that amino acids can regulate cellular signaling in addition to their role as biosynthetic substrates. Using lymphoid cancer cells as a model, we found that asparagine depletion acutely reduces the expression of c-MYC protein without changing its mRNA expression. Furthermore, asparagine depletion inhibits the translation of MYC mRNA without altering the rate of MYC protein degradation. Of interest, the inhibitory effect on MYC mRNA translation during asparagine depletion is not due to the activation of the general controlled nonderepressible 2 (GCN2) pathway and is not a consequence of the inhibition of global protein synthesis. In addition, both the 5' and 3' untranslated regions (UTRs) of MYC mRNA are not required for this inhibitory effect. Finally, using a MYC-driven mouse B cell lymphoma model, we found that shRNA inhibition of asparagine synthetase (ASNS) or pharmacological inhibition of asparagine production can significantly reduce the MYC protein expression and tumor growth when environmental asparagine becomes limiting. Since MYC is a critical oncogene, our results uncover a molecular connection between MYC mRNA translation and asparagine bioavailability and shed light on a potential to target MYC oncogene post-transcriptionally through asparagine restriction.
Topics: Mice; Animals; Asparagine; Biological Availability; Genes, myc; Proto-Oncogene Proteins c-myc; Neoplasms; Amino Acids; 3' Untranslated Regions
PubMed: 36182969
DOI: 10.1038/s41388-022-02474-9 -
Biochemistry May 2011Asparagine-linked glycosylation involves the sequential assembly of an oligosaccharide onto a polyisoprenyl donor, followed by the en bloc transfer of the glycan to... (Review)
Review
Asparagine-linked glycosylation involves the sequential assembly of an oligosaccharide onto a polyisoprenyl donor, followed by the en bloc transfer of the glycan to particular asparagine residues within acceptor proteins. These N-linked glycans play a critical role in a wide variety of biological processes, such as protein folding, cellular targeting and motility, and the immune response. In the past decade, research in the field of N-linked glycosylation has achieved major advances, including the discovery of new carbohydrate modifications, the biochemical characterization of the enzymes involved in glycan assembly, and the determination of the biological impact of these glycans on target proteins. It is now firmly established that this enzyme-catalyzed modification occurs in all three domains of life. However, despite similarities in the overall logic of N-linked glycoprotein biosynthesis among the three kingdoms, the structures of the appended glycans are markedly different and thus influence the functions of elaborated proteins in various ways. Though nearly all eukaryotes produce the same nascent tetradecasaccharide (Glc(3)Man(9)GlcNAc(2)), heterogeneity is introduced into this glycan structure after it is transferred to the protein through a complex series of glycosyl trimming and addition steps. In contrast, bacteria and archaea display diversity within their N-linked glycan structures through the use of unique monosaccharide building blocks during the assembly process. In this review, recent progress toward gaining a deeper biochemical understanding of this modification across all three kingdoms will be summarized. In addition, a brief overview of the role of N-linked glycosylation in viruses will also be presented.
Topics: Asparagine; Bacteria; Carbohydrate Sequence; Glycosylation; Models, Molecular; Molecular Sequence Data; Viruses
PubMed: 21506607
DOI: 10.1021/bi200346n -
Proceedings of the National Academy of... Oct 2022-glycosylation is a common posttranslational modification of secreted proteins in eukaryotes. This modification targets asparagine residues within the consensus...
-glycosylation is a common posttranslational modification of secreted proteins in eukaryotes. This modification targets asparagine residues within the consensus sequence, N-X-S/T. While this sequence is required for glycosylation, the initial transfer of a high-mannose glycan by oligosaccharyl transferases A or B (OST-A or OST-B) can lead to incomplete occupancy at a given site. Factors that determine the extent of transfer are not well understood, and understanding them may provide insight into the function of these important enzymes. Here, we use mass spectrometry (MS) to simultaneously measure relative occupancies for three -glycosylation sites on the N-terminal IgV domain of the recombinant glycoprotein, hCEACAM1. We demonstrate that addition is primarily by the OST-B enzyme and propose a kinetic model of OST-B -glycosylation. Fitting the kinetic model to the MS data yields distinct rates for glycan addition at most sites and suggests a largely stochastic initial order of glycan addition. The model also suggests that glycosylation at one site influences the efficiency of subsequent modifications at the other sites, and glycosylation at the central or N-terminal site leads to dead-end products that seldom lead to full glycosylation of all three sites. Only one path of progressive glycosylation, one initiated by glycosylation at the C-terminal site, can efficiently lead to full occupancy for all three sites. Thus, the hCEACAM1 domain provides an effective model system to study site-specific recognition of glycosylation sequons by OST-B and suggests that the order and efficiency of posttranslational glycosylation is influenced by steric cross-talk between adjoining acceptor sites.
Topics: Asparagine; Glycoproteins; Glycosylation; Hexosyltransferases; Mannose; Polysaccharides; Transferases
PubMed: 36251991
DOI: 10.1073/pnas.2202992119 -
Journal of Separation Science Dec 2022Human serum N-linked glycans expression levels change during the disease progression. The low abundance, structural diversity, and coexisting matrices hinder their...
Human serum N-linked glycans expression levels change during the disease progression. The low abundance, structural diversity, and coexisting matrices hinder their detection in mass spectrometry analysis. Considering the hydrophilic nature of N-glycans, cellulose/polymer (1,2-Epoxy-5-hexene) nanohybrid is fabricated with oxirane groups functionalized of asparagine to develop solid phase extraction based hydrophilic interaction liquid chromatography sorbent (cellulose/1,2-Epoxy-5-hexene/asparagine). The morphology, elemental analysis, and surface properties are studied through scanning electron microscopy, energy dispersive X-ray spectroscopy, and Fourier-transform infrared spectroscopy. The large surface area of cellulose/polymer nanohybrid (2.09 × 10 m /g) facilitates the high density of asparagine immobilization resulting in better hydrophilic interaction liquid chromatography enrichment under optimized conditions. The enrichment capability of nanohybrid/asparagine is assessed by the N-Linked glycans released from ovalbumin and immunoglobulin G where 23 and 13 N-glycans are detected respectively. The nanohybrid/asparagine shows selectivity of 1:1200 with spiked bovine serum albumin and sensitivity down to 100 attomole. Human serum profiling for N-glycans identifies 52 glycan structures. This new enrichment strategy enriches serum N-linked glycans in the presence of salts, proteins, endogenous serum peptides, and so forth.
Topics: Humans; Cellulose; Polymers; Asparagine
PubMed: 36168850
DOI: 10.1002/jssc.202200179 -
International Journal of Molecular... May 2022Complex asparagine-linked glycosylation plays key roles in cellular functions, including cellular signaling, protein stability, and immune response. Previously, we...
Complex asparagine-linked glycosylation plays key roles in cellular functions, including cellular signaling, protein stability, and immune response. Previously, we characterized the appearance of a complex asparagine-linked glycosylated form of lysosome-associated membrane protein 1 (LAMP1) in the cerebellum of mice. This LAMP1 form was found on activated microglia, and its appearance correlated both spatially and temporally with cerebellar Purkinje neuron loss. To test the importance of complex asparagine-linked glycosylation in NPC1 pathology, we generated NPC1 knock-out mice deficient in MGAT5, a key Golgi-resident glycosyl transferase involved in complex asparagine-linked glycosylation. Our results show that mice were smaller than mice, and exhibited earlier NPC1 disease onset and reduced lifespan. Western blot and lectin binding analyses of cerebellar extracts confirmed the reduction in complex asparagine-linked glycosylation, and the absence of the hyper-glycosylated LAMP1 previously observed. Western blot analysis of cerebellar extracts demonstrated reduced calbindin staining in mice compared to mutant mice, and immunofluorescent staining of cerebellar sections indicated decreased levels of Purkinje neurons and increased astrogliosis in mice. Our results suggest that reduced asparagine-linked glycosylation increases NPC1 disease severity in mice, and leads to the hypothesis that mutations in genes involved in asparagine-linked glycosylation may contribute to disease severity progression in individuals with NPC1. To examine this with respect to we analyzed 111 NPC1 patients for two SNPs associated with multiple sclerosis; however, we did not identify an association with NPC1 phenotypic severity.
Topics: Animals; Asparagine; Glycosylation; Humans; Mice; Mice, Inbred BALB C; N-Acetylglucosaminyltransferases; Niemann-Pick Disease, Type C
PubMed: 35563467
DOI: 10.3390/ijms23095082 -
Methods (San Diego, Calif.) Apr 2022Deamidation of asparagine and glutamine alters protein structures and affects the chemical and biological properties of proteins. Protein deamidation has been... (Review)
Review
Deamidation of asparagine and glutamine alters protein structures and affects the chemical and biological properties of proteins. Protein deamidation has been demonstrated to be associated with protein folding, enzymatic activity, and degradation, as well as aging, cancer, and neurodegenerative diseases. To gain a better understanding on the biological roles of protein deamidation in aging and diseases, mass spectrometry (MS) has been employed in the identification of deamidated protein species and comprehensive characterization of deamidation sites. Three main MS approaches, top-down, middle-down, and bottom-up have been applied in the study of protein deamidation with high sensitivity, throughput, and accuracy. In this review, we discuss the application of top-down and middle-down MS in the study of protein deamidation, including sample preparation methods, separation strategies, MS and MS/MS techniques and data analysis. The advantages and drawbacks of these two approaches are also discussed and compared with those of the bottom-up method. The development of top-down and middle-down MS methods provides new strategies for protein deamidation analysis and gives new insights into the biological significance of protein deamidation in diseases.
Topics: Asparagine; Glutamine; Protein Folding; Proteins; Tandem Mass Spectrometry
PubMed: 32791336
DOI: 10.1016/j.ymeth.2020.08.002 -
Shokuhin Eiseigaku Zasshi. Journal of... 2018New analytical methods for the determination of free asparagine (Asn), which is a precursor of acrylamide, in grains were developed using LC-MS and LC-MS/MS. Asn was...
New analytical methods for the determination of free asparagine (Asn), which is a precursor of acrylamide, in grains were developed using LC-MS and LC-MS/MS. Asn was extracted from a sample with 5% (w/v) aqueous trichloroacetic acid solution, appropriately diluted with 0.1% (v/v) formic acid solution, and then analyzed by LC-MS or LC-MS/MS. HPLC separation was performed by isocratic elution on a Penta Fluoro Phenyl (PFP) column using 0.1% (v/v) formic acid and acetonitrile mixture as the mobile phase. The calibration curve was linear in the range of 0.005-0.1 μg/mL. The mean recoveries from potato starch, non-glutinous rice flour and whole wheat flour ranged from 95.4 to 100.9%, repeatability (RSD) ranged from 0.9 to 6.0%, and within-laboratory reproducibility (RSDwr) ranged from 2.8 to 7.1%. Limits of quantitation (LOQs) were 7 mg/kg for potato starch, and 5 mg/kg for non-glutinous rice flour. In addition, an inter-laboratory study was performed in 10 laboratories using 5 kinds of grains (non-glutinous brown rice flour, corn flour, strong flour, whole wheat flour, and whole rye flour), which naturally contained free asparagine. The HORRAT values ranged from 0.4 to 1.0. These results are within the range of the procedural manual of the Codex Alimentarius Commission, confirming the effectiveness of the developed procedures.
Topics: Asparagine; Chromatography, Liquid; Edible Grain; Flour; Food Analysis; Reproducibility of Results; Tandem Mass Spectrometry
PubMed: 30429425
DOI: 10.3358/shokueishi.59.248 -
Glycobiology Apr 2019Asparagine-linked (N-linked) glycosylation is one of the most common protein modification reactions in eukaryotic cells, occurring upon the majority of proteins that... (Review)
Review
Asparagine-linked (N-linked) glycosylation is one of the most common protein modification reactions in eukaryotic cells, occurring upon the majority of proteins that enter the secretory pathway. X-ray crystal structures of the single subunit OSTs from eubacterial and archaebacterial organisms revealed the location of donor and acceptor substrate binding sites and provided the basis for a catalytic mechanism. Cryoelectron microscopy structures of the octameric yeast OST provided substantial insight into the organization and assembly of the multisubunit oligosaccharyltransferases. Furthermore, the cryoelectron microscopy structure of a complex consisting of a mammalian OST complex, the protein translocation channel and a translating ribosome revealed new insight into the mechanism of cotranslational glycosylation.
Topics: Asparagine; Cryoelectron Microscopy; Crystallography, X-Ray; Eukaryotic Cells; Glycosylation; Hexosyltransferases; Humans; Membrane Proteins; Models, Molecular; Prokaryotic Cells; Protein Conformation
PubMed: 30312397
DOI: 10.1093/glycob/cwy093 -
The Journal of Physical Chemistry. B Oct 2014Asparagine-linked carbohydrates profoundly impact glycoprotein folding, stability, and structure. However, the "glycosylation code" that relates these effects to protein...
Asparagine-linked carbohydrates profoundly impact glycoprotein folding, stability, and structure. However, the "glycosylation code" that relates these effects to protein sequence remains unsolved. We report atomically detailed replica exchange molecular dynamics simulations in explicit solvent that systematically investigate the impact of glycosylation upon peptides with the central sequon Pro-Asn-Gly/Ala-Thr-Trp/Ala. These simulations suggest that the effects of glycosylation may be quite sensitive to steric crowding by the side chain immediately following the glycosylation site but less sensitive to stacking interactions with the aromatic Trp residue. In addition, we compare our simulated ensembles with the known structures for full length glycoproteins. These structures corroborate the simulations and also suggest a remarkable consistency between the intraprotein and protein-glycan interactions of natural glycoproteins. Moreover, our analysis highlights the significance of left-handed conformations for compact β-hairpins at glycosylation sites. In summary, these studies elucidate basic biophysical principles for the glycosylation code.
Topics: Amino Acid Sequence; Asparagine; Computational Biology; Databases, Protein; Glycoproteins; Glycosylation; Molecular Dynamics Simulation; Peptides; Protein Structure, Secondary
PubMed: 25188817
DOI: 10.1021/jp508535f