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21.
Kazuki Nakajima Emi Ito Kazuaki Ohtsubo Ken Shirato Rina Takamiya Shinobu Kitazume Takashi Angata Naoyuki Taniguchi 《Molecular & cellular proteomics : MCP》2013,12(9):2468-2480
Nucleotide sugars are the donor substrates of various glycosyltransferases, and an important building block in N- and O-glycan biosynthesis. Their intercellular concentrations are regulated by cellular metabolic states including diseases such as cancer and diabetes. To investigate the fate of UDP-GlcNAc, we developed a tracing method for UDP-GlcNAc synthesis and use, and GlcNAc utilization using 13C6-glucose and 13C2-glucosamine, respectively, followed by the analysis of mass isotopomers using LC-MS.Metabolic labeling of cultured cells with 13C6-glucose and the analysis of isotopomers of UDP-HexNAc (UDP-GlcNAc plus UDP-GalNAc) and CMP-NeuAc revealed the relative contributions of metabolic pathways leading to UDP-GlcNAc synthesis and use. In pancreatic insulinoma cells, the labeling efficiency of a 13C6-glucose motif in CMP-NeuAc was lower compared with that in hepatoma cells.Using 13C2-glucosamine, the diversity of the labeling efficiency was observed in each sugar residue of N- and O-glycans on the basis of isotopomer analysis. In the insulinoma cells, the low labeling efficiencies were found for sialic acids as well as tri- and tetra-sialo N-glycans, whereas asialo N-glycans were found to be abundant. Essentially no significant difference in secreted hyaluronic acids was found among hepatoma and insulinoma cell lines. This indicates that metabolic flows are responsible for the low sialylation in the insulinoma cells. Our strategy should be useful for systematically tracing each stage of cellular GlcNAc metabolism.Protein glycosylation, which is the most abundant post-translational modification, has important roles in many biological processes by modulating conformation and stability, whereas its dysregulation is associated with various diseases such as diabetes and cancer (1, 2). Glycosylation is regulated by various factors including glucose metabolism, the availability and localization of nucleotide sugars, and the expression and localization of glycosyltransferases (3, 4). Thus, ideally all of these components should be considered when detecting changes in a dynamic fashion; namely, it is necessary not only to take a snapshot but also to make movies of the dynamic changes in glycan metabolism.Glucose is used by living cells as an energy source via the glycolytic pathway as well as a carbon source for various metabolites including nucleotide sugars (e.g. UDP-GlcNAc and CMP-NeuAc). These nucleotide sugars are transported into the Golgi apparatus, and added to various glycans on proteins. UDP-GlcNAc is the donor substrate for N-acetylglucosaminyl (GlcNAc)1 transferases; alternatively, it is used in the cytosol for O-GlcNAc modification (i.e. O-GlcNAcylation) of intracellular proteins (5). The UDP-GlcNAc synthetic pathway is complex as it is a converging point of glucose, nucleotide, fatty acid and amino acid metabolic pathways. Thus, the metabolic flow of glucose modulates the branching patterns of N-glycans via UDP-GlcNAc concentrations because many of the key GlcNAc transferases that determine the branching patterns have widely different Km values for UDP-GlcNAc ranging from 0.04 mm to 11 mm (6, 7). Indeed, it was demonstrated that the branching formation of N-glycans in T cells is stimulated by the supply from the hexosamine pathway, whereby it regulates autoimmune reactions promoted by T cells (8).UDP-GlcNAc is also used for the synthesis of CMP-NeuAc, the donor substrate for sialyltransferases (9). The CMP-NeuAc concentration is controlled by the feedback inhibition of UDP-GlcNAc epimerase/ManNAc kinase by the final product CMP-NeuAc, and hence a high CMP-NeuAc level reduces metabolic flow in CMP-NeuAc de novo synthesis (10). However, there is still only limited information about how the levels of nucleotide sugars dynamically change in response to the environmental cues, and how such changes are reflected in the glycosylation of proteins.Stable isotope labeling is a promising approach to quantify metabolic changes in response to external cues (11, 12). For example, the use of nuclear magnetic resonance to obtain isotopomer signals of metabolically labeled molecules has been applied to trace the flux in glycolysis and fatty acid metabolism (13). An approach based on the mass isotopomers of labeled metabolites with 13C6-glucose has been developed to monitor the UDP-GlcNAc synthetic pathway (13–15). The method based on the labeling ratio of each metabolite related to UDP-GlcNAc synthesis has clarified the contribution of each metabolic pathway (14). Moseley reported a novel deconvolution method for modeling UDP-GlcNAc mass isotopomers (15).Previous studies into the use of nucleotide sugars in glycosylation have relied on the specific detection of metabolically radiolabeled glycans (16). It is possible not only to deduce the glycan structures but also to trace their relative contributions to glycan synthesis without MS. On the other hand, mass isotopomer analysis of glycans labeled with stable isotope provides the ratios of labeled versus unlabeled molecules from MS spectra and structural details of the glycans. However, there are only a limited number of publications reporting the application of stable isotope labeling of glycans for monitoring the dynamics of glycans (17). To date, there have been no reports describing a systematic method for tracing cellular GlcNAc biosynthesis and use based on mass isotopomer analysis.The aim of this study was to extend our knowledge of the synthesis and metabolism of UDP-GlcNAc as well as its use in the synthesis of CMP-NeuAc, N- and O-glycans. We recently developed a conventional HPLC method for simultaneous determination of nucleotide sugars including unstable CMP-NeuAc (18). We first established an LC-MS method for isotopomer analysis of 13C6-glucose labeled nucleotide sugars for tracing UDP-GlcNAc metabolism from synthesis to use, because previous methods were not suitable for estimating UDP-GlcNAc use in CMP-NeuAc de novo synthesis (15). We also established a method for isotopomer analysis of labeled N- and O-glycan to monitor the metabolic flow of hexosamine into glycans. Using these two methods, we demonstrated the differences in the use of hexosamines between hepatoma and pancreatic insulinoma cell lines. Our approach may be useful for identifying a metabolic “bottleneck” that governs the turnover speed and patterns of cellular glycosylation, which may be relevant for various applications including glycoprotein engineering and discovery of disease biomarkers. 相似文献
22.
Lance G. Woolaver Rina K. Nichols Eugene S. Morton Bridget J. M. Stutchbury 《Conservation Genetics》2013,14(3):559-571
Many island avian populations are of conservation interest because they have a higher risk of extinction than mainland populations. Susceptibility of island birds to extinction is primarily related to human induced change through habitat loss, persecution, and introduction of exotic species, in combination with genetic factors. We used microsatellite profiles from 11 loci to assess genetic diversity and relatedness in the critically endangered hawk Buteo ridgwayi endemic to the island of Hispaniola in the Caribbean. Using samples collected between 2005 and 2009, our results revealed a relatively high level of heterozygosity, evidence of a recent genetic bottleneck, and the occurrence of inbreeding within the population. Pair relatedness analysis found 4 of 7 sampled breeding pairs to be related similar to that of first cousin or greater. Pedigree estimates indicated that up to 18 % of potential pairings would be between individuals with relatedness values similar to that of half-sibling. We discuss our findings in the context of conservation genetic management suggesting both carefully managed translocations and the initiation of a captive population as a safeguard of the remaining genetic diversity. 相似文献
23.
24.
Lovasoa Rina Raharinaivo Henitsoa Jaonalison Jamal Mahafina Dominique Ponton 《Zeitschrift fur angewandte Ichthyologie》2020,36(4):402-413
In order to provide biological evidence of the real impact of mosquito seine nets in southwestern Madagascar, an efficient procedure for determining the size at maturity of small-sized tropical fishes was developed. The fishes caught by two small-scale fishermen were studied between October 2017 and April 2018. One catch per day was analyzed three days per month during the full-moon period. In the laboratory, fishes were all sorted by morphospecies, photographed and measured. One individual per morphospecies was selected for being identified using CO1 DNA barcoding. A total of 34,051 individual fishes belonging to 144 DNA bacoded species from 48 families was obtained from 42 samples, 467 individuals from 22 morphospecies that had not been successfully barcoded were excluded from the analyses. The macroscopic observations of 8,143 individuals between 0.7 and 10 cm SL indicated the proportion of individuals with clearly observable gonads was 15% only.Among the 144 species identified via DNA barcoding, 83 consisted of individuals that were all without clearly observable gonads, seven of individuals that were all with clearly observable gonads and 54 included of individuals with and without clearly observable gonads. As the determination of L50 using logistic general linear models failed for most species, the minimum size at maturity was retained to determine the proportion of juveniles and adults for these 54 species. Compared to the data available in FishBase, the minimum size at maturity appears more adequate to discrimine juvenile from adult fish of small-sized tropical species. 相似文献
25.
Pumtiwitt C. McCarthy Rina Saksena Dwight C. Peterson Che-Hung Lee Yanming An John F. Cipollo Willie F. Vann 《Glycoconjugate journal》2013,30(9):857-870
Vaccination with meningococcal glycoconjugate vaccines has decreased the incidence of invasive meningitis worldwide. These vaccines contain purified capsular polysaccharides attached to a carrier protein. Because of derivatization chemistries used in the process, conjugation of polysaccharide to protein often results in heterogeneous mixtures. Well-defined vaccines are needed to determine the relationship between vaccine structure and generated immune response. Here, we describe efforts to produce well-defined vaccine candidates by chemoenzymatic synthesis. Chemically synthesized lactosides were substrates for recombinant sialyltransferase enzymes from Camplyobacter jejuni and Neisseria meningitidis serogroup C. These resulting oligosialic acids have the same α(2-9) sialic acid repeat structure as Neisseria polysaccharide capsule with the addition of a conjugatable azide aglycon. The degree of polymerization (DP) of carbohydrate products was controlled by inclusion of the inhibitor CMP-9-deoxy-NeuNAc. Polymers with estimated DP?<?47 (median DP 25) and DP?<?100 (median DP 51) were produced. The receptor binding domain of the tetanus toxin protein (TetHc) was coupled as a carrier to the enzymatically synthesized oligosialic acids. Recombinant TetHc was derivatized with an alkyne squarate. Protein modification sites were determined by trypsin proteolysis followed by LC/MS-MSE analysis of peptides. Oligosialic acid azides were conjugated to modified TetHc via click chemistry. These chemoenzymatically prepared glycoconjugates were reactive in immunoassays with specific antibodies against either group C polysaccharide or TetHc. Sera of mice immunized with oligosialic acid-TetHc glycoconjugates contained much greater levels of polysaccharide-reactive IgG than the sera of control mice receiving unconjugated oligosialic acids. There was no apparent difference between glycoconjugates containing oligosaccharides of DP?<?47 and DP?<?100. These results suggest that chemoenzymatic synthesis may provide a viable method for making defined meningococcal vaccine candidates. 相似文献
26.
Emma Smith Rina Vekaria Katherine A. Brown Colin Longstaff 《Molecular and cellular biochemistry》2013,382(1-2):193-201
A wide range of equilibrium and kinetic constants exist for the interaction of prothrombin and other coagulation factors with various model membranes from a variety of techniques. We have investigated the interaction of prothrombin with pure dioleoylphosphatidylcholine (DOPC) membranes and dioleoylphosphatidlyserine (DOPS)-containing membranes (DOPC:DOPS, 3:1) using surface plasmon resonance (SPR, with four different model membrane presentations) in addition to isotheral titration calorimetry (ITC, with suspensions of phospholipid vesicles) and ELISA methods. Using ITC, we found a simple low-affinity interaction with DOPC:DOPS membranes with a K D = 5.1 μM. However, ELISA methods using phospholipid bound to microtitre plates indicated a complex interaction with both DOPC:DOPS and DOPC membranes with K D values of 20 and 58 nM, respectively. An explanation for these discrepant results was developed from SPR studies. Using SPR with low levels of immobilised DOPC:DOPS, a high-affinity interaction with a K D of 18 nM was obtained. However, as phospholipid and prothrombin concentrations were increased, two distinct interactions could be discerned: (i) a kinetically slow, high-affinity interaction with K D in the 10?8 M range and (ii) a kinetically rapid, low-affinity interaction with K D in the 10?6 M range. This low affinity, rapidly equilibrating, interaction dominated in the presence of DOPS. Detailed SPR studies supported a heterogeneous binding model in agreement with ELISA data. The binding of prothrombin with phospholipid membranes is complex and the techniques used to measure binding will report K D values reflecting the mixture of complexes detected. Existing data suggest that the weaker rapid interaction between prothrombin and membranes is the most important in vivo when considering the activation of prothrombin at the cell surface. 相似文献
27.
Phospholipase D (PLD) and its product phosphatidic acid (PA) are incorporated in a complex metabolic network in which the individual PLD isoforms are suggested to regulate specific developmental and stress responses, including plant programmed cell death (PCD). Despite the accumulating knowledge, the mechanisms through which PLD/PA operate during PCD are still poorly understood. In this work, the role of PLDα1 in PCD and the associated caspase-like proteolysis, ethylene and hydrogen peroxide (H2O2) synthesis in tomato suspension cells was studied. Wild-type (WT) and PLDα1-silenced cell lines were exposed to the cell death-inducing chemicals camptothecin (CPT), fumonisin B1 (FB1) and CdSO4. A range of caspase inhibitors effectively suppressed CPT-induced PCD in WT cells, but failed to alleviate cell death in PLDα1-deficient cells. Compared to WT, in CPT-treated PLDα1 mutant cells, reduced cell death and decreased production of H2O2 were observed. Application of ethylene significantly enhanced CPT-induced cell death both in WT and PLDα1 mutants. Treatments with the PA derivative lyso-phosphatidic acid and mastoparan (agonist of PLD/PLC signalling downstream of G proteins) caused severe cell death. Inhibitors, specific to PLD and PLC, remarkably decreased the chemical-induced cell death. Taken together with our previous findings, the results suggest that PLDα1 contributes to caspase-like-dependent cell death possibly communicated through PA, reactive oxygen species and ethylene. The dead cells expressed morphological features of PCD such as protoplast shrinkage and nucleus compaction. The presented findings reveal novel elements of PLD/PA-mediated cell death response and suggest that PLDα1 is an important factor in chemical-induced PCD signal transduction. 相似文献
28.
Akemi Shodai Toshifumi Morimura Akemi Ido Tsukasa Uchida Takashi Ayaki Rina Takahashi Soichiro Kitazawa Sakura Suzuki Mikako Shirouzu Takanori Kigawa Yutaka Muto Shigeyuki Yokoyama Ryosuke Takahashi Ryo Kitahara Hidefumi Ito Noriko Fujiwara Makoto Urushitani 《The Journal of biological chemistry》2013,288(21):14886-14905
Aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) is a pathological signature of amyotrophic lateral sclerosis (ALS). Although accumulating evidence suggests the involvement of RNA recognition motifs (RRMs) in TDP-43 proteinopathy, it remains unclear how native TDP-43 is converted to pathogenic forms. To elucidate the role of homeostasis of RRM1 structure in ALS pathogenesis, conformations of RRM1 under high pressure were monitored by NMR. We first found that RRM1 was prone to aggregation and had three regions showing stable chemical shifts during misfolding. Moreover, mass spectrometric analysis of aggregated RRM1 revealed that one of the regions was located on protease-resistant β-strands containing two cysteines (Cys-173 and Cys-175), indicating that this region served as a core assembly interface in RRM1 aggregation. Although a fraction of RRM1 aggregates comprised disulfide-bonded oligomers, the substitution of cysteine(s) to serine(s) (C/S) resulted in unexpected acceleration of amyloid fibrils of RRM1 and disulfide-independent aggregate formation of full-length TDP-43. Notably, TDP-43 aggregates with RRM1-C/S required the C terminus, and replicated cytopathologies of ALS, including mislocalization, impaired RNA splicing, ubiquitination, phosphorylation, and motor neuron toxicity. Furthermore, RRM1-C/S accentuated inclusions of familial ALS-linked TDP-43 mutants in the C terminus. The relevance of RRM1-C/S-induced TDP-43 aggregates in ALS pathogenesis was verified by immunolabeling of inclusions of ALS patients and cultured cells overexpressing the RRM1-C/S TDP-43 with antibody targeting misfolding-relevant regions. Our results indicate that cysteines in RRM1 crucially govern the conformation of TDP-43, and aberrant self-assembly of RRM1 at amyloidogenic regions contributes to pathogenic conversion of TDP-43 in ALS. 相似文献
29.
30.
Fulton P. Rivera Anicia M. Medina Sandra Bezada Roberto Valencia María Bernal Rina Meza Ryan C. Maves Theresa J. Ochoa 《PloS one》2013,8(4)
Secretory diarrhea caused by cholera toxin (CT) is initiated by binding of CT’s B subunit (CTB) to GM1-ganglioside on the surface of intestinal cells. Lactoferrin, a breast milk glycoprotein, has shown protective effect against several enteropathogens. The aims of this study were to determine the effect of bovine-lactoferrin (bLF) on CT-induced intestinal fluid accumulation in mice, and the interaction between bLF and CT/CTB with the GM1-ganglioside receptor. Fluid accumulation induced by CT was evaluated in the mouse ileal loop model using 56 BALB/c mice, with and without bLF added before, after or at the same time of CT administration. The effect of bLF in the interaction of CT and CTB with GM1-ganglioside was evaluated by a GM1-enzyme-linked immunosorbent assay. bLF decreased CT-induced fluid accumulation in the ileal loop of mice. The greatest effect was when bLF was added before CT (median, 0.066 vs. 0.166 g/cm, with and without bLF respectively, p<0.01). We conclude that bLF decreases binding of CT and CTB to GM1-ganglioside, suggesting that bLF suppresses CT-induced fluid accumulation by blocking the binding of CTB to GM1-ganglioside. bLF may be effective as adjunctive therapy for treatment of cholera diarrhea. 相似文献