Discrimination of cellular developmental states focusing on glycan transformation and membrane dynamics by using BODIPY-tagged lactosyl ceramides.

Glycosphingolipids (GSLs) are a group of molecules composed of a hydrophilic glycan part and a hydrophobic ceramide creating a diverse family. GSLs are de novo synthesised from ceramides at the endoplasmic reticulum and Golgi apparatus, and transported to the outer surface of the plasma membrane. It has been known that the glycan structures of GSLs change reflecting disease states.

Synthesis and structural investigation of a series of mannose-containing oligosaccharides using mass spectrometry.

A series of compounds associated with naturally occurring and biologically relevant glycans consisting of α-mannosides were prepared and analyzed using collision-induced dissociation (CID), energy-resolved mass spectrometry (ERMS), and H nuclear magnetic resonance spectroscopy. The CID experiments of sodiated species of disaccharides and ERMS experiments revealed that the order of stability of mannosyl linkages was as follows: 6-linked > 4-linked ≧ 2-linked > 3-linked mannosyl residues. Analysis of linear trisaccharides revealed that the order observed in disaccharides could be applied to higher glycans.

Reactivation of hyperglycemia-induced hypocretin (HCRT) gene silencing by N-acetyl-d-mannosamine in the orexin neurons derived from human iPS cells.

Orexin neurons regulate critical brain activities for controlling sleep, eating, emotions, and metabolism, and impaired orexin neuron function results in several neurologic disorders. Therefore, restoring normal orexin function and understanding the mechanisms of loss or impairment of orexin neurons represent important goals. As a step toward that end, we generated human orexin neurons from induced pluripotent stem cells (hiPSCs) by treatment with N-acetyl-d-mannosamine (ManNAc) and its derivatives.

Non-enzymatic reaction of glycosyl oxazoline with peptides.

Recently, a number of chemoenzymatic strategies have been explored for achieving preparation of homogeneous glycopeptides and glycoproteins, especially by using endoglycanases and glycosyl oxazolines. However, concomitant occurrence of non-enzymatic reactions has been reported, but no further characterization of the byproducts was conducted. In this work, we made an attempt to identify the side product by using model substrates.

Diastereomeric resolution directed towards chirality determination focussing on gas-phase energetics of coordinated sodium dissociation.

Defining chiral centres is addressed by introducing a pair of chiral auxiliary groups. Ions of diastereomeric pairs of molecules could be distinguished utilising energy-resolved mass spectrometry, and the applicability of the method to a series of compounds carrying amine, carboxylic acid, alcohol, and all the amino acids was verified. The method was further strengthened by distinguishing diastereomeric ions that did not undergo fragmentation.

Endoplasmic Reticulum (ER)-Targeted, Galectin-Mediated Retrograde Transport by Using a HaloTag Carrier Protein.

Investigations into metabolic processes within the cell have often relied on genetic methods such as forced expression and knockout or knockdown techniques. An alternative approach would be introducing a molecule into the desired location inside the cell. To translocate compounds from outside cells into the endoplasmic reticulum (ER), we constructed a delivery carrier protein.

Hydrophobic Tagged Dihydrofolate Reductase for Creating Misfolded Glycoprotein Mimetics.

In the endoplasmic reticulum (ER), nascent glycoproteins that have not acquired the native conformation are either repaired or sorted for degradation by specific quality-control systems composed by various proteins. Among them, UDP-glucose:glycoprotein glucosyltransferase (UGGT) serves as a folding sensor in the ER. However, the molecular mechanism of its recognition remains obscure.

Profiling Aglycon-Recognizing Sites of UDP-glucose:glycoprotein Glucosyltransferase by Means of Squarate-Mediated Labeling.

Because of its ability to selectively glucosylate misfolded glycoproteins, UDP-glucose:glycoprotein glucosyltransferase (UGGT) functions as a folding sensor in the glycoprotein quality control system in the endoplasmic reticulum (ER). The unique property of UGGT derives from its ability to transfer a glucose residue to N-glycan moieties of incompletely folded glycoproteins. We have previously discovered nonproteinic synthetic substrates of this enzyme, allowing us to conduct its high-sensitivity assay in a quantitative manner.

The relationship between glycan structures and expression levels of an endoplasmic reticulum-resident glycoprotein, UDP-glucose: Glycoprotein glucosyltransferase 1.

In this article, we report a relationship between glycan structures and expression levels of a recombinant ER-resident glycoprotein, uridine 5'-diphosphate-glucose: glycoprotein glucosyltransferase (UGGT1). The function of glycan structures attached to a glycoprotein is actively studied; however, the glycan structures of recombinant, and not endogenous, glycoproteins have not been examined. In this study, we indicate a relationship between the glycan structure and the level of protein expression.

Synthetic study of 3-fluorinated sialic acid derivatives.

Sialic acid derivatives, analogs, and their conjugates are expected to be pharmaceutical candidates such as anti-influenza drugs and also useful probes for investigating the biological role of glycoconjugates. Derivatives of 3-fluorinated sialic acid (3-F-Sia) have been found to be excellent probes in investigating functions and mechanisms of a series of proteins. Here, we describe the syntheses of 3-F-Sia derivatives, which are useful in making biologically important conjugate probes.

Glycan structure and site of glycosylation in the ER-resident glycoprotein, uridine 5'-diphosphate-glucose: glycoprotein glucosyltransferases 1 from rat, porcine, bovine, and human.

Here we report glycan structures and their position of attachment to a carrier protein, uridine 5'-diphosphate-glucose: glycoprotein glucosyltransferase (UGGT1), as detected using tandem mass spectrometry. UGGT1 acts as a folding sensor of newly synthesized glycosylated polypeptides in the endoplasmic reticulum, and the transferase itself is known to be glycosylated. The structure of glycan attached to UGGT1, however, has not been investigated.

Syntheses of lactosyl ceramide analogues carrying novel bifunctional BODIPY dyes directed towards the differential analysis of multiplexed glycosphingolipids by MS/MS using iTRAQ.

Lactosyl ceramide analogues carrying novel bifunctional BODIPY-based fluorescent tags were designed and synthesised for live cell imaging. Addition of azide functionality on the fluorophore facilitated isobaric tagging for quantitative multiplexed analysis of biomolecules based on tandem mass spectrometry.

Both isoforms of human UDP-glucose:glycoprotein glucosyltransferase are enzymatically active.

Being recognized as an important constituent of the glycoprotein folding cycle, uridine diphosphate-glucose:glycoprotein glucosyltransferase (UGGT) has been a subject of intense study. Up to now, it is two isoforms, UGGT1 and 2 have been identified, which share ∼ 50% amino acid identity. UGGT1 is a well-documented enzyme which functions as a folding sensor in the endoplasmic reticulum, by the virtue of its ability to transfer a glucose residue to non-glucosylated high-mannose-type glycans of immature glycoproteins exhibiting non-native conformation.

Stereospecific generation and analysis of α- and β-hemiacetals of monosaccharides in gas phase.

A series of Boc-protected 4-aminobutyl α- and β-glycosides of commonly found neutral monosaccharides were synthesized. The sodium adducted ions of these individual molecules were used in producing corresponding α- and β-anomers of hemiacetal species under collision-induced dissociation (CID) conditions. The Boc group was successfully removed under CID conditions producing 4-aminobutyl glycosides, which were then used as the precursors.

Analysis of the cellular dynamics of fluorescently tagged glycosphingolipids by using a nanoliquid chromatography-tandem mass spectrometry platform.

Despite the increasing biological interests on glycoconjugates, the synthetic mechanism of oligosaccharides has not yet been revealed except for the enzymes involved. To clarify the synthetic events that occur inside cells, spatiotemporal analysis of fluorescently tagged glycosphingolipids was carried out. Transformation of the incorporated lactosylceramide analogue carrying 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl group (BODIPY fluorophore) was analyzed using nanoLC-fluorescence detection-nanoelectrospray ionization-mass spectrometry.

Multi-stage mass spectrometric information obtained by deconvolution of energy-resolved spectra acquired by triple-quadrupole mass spectrometry.

Triple-quadrupole mass spectrometry (TQ-MS) provides the capability to carry out collision-induced dissociation (CID) and it offers advantages in quantification when connected with high-performance liquid chromatography through an electrospray ionization interface. However, although TQ-MS provides information on partial structures through the analysis of product ions obtained by CID experiments, the method only provides single-stage CID experiments, which limits the detailed structural information that can be obtained. Herein, a method of overcoming this limitation of TQ-MS is described.

Fluorescence-monitored zero dead-volume nanoLC-microESI-QIT-TOF MS for analysis of fluorescently tagged glycosphingolipids.

An analysis of the glycan processing event is of particular importance to understand the nontemplate dependent synthetic mechanism of the multiple glycosylation reactions taking place in the Golgi apparatus in connection with the post-translational modification of biomolecules. In our efforts to address the issue, we constructed an analysis platform using nano-liquid chromatography (LC), which also worked as a spray tip, with an optical-fiber-based blue (470 nm) light emitting diode (LED)-induced fluorescence (520 nm) detector coupled with a microelectrospray ionization (ESI)-quadrupole ion trap (QIT)-time of flight (TOF) mass spectrometer (MS). This system was designed to enable both quantitative and qualitative analyses of fluorescently tagged molecules such as BODIPY-tagged lactosylceramide.

Analysis of a series of isomeric oligosaccharides by energy-resolved mass spectrometry: a challenge on homobranched trisaccharides.

Glycans exist as part of glycoproteins and glycolipids, which are involved in a variety of biological functions. The analysis of glycan structures, particularly that of structural isomers, is fundamentally important since isomeric glycans often show distinct functions; however, a method for their structural elucidation has not yet been established. Anomeric configurations, linkage positions and branching are the major factors in glycans and their alteration results in a large diversity of glycan structures.

N-Hexyl-4-aminobutyl glycosides for investigating structures and biological functions of carbohydrates.

The potential applications of N-hexyl-4-aminobutyl glycosides in the mass spectrometric investigation of glycan structure and in the investigation of glycan functions were studied. Under collision-induced dissociation (CID) conditions, sodiated glycosides carrying N-hexyl-4-aminobutyl groups effectively produced a hemiacetal species (C-ions), which is important in mass-spectrometry-based structural investigation. The usefulness of N-hexyl-4-aminobutyl glycosides in biological analysis was also confirmed by obtaining a binding constant for the binding of dipyrrometheneboron difluoride C3-labeled N-hexyl-4-aminobutyl beta-lactoside with an Erythrina cristagalli lectin, and by visualizing cellular organelles using a more hydrophobic BODIPY-labeled compound.

Analysis of behavior of sodiated sugar hemiacetals under low-energy collision-induced dissociation conditions and application to investigating mutarotation and mechanism of a glycosidase.

Analysis of anomericity is one of the most important issues in the structure elucidation of carbohydrates. Mass spectrometry (MS)-based methods are of particular interest and important to address the issue related to resolving anomericity of monosaccharide units in a glycan. However, direct analysis of hemiacetals has not been possible by MS because of the nonavailability of information regarding the gas-phase behavior of such ion species.

Ion-trap mass spectrometry unveils the presence of isomeric oligosaccharides in an analyte: stage-discriminated correlation of energy-resolved mass spectrometry.

Mass spectrometry, especially tandem mass spectrometry, has been widely used in the field of analytical sciences for handling biological and chemical samples. The technique resolves molecular and fragment ions based on the mass to charge ratio. Energy-resolved mass spectrometry (ERMS) further provides an activation energy-related factor in the dissociation reaction.

Energy-resolved structural details obtained from gangliosides.

Gangliosides, a family of glycosphingolipids (GSLs) that comprise sialic acid residue(s), are an important class of molecules that exist on the outer surface of the plasma membrane. To assess the functions of a particular series of gangliosides that play important roles in brain functions, their structures and localizations need to be investigated. We studied the structures of these gangliosides by collision-induced dissociation using quadrupole ion-trap mass spectrometry.

Sequential enzymatic glycosyltransfer reactions on a microfluidic device: Synthesis of a glycosaminoglycan linkage region tetrasaccharide.

A microfluidic chip carrying three reaction chambers was designed and constructed to examine sequential multiple enzymatic reactions. The synthesis of oligosaccharides in living cells is carried out in the Golgi apparatus where multiple enzymes such as glycosidase and glycosyltransferases act on a variety of substrates to generate glycoconjugates that include glycolipids and glycoproteins. The regulatory mechanism of the process however remains unknown.

High-yielding and controlled dissociation of glycosides producing B- and C-ion species under collision-induced dissociation MS/MS conditions and use in structural determination.

Collision-induced dissociation (CID) in mass spectrometry is a powerful technique with which to understand gas-phase chemical reactions. A mass spectrometer is used to carry out the reaction, isolation, and analysis. On the other hand, structural analysis of glycan structures is of extreme importance in the analysis of biomolecules, such as glycoproteins and glycolipids.

Discrimination of 16 structural isomers of fucosyl galactoside based on energy-resolved mass spectrometry.

Glycans, a family of compounds often attached to proteins and ceramides, are diverse molecules involved in a wide range of biological functions. Their structural analysis is necessary and is often carried out at the microscale level. Methods based on mass spectrometry are therefore used, although they do not provide information regarding isomeric structures often found in glycan structures.