Investigation of the Protective Effect for GcMAF by a Glycosidase Inhibitor and the Glycan Structure of Gc Protein.

O-linked α-N-acetylgalactosamine (α-GalNAc) in the Gc protein is essential for macrophage activation; thus, the GalNAc-attached form of Gc protein is called Gc macrophage activating factor (GcMAF). O-linked glycans in Gc proteins from human plasma mainly consist of trisaccharides. GcMAF is produced when glycans on the Gc protein are hydrolyzed by α-Sia-ase and ß-Gal-ase, leaving an α-GalNAc. Upon hydrolysis of α-GalNAc present on GcMAF, the protein loses the macrophage-activating effect. In contrast, our synthesized pyrrolidine-type iminocyclitol possessed strong in vitro α-GalNAc-ase inhibitory activity. In this study, we examined the protective effects of iminocyclitol against GcMAF via inhibition of α-GalNAc-ase activity. Detailed mass spectrometric analyses revealed the protective effect of the inhibitor on GcMAF. Furthermore, structural information regarding the glycosylation site and glycan structure was obtained using tandem mass spectrometric (MS/MS) analysis of the glycosylated peptides after tryptic digestion.

Temporal analysis of localization and trafficking of glycolipids.

Glycolipid metabolism occurs in the Golgi apparatus, but the detailed mechanisms have not yet been elucidated. We used fluorescently labeled glycolipids to analyze glycolipid composition and localization changes and shed light on glycolipid metabolism. In a previous study, the fatty chain of lactosyl ceramide was fluorescently labeled with BODIPY (LacCer-BODIPY) before being introduced into cultured cells to analyze the cell membrane glycolipid recycling process. However, imaging analysis of glycolipid recycling is difficult because of limited spatial resolution. Therefore, we examined the microscopic conditions that allow the temporal analysis of LacCer-BODIPY trafficking and localization. We observed that the glycolipid fluorescent probe migrated from the cell membrane to intracellular organelles before returning to the cell membrane. We used confocal microscopy to observe co-localization of the glycolipid probe with endosomes and Golgi markers, demonstrating that it recycles mainly through the trans-Golgi network (TGN). Here, a glycolipid recycling pathway was observed that did not require the lipids to pass through the lysosome.

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. We envisioned that analysing the glycan pattern of GSLs enables distinguishing diseases. For this purpose, we utilised a fluorescently tagged compound, LacCerBODIPY (1). At first, compound 1 was taken up by cultured PC12D cells and transformed into various GSLs. As a result, changes in the GSL patterns of differentiation states of the cells were successfully observed by using an analysis platform, nano-liquid chromatography (LC)-fluorescence detection (FLD)-electrospray ionisation (ESI)-mass spectrometry (MS), which could quantify and provide molecular ions simultaneously. We found that compound 1 remained for about 10 min on the plasma membrane before it was converted into other GSLs. We therefore investigated a more rapid way to discriminate different cellular states by fluorescence recovery after photobleaching, which revealed that it is possible to distinguish the differentiation states as well.

Identification of genes required for neural-specific glycosylation using functional genomics.

Glycosylation plays crucial regulatory roles in various biological processes such as development, immunity, and neural functions. For example, α1,3-fucosylation, the addition of a fucose moiety abundant in Drosophila neural cells, is essential for neural development, function, and behavior. However, it remains largely unknown how neural-specific α1,3-fucosylation is regulated. In the present study, we searched for genes involved in the glycosylation of a neural-specific protein using a Drosophila RNAi library. We obtained 109 genes affecting glycosylation that clustered into nine functional groups. Among them, members of the RNA regulation group were enriched by a secondary screen that identified genes specifically regulating α1,3-fucosylation. Further analyses revealed that an RNA-binding protein, second mitotic wave missing (Swm), upregulates expression of the neural-specific glycosyltransferase FucTA and facilitates its mRNA export from the nucleus. This first large-scale genetic screen for glycosylation-related genes has revealed novel regulation of fucTA mRNA in neural cells.

Cellular uptake of liposome consisting mainly of glucocerebroside from the starfish Asterias amurensis into Caco-2 cells.

Glucocerebroside (GlcCer) is a group of compounds consisting of ß-linked glucose and ceramide with various chain lengths, some of which possess anti-tumor activity and improve skin barrier function for atopic patients when administered orally. The amphiphilic GlcCer molecules are generally easy to aggregate in aqueous solution and result in low absorption in the gut, which can be improved by forming a liposome. With a recognition that a relatively large amount of GlcCer is contained in the starfish and is being discarded, we prepared a liposome consisting mainly of GlcCer (over 95%) with 100 nm in diameter. The adsorption efficiency of the liposome into cultured Caco-2 cells was investigated by live-cell imaging using fluorescently labeled liposomes. We found an immediate internalization of GlcCer-liposome on exposure without significant accumulation on the plasma membrane. The membrane fluidity was transiently affected as evidenced by fluorescence recovery after photobleaching (FRAP) experiments without no significant cellular damage, which indicates a liposome with high content of GlcCer might be useful as the carrier of dietary and/or drug molecules.

Surface Modification of Porous Silica Particles with Carbohydrate Scaffolds as Receptor Components for Molecular Recognition.

Invited for this month's cover is the group of Osamu Kanie and co-workers at Tokai University. The cover picture shows the interaction of an incoming molecule and a receptor surface as the first process of a molecular sensing mechanism. For this, porous silica particles were decorated with protected carbohydrates to create artificial pocket-like structures on its surface. More information can be found in the Research Article by O. Kanie and co-workers.

Surface Modification of Porous Silica Particles with Carbohydrate Scaffolds as Receptor Components for Molecular Recognition.

Biosensors play a pivotal role in diagnosis through specific receptor-ligand interactions. However, receptor biomolecules such as proteins have inevitable stability issues due to denaturation. Alternatively, utilization of the small molecules multivalently presented on porous silica particles as receptor components is considered to address the stability issues. A series of receptor components was synthesized using carbohydrates as a chiral scaffold to support multiple functional groups. The synthesized molecules were attached on the porous silica particles. Raman spectral analyses of single silica particles in the presence of nitrobenzene derivatives revealed a specific interaction with 4-nitrophenol among others using a confocal Raman microscope. Chiral selective recognition was also accomplished for (1S,3S)- and (1R,3R)-2-amino-1-(4-nitrophenyl)-1,3-propanediols. Protected carbohydrate derivatives are shown to be useful as receptor components on porous silica particles.

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. A spectrum obtained by energy-resolved mass spectrometry (ERMS) was used to deconvolute the fragmentation process, with a Galili-antigenic trisaccharide derivative being used as an example. A replot of the ERMS data showing the ratios of the product ions to the precursor ion resulted in a descriptive graph. Analysis of the sum of the ratios of individual product ions to the precursor ion at specific CID energies revealed that the members of a series of product ions were related to each other. The obtained relationships and the m/z values of the product ions provided information on the fragmentation process taking place during the dissociation, indicating that the ERMS spectrum obtained by TQ-MS contained equivalent information to that obtainable by multi-stage MS/MS (MS(n); n≥2). This method may allow users of triple-quadrupole mass spectrometers to obtain MS(n)-type information by performing a single ERMS experiment, which is even advantageous over quadrupole ion trap (QIT)-MS/MS because CID experiments on individual first-generation product ions are not required.

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. We addressed this issue by using stage-discriminated energy-resolved mass spectrometry (ERMS) at the stages of MS(n) and MS(n+1) and showed that such analysis can be made. This was achieved by proving that individual anomers can be identified and that the equilibrium of sodium adducted ion species of alpha- and beta-anomers can be negated in the gas phase under collision-induced dissociation (CID) conditions. On the basis of these results, we could 1) observe the mutarotation of lactose and 2) speculate the hydrolysis mechanism of endo-glycosylceramidase by using mass spectrometry.

A unique structural distribution pattern discovered for the cerebrosides from starfish Asterias amurensis.

Cerebroside is an important family of the mono-glycosylated ceramides involved in the larger family of glycosphingolipid and sulfatide. Cerebroside is synthesized from ceramide by the transfer of glucose from UDP-glucose, and degraded back to ceramide, which plays an important role at the epidermis protecting interior of the body as a barrier. Because cerebroside is regarded as the source molecule of ceramide and is amphiphilic in nature, cerebroside is considered valuable as the ingredient of cosmetic lotion. Various sources can be considered as raw material of cerebrosides. Starfish is considered as one of such potent source. However, the structure of the ceramide part of cerebroside is not fully investigated. Therefore, the individual structures of cerebroside molecules need to be identified including sphingosine and fatty acyl group composition to assess the potential of the molecule. We investigated and determined the structures of cerebrosides in starfish Asterias amurensis using LC-MS, GC-MS, tandem mass spectrometry (MS/MS), and 1H NMR. We also discovered a characteristic structure distribution that was divided into three major groups: 1) a group composed of a relatively long sphingosine (C22) and a short length of fatty acyl group (less than C16), 2) a group composed of a typical C18 sphingosine and long fatty acyl groups (greater than C23), and 3) a group composed of C18 sphingosine and fatty acyl groups with their length less than C18. The calculated Log P values of cerebrosides ranging from 9 to 11 covered about 80% of the molecules that were in the range of those used in cosmetics, thus showing the potential usefulness of starfish Asterias amurensis as a source of raw material for cerebrosides.

Stereoselective trimethylsilylation of α- and ß-galactopyranoses.

Trimethylsilylation of the anomeric hydroxyl groups of tetra-O-benzyl and tetra-O-acetyl galactopyranoses was investigated. Stereoselective formation of ß-trimethylsilyl glycoside (ß-TMS glycoside) of benzyl protected compound was achieved using N-trimethylsilyl diethylamine. In the course of the investigation of the selective synthesis of TMS galactosides using TMS-imidazole, we observed the formation of an intermediate, which was converted predominantly into α-TMS glycoside after silica gel column chromatography. A reaction of acetylated compound using TMS-trifluoromethanesulfonate-2,6-lutindine selectively yielded α-TMS glycoside.

Structural analysis of a novel lipooligosaccharide (LOS) from Rhodobacter azotoformans.

Lipopolysaccharides (LPS) are components of the Gram-negative bacterial cell surface that stimulate the host innate immune system through the Toll-like receptor (TLR) 4-MD-2 complex. Rhodobacter sp. have been reported to produce LPS that lack endotoxic activity, and instead act as antagonists of other endotoxins. In this report, we focused on LPS, especially the lipooligosaccharide (LOS) fraction produced by Rhodobacter azotoformans that shows production of IL-8, but has an inverse correlation with IL-6 production. We analyzed their molecular structure by using mass spectrometry and nuclear magnetic resonance spectroscopy and report a novel LOS consisting of a shorter glycan structure containing glucuronic acid but not heptoses. A novel glycan structure, Glcα(1 → 4)GlcAα(1 → 4)KDOα(2 → 4)[Glcα(1 → 5)]KDOα(2 → 6)[4-phosphate]GlcNß(1 → 6) GlcNα1-phosphate, was proposed using NMR methods. The structure was consistent with one obtained based on MS. The MS analysis further revealed the existence of structural variation caused by extension with hexoses. The acyl composition in lipid A was suggested to contain three C14 fatty acyl chains (3-OH-14:0 or 3-oxo-14:0 at N2 of GlcN-1, 3-OH-14:0 at N2 of GlcN-2, that carried another 14:1 Δ7 on its ß-hydroxyl group) and two C10 fatty acyl chains (3-OH-10:0 at O3 of both GlcN), which are same as those found in lipid A from Rhodobacter sphaeroides.

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. A microchip-based analysis platform may provide a valuable tool with which to address the issue by mimicking the Golgi function. We thus examined 3 sequential glycosyltransfer reactions on a chip, and succeeded in the synthesis of a tetrasaccharide using immobilized enzymes. Also, the kinetic parameters for a recently identified glycosyltransferase, proteoglycan GalT-I, were obtained for the first time.

Comparative RP-HPLC for rapid identification of glycopeptides and application in off-line LC-MALDI-MS analysis.

Despite the increasing attention being paid to the functions of glycoproteins, their structural analysis is still difficult and hinders functional investigations. Structural analysis of post-translationally modified proteins is thought to be achieved using methods frequently utilized in proteomics research; however, the same methods cannot be used for glycosylated proteins. One of the difficulties associated with the physiochemical properties of glycopeptides and peptides is that the detection of the former is considerably more difficult, because of the existence of glycoforms that increase molecular weight and reduces quantities of individual species. Thus, difficulties are often faced in finding glycopeptide(s) by using MS when analyzing peaks (or fractions) obtained after proteolytic digestion and HPLC. One simple yet difficult solution to this problem would be to develop a purification method that provides better resolution. Our intention has been to address this issue by using a combination of conventional methods. We found that a method consisting of a combination of rough fractionation using a reverse-phase cartridge column under acidic conditions and comparative RP-HPLC, where the two chromatograms obtained using phosphate and borate buffers under basic conditions were compared, is effective for MS-based structural analysis. The applicability of the method in glycoprotein analysis was examined using various samples including ribonuclease B (RNase B), IgG1, ovalbumin (OVA), and asialo fetuin (ASF). The results suggest that the method is useful in the analysis of glycoproteins.

Evaluation of reversed-phase nano liquid chromatography conditions by using reversed-phase thin layer chromatography based on Hansen solubility parameters for the analysis of amphiphilic glycosylsphingolipid transformations.

Pulse chase analysis is often used in investigating dynamics of cellular substances. Fluorescently labeled lactosyl sphingosine molecule is useful in chasing its transformation, however the analysis of such metabolites in attomole level is of extreme difficult due to the presence of large amount of endogenous amphiphilic molecules such as glycosphingolipids, sphingomyerin, and glycerophospholipids. Nano LC suites for analyzing the attomole scale metabolites, therefore removal of endogenous substances prior to nano LC and finding appropriate nano LC conditions are necessary. Thus, we focused on the solubility of fluorescent BODIPY-labeled lactosylsphingosine (Lac-Sph-BODIPY) to identify suitable solvents to remove endogenous compounds. In this study, we evaluated solvents by using C18 thin layer chromatography (RP TLC). The mobility (Rf) of Lac-Sph-BODIPY against several solvent mixtures on RP TLC were plotted against polarity and hydrogen bonding capability followed by Hansen solubility parameters (HSPs). The optimum solvent mixture with Rf = 0.3 ±â€¯0.1 was chosen for elimination of endogenous phospholipids on a ZrO2-SiO2 cartridge column and subsequent separation by nano LC. Efficient removal of endogenous phospholipids was demonstrated, and good resolution in nano LC analysis of Lac-Sph-BODIPY extracted from Chinese hamster ovary (CHO)-K1 cells was achieved. It was also shown that the amount of exogenously added compound was important in the investigation of metabolites using cultured cells.

A Versatile Synthetic Strategy for the Preparation and Discovery of New Iminocyclitols as Inhibitors of Glycosidases.

A series of iminocyclitols was prepared using a versatile synthetic strategy, and their inhibition of glycosidases was evaluated using capillary electrophoresis. The study has demonstrated that remarkable specificities in enzyme inhibition can be achieved with small modifications on the aglycon side chain and the ring nitrogen. Among the compounds synthesized, (2R,3R,4R,5R)-N-methyl-2-(acetamidomethyl)-3,4-dihydroxy-5-(hydroxymethyl)pyrrolidine was found to be very potent against ß-N-acetylhexosaminidase P with the Ki value of 80 nM.

Electrophoretically mediated microscale reaction of glycosidases: kinetic analysis of some glycosidases at the nanoliter scale.

Capillary electrophoresis (CE) is one of the extremely important analytical techniques known for its high sensitivity and resolution. We have investigated electrophoretically mediated microanalysis (EMMA) for the assay of some native glycosidases. Under optimized conditions, the enzymatic reactions of alpha-glucosidase, beta-galactosidase and beta-N-acetylglucosaminidase were carried out, and the Michaelis constants were obtained. The current method may have advantages over traditional assay methods, especially in terms of the amount of enzyme and substrate required for a reaction.

Analysis of pyridylaminated oligosaccharides using liquid chromatography-mass spectrometry with a monolithic capillary column.

We examined the utility of a monolithic capillary column in the analysis of pyridylaminated oligosaccharides. Fluorescence detection and mass spectrometry were used to monitor a series of oligosaccharides. Although the total-ion chromatogram appeared similar to that obtained with fluorescence detection, the sensitivity of this technique was limited, especially in the case of smaller oligosaccharides. This limitation was overcome by applying selected ion current monitoring. Further, the capillary column also exhibited good reproducibility. We showed that the retention times obtained by using the monolithic capillary column could be converted into the standard data to enable comparison of the experimental data with the existing data. Furthermore, our studies revealed an important difference in the separation profile, i.e., the monolithic capillary column could resolve smaller oligosaccharides to a greater extent.

Insight into the regulation of glycan synthesis in Drosophila chaoptin based on mass spectrometry.

BACKGROUND: A variety of N-glycans attached to protein are known to involve in many important biological functions. Endoplasmic reticulum (ER) and Golgi localized enzymes are responsible to this template-independent glycan synthesis resulting glycoforms at each asparagine residues. The regulation mechanism such glycan synthesis remains largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: In order to investigate the relationship between glycan structure and protein conformation, we analyzed a glycoprotein of Drosophila melanogaster, chaoptin (Chp), which is localized in photoreceptor cells and is bound to the cell membrane via a glycosylphosphatidylinositol anchor. Detailed analysis based on mass spectrometry revealed the presence of 13 N-glycosylation sites and the composition of the glycoform at each site. The synthetic pathway of glycans was speculated from the observed glycan structures and the composition at each N-glycosylation site, where the presence of novel routes were suggested. The distribution of glycoforms on a Chp polypeptide suggested that various processing enzymes act on the exterior of Chp in the Golgi apparatus, although virtually no enzyme can gain access to the interior of the horseshoe-shaped scaffold, hence explaining the presence of longer glycans within the interior. Furthermore, analysis of Chp from a mutant (RNAi against dolichyl-phosphate alpha-d-mannosyltransferase), which affects N-glycan synthesis in the ER, revealed that truncated glycan structures were processed. As a result, the distribution of glycoforms was affected for the high-mannose-type glycans only, whereas other types of glycans remained similar to those observed in the control and wild-type. CONCLUSIONS/SIGNIFICANCE: These results indicate that glycan processing depends largely on the backbone structure of the parent polypeptide. The information we obtained can be applied to other members of the LRR family of proteins.