Simultaneous determination of nucleotide sugars with ion-pair reversed-phase HPLC

Nakajima, Kazuki; Kitazume, Shinobu; Angata, Takashi; Fujinawa, Reiko; Ohtsubo, Kazuaki; Miyoshi, Eiji; Taniguchi, Naoyuki

doi:10.1093/glycob/cwq044

Cited by 131 publications

(129 citation statements)

References 36 publications

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“…The amounts of GDP-Man, UDP-Glc, and UDP-GlcNAc were simultaneously measured in samples by HPLC coupled with UV detection, as described previously (27). Three nucleotide sugars were also analyzed by ion-pair, reversed-phase liquid chromatography electrospray ionization-MS/MS, as described previously (39).…”

Section: Methodsmentioning

confidence: 99%

“…The intracellular pool of the nucleotide sugars UDP-GlcNAc, GDPMan, and UDP-Glc, which are essential precursors for the biosynthesis of DLOs, is regulated by glucose metabolism (27). To examine whether a correlation exists between the release of POSs and nucleotide sugar levels under low-glucose conditions, the amounts of UDP-GlcNAc, GDP-Man, and UDP-Glc were quantitated at various time points in MEFs shifted from highglucose to low-glucose conditions.…”

Section: Dlo Intermediates Synthesized Under Low-glucose Conditions Arementioning

confidence: 99%

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Metabolically programmed quality control system for dolichol-linked oligosaccharides

Harada¹,

Nakajima

Masahara-Negishi³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The glycolipid Glc 3 Man 9 GlcNAc 2 -pyrophosphate-dolichol serves as the precursor for asparagine (N)-linked protein glycosylation in mammals. The biosynthesis of dolichol-linked oligosaccharides (DLOs) is arrested in low-glucose environments via unknown mechanisms, resulting in abnormal N-glycosylation. Here, we show that under glucose deprivation, DLOs are prematurely degraded during the early stages of DLO biosynthesis by pyrophosphatase, leading to the release of singly phosphorylated oligosaccharides into the cytosol. We identified that the level of GDP-mannose (Man), which serves as a donor substrate for DLO biosynthesis, is substantially reduced under glucose deprivation. We provide evidence that the selective shutdown of the GDP-Man biosynthetic pathway is sufficient to induce the release of phosphorylated oligosaccharides. These results indicate that glucoseregulated metabolic changes in the GDP-Man biosynthetic pathway cause the biosynthetic arrest of DLOs and facilitate their premature degradation by pyrophosphatase. We propose that this degradation system may avoid abnormal N-glycosylation with premature oligosaccharides under conditions that impair efficient DLO biosynthesis.

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Section: Methodsmentioning

confidence: 99%

Section: Dlo Intermediates Synthesized Under Low-glucose Conditions Arementioning

confidence: 99%

Metabolically programmed quality control system for dolichol-linked oligosaccharides

Harada¹,

Nakajima

Masahara-Negishi³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Nucleotide sugars were extracted and analyzed by ion-pair HPLC analysis as described by Nakajima et al 25 and in supplemental Methods.…”

Section: Quantification Of Sugar Nucleotidesmentioning

confidence: 99%

The origin and function of platelet glycosyltransferases

Wandall

Rumjantseva

Sørensen

et al. 2012

Blood

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Platelets are megakaryocyte subfragments that participate in hemostatic and host defense reactions and deliver proand antiangiogenic factors throughout the vascular system. Although they are anucleated cells that lack a complex secretory apparatus with distinct Golgi/ endoplasmic reticulum compartments, past studies have shown that platelets have glycosyltransferase activities. In the present study, we show that members of 3 distinct glycosyltransferase families are found within and on the surface of platelets. Immunocytology and flow cytometry results indicated that megakaryocytes package these Golgi-derived glycosyltransferases into vesicles that are sent via proplatelets to nascent platelets, where they accumulate. These glycosyltransferases are active, and intact platelets glycosylate large exogenous substrates. Furthermore, we show that activation of platelets results in the release of soluble glycosyltransferase activities and that platelets contain sufficient levels of sugar nucleotides for detection of glycosylation of exogenously added substrates. Therefore, the results of the present study show that blood platelets are a rich source of both glycosyltransferases and donor sugar substrates that can be released to function in the extracellular space. This plateletglycosylation machinery offers a pathway to a simple glycoengineering strategy improving storage of platelets and may serve hitherto unknown biologic functions. (Blood. 2012;120(3):626-635) IntroductionGlycosylation of proteins and lipids has a wide range of biologic functions. 1 The glycosylation apparatus of nucleated cells is primarily located in the secretory pathway throughout the endoplasmic reticulum-Golgi stacks and consists of more than 200 glycosyltransferases, most of which are type II transmembrane-anchored proteins with distinct localization. Their topology within these compartments is directed by signal motifs contained in their stem, transmembrane, and cytoplasmic domains. 2,3 Golgi-located membrane-bound glycosyltransferases are believed to be retained in the appropriate compartments by coat protein I-mediated retrograde transport 3 and to be released and secreted only after proteolytic cleavage in their stalk regions to yield soluble, catalytically active truncated enzymes lacking their N-terminal transmembrane segment. 4 Several previous studies have described unusual subcellular localization of glycosyltransferases outside of their normal confinement to the endoplasmic reticulum-Golgi (referred to herein as ectopic localization). Methodologic ambiguities, however, have cast doubt on these findings. 5 One notable exception concerns reports of glycosyltransferase activities associated with blood platelets. [6][7][8][9][10][11] Platelets are anucleated megakaryocyte subfragments that participate in hemostatic, inflammatory, and host defense reactions. 12 Moreover, platelets deliver pro-and antiangiogenic factors throughout the vascular system. 12 Glycosyltransferase activities in platelets were described 30 years ago and are propo...

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“…We examined this possibility by comparing UDPGlcNAc content between lig and the wild type and by adding UDP-GlcNAc to the lig mutant. We first applied the methods of Nakajima et al (2010) and Carpita and Delmer (1981) for HPLC analysis of nucleotide sugars, but neither of them gave a resolution sufficiently high to isolate UDP-GlcNAc. Then we modified the latter method by changing the pH of the buffers and finally succeeded in separation and quantification of UDP-GlcNAc (see Supplemental Figure 1 online).…”

Section: Deficiency Of Udp-glcnac In the Lig Mutantmentioning

confidence: 99%

“…Frozen samples were disrupted with a bead-type cell disrupter (MS-100; Tomy Seiko) and then homogenized with an ultrasonic disrupter (UD-200; Tomy Seiko) in 1 mL extraction buffer containing 75% ethanol and 25% sodium-phosphate buffer saline (10 mM sodium phosphate and 0.15 M NaCl, pH 7.4) on ice. After addition of cytidine-59-monophospho-N-acetyl-D-neuraminic acid, which plants usually do not contain, as an internal standard, nucleotide sugars were purified with Envi-Carb SPE column (250 mg/6 mL column; Supelco) according to of Nakajima et al (2010), lyophilized, and stored at 270°C.…”

Section: Determination Of Udp-glcnac and Udp-galnac By Hplcmentioning

confidence: 99%

A Missense Mutation in the Glucosamine-6-Phosphate N-Acetyltransferase–Encoding Gene Causes Temperature-Dependent Growth Defects and Ectopic Lignin Deposition in Arabidopsis

et al. 2012

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To study the regulatory mechanisms underlying lignin biosynthesis, we isolated and characterized lignescens (lig), a previously undescribed temperature-sensitive mutant of Arabidopsis thaliana that exhibits ectopic lignin deposition and growth defects under high-temperature conditions. The lig mutation was identified as a single base transition in GNA1 encoding glucosamine-6-phosphate N-acetyltransferase (GNA), a critical enzyme of UDP-N-acetylglucosamine (UDP-GlcNAc) biosynthesis. lig harbors a glycine-to-serine substitution at residue 68 (G68S) of GNA1. Enzyme activity assays of the mutant protein (GNA1 G68S ) showed its thermolability relative to the wild-type protein. The lig mutant exposed to the restrictive temperature contained a significantly smaller amount of UDP-GlcNAc than did the wild type. The growth defects and ectopic lignification of lig were suppressed by the addition of UDP-GlcNAc. Since UDP-GlcNAc is an initial sugar donor of N-glycan synthesis and impaired N-glycan synthesis is known to induce the unfolded protein response (UPR), we examined possible relationships between N-glycan synthesis, UPR, and the lig phenotype. N-glycans were reduced and LUMINAL BINDING PROTEIN3, a typical UPR gene, was expressed in lig at the restrictive temperature. Furthermore, treatment with UPR-inducing reagents phenocopied the lig mutant. Our data collectively suggest that impairment of N-glycan synthesis due to a shortage of UDP-GlcNAc leads to ectopic lignin accumulation, mostly through the UPR.

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Simultaneous determination of nucleotide sugars with ion-pair reversed-phase HPLC

Cited by 131 publications

References 36 publications

Metabolically programmed quality control system for dolichol-linked oligosaccharides

Metabolically programmed quality control system for dolichol-linked oligosaccharides

The origin and function of platelet glycosyltransferases

A Missense Mutation in the Glucosamine-6-Phosphate N-Acetyltransferase–Encoding Gene Causes Temperature-Dependent Growth Defects and Ectopic Lignin Deposition in Arabidopsis

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