Multifunctional Cell Regulation Activities of the Mussel Lectin SeviL: Induction of Macrophage Polarization toward the M1 Functional Phenotype.

SeviL, a galactoside-binding lectin previously isolated from the mussel Mytilisepta virgata, was demonstrated to trigger apoptosis in HeLa ovarian cancer cells. Here, we show that this lectin can promote the polarization of macrophage cell lines toward an M1 functional phenotype at low concentrations. The administration of SeviL to monocyte and basophil cell lines reduced their growth in a dose-dependent manner. However, low lectin concentrations induced proliferation in the RAW264.7 macrophage cell line, which was supported by the significant up-regulation of TOM22, a component of the mitochondrial outer membrane. Furthermore, the morphology of lectin-treated macrophage cells markedly changed, shifting from a spherical to an elongated shape. The ability of SeviL to induce the polarization of RAW264.7 cells to M1 macrophages at low concentrations is supported by the secretion of proinflammatory cytokines and chemokines, as well as by the enhancement in the expression of IL-6- and TNF-α-encoding mRNAs, both of which encode inflammatory molecular markers. Moreover, we also observed a number of accessory molecular alterations, such as the activation of MAP kinases and the JAK/STAT pathway and the phosphorylation of platelet-derived growth factor receptor-α, which altogether support the functional reprogramming of RAW264.7 following SeviL treatment. These results indicate that this mussel ß-trefoil lectin has a concentration-dependent multifunctional role in regulating cell proliferation, phenotype, and death in macrophages, suggesting its possible involvement in regulating hemocyte activity in vivo.

Exposure of silver nanocolloids causes glycosylation disorders and embryonic deformities in medaka.

Silver nanomaterials such as silver nanocolloids (SNC) contribute to environmental pollution and have adverse ecological effects on aquatic organisms. In particular, chemical exposure of fish during embryogenesis leads to deformities and puts the population at risk. Although glycans and glycosylation are known to be important for proper morphology in embryogenesis, little glycobiology-based research has examined morphological disorders caused by environmental pollutants. This study addressed the glycobiological effects of SNC exposure on medaka embryogenesis. After exposure of medaka embryos to SNC, deformities such as small heads and deformed eyes were observed. The expression of five glycan-related genes (alg2, gnsb, b4galt2, b3gat1a, and b3gat2) was significantly altered, with changes depending on the embryonic stage at exposure, with more severe deformities with exposure at earlier stages. In situ hybridization analyses indicated that the five genes were expressed mainly in the head region; exposure of SNC suppressed alg2 and gnsb and enhanced b4galt2 and b3gat1a expression relative to controls on day 7. Loss (siRNA)- and gain (RNA overexpression)-of-function experiments confirmed that alg2, gnsb, and b4galt2 are essential for embryogenesis. The effects of SNC exposure on glycan synthesis were estimated by glycan structure analysis. In the medaka embryo, high mannose-type glycans were dominant, and SNC exposure altered glycan synthesis. The alteration was more significant when exposure occurred at an early stage of medaka embryogenesis. Thus, SNC exposure causes embryonic deformities in medaka embryos through disordered glycosylation.

Structural analysis of N-glycans in medaka gut exposed to silver and titanium dioxide nanoparticles.

Nanomaterials are in general use in a broad range of industries. However, there are concerns that their intense use leads to heavy damage to the aquatic environment, and their discharge harms many aquatic organisms. N-Glycans are widely distributed in eukaryotic organisms and are intimately involved in most life phenomena. However, little is known about N-glycans in aquatic organisms exposed to nanomaterials. In this study, we investigated how nanomaterials affect N-glycans in the gut of adult female medaka. We found that silver nanoparticles exposure had little effect on gut N-glycans, whereas titanium dioxide nanoparticles (TiO2NPs) exposure increased the relative levels of several N-glycans in comparison with control. Structural analysis showed high levels of N-glycans of the high-mannose type, of which five N-glycans were free N-glycans with one ß-N-acetylglucosamine residue on the reducing end. The levels of free N-glycans are closely related to protein quality control in the endoplasmic reticulum and cytosol. Our results suggest that TiO2NPs exposure increases the levels of misfolded glycoproteins, resulting in generation of considerable amounts of free N-glycans. Our findings also suggest that TiO2NPs exposure suppresses cytosolic α-mannosidase trimming. This study provides new evidence for the effect of TiO2NPs on medaka gut from the aspect of environmental glycobiology.

Effects of silver nanocolloids on plant complex type N-glycans in Oryza sativa roots.

Silver nanomaterials have been mainly developed as antibacterial healthcare products worldwide, because of their antibacterial activity. However, there is little data regarding the potential risks and effects of large amounts of silver nanomaterials on plants. In contrast, N-glycans play important roles in various biological phenomena, and their structures and expressions are sensitive to ambient environmental changes. Therefore, to assesse the effects of silver nanomaterials, we focused on the correlation between N-glycans and the effects of silver nanomaterials in plants and analyzed N-glycan structures in Oryza sativa seedlings exposed to silver nanocolloids (SNCs). The phenotype analysis showed that the shoot was not affected by any SNC concentrations, whereas the high SNC exposed root was seriously damaged. Therefore, we performed comparative N-glycan analysis of roots. As a result, five of total N-glycans were significantly increased in SNC exposed roots, of which one was a free-N-glycan with one beta-N-acetylglucosamine residue at the reducing end. Our results suggest that the transition of plant complex type N-glycans, including free-N-glycans, was caused by abnormalities in O. sativa development, and free-N-glycan itself has an important role in plant development. This study originally adapted glycome transition analysis to environmental toxicology and proposed a new category called "Environmental glycobiology".

Glucose deprivation regulates the progranulin-sortilin axis in PC12 cells.

Progranulin (PGRN) is a growth factor implicated in several neurodegenerative diseases, such as frontotemporal lobar degeneration. Despite its important role in the central nervous system (CNS), the mechanisms controlling PGRN expression in the CNS are largely unknown. Recent evidence, however, suggested that several stressors, such as hypoxia, acidosis, or oxidative stress, induce PGRN expression. The present study was mainly aimed at determining whether and, if so, how glucose deprivation affects PGRN expression in PC12 cells. Initially, it was found that glucose deprivation gradually induced PGRN gene expression in PC12 cells. To elucidate the underlying molecular mechanisms, several intracellular signalings that were modified in response to glucose deprivation were examined. Both adenosine monophosphate kinase (AMPK) activation and changes in osmotic pressure, which are modified by extracellular glucose concentration, had no effect on PGRN gene expression; on the other hand, p38 activation in response to glucose deprivation played an important role in inducing PGRN gene expression. It was also found that expression of sortilin, a PGRN receptor implicated in PGRN endocytosis, was dramatically reduced by glucose deprivation. In contrast to glucose-dependent regulation of PGRN gene expression, AMPK activation played a central role in reducing sortilin expression. Overall, the present study suggests that the PGRN-sortilin axis is modulated by glucose deprivation via two distinct mechanisms. As PGRN is neuroprotective, this system may represent a new neuroprotective mechanism activated by glucose deprivation in the CNS.

N-glycan transition of the early developmental stage in Oryza sativa.

N-Glycosylation is one of the post-translational modifications. In animals, N-glycans linked to proteins function in cell-cell recognition, sorting, transport, and other biological phenomena. However, in plants, N-glycan-mediated biological functions remain obscure. In a previous study, we showed that the main type of N-glycan transition is from the paucimannosidic to complex type before and after germination in Oryza sativa, suggesting that transitions of N-glycan, including those of glycoproteins and glycosyltransferases, are closely associated with plant growth. To further elucidate the relationship between N-glycan structure and plant growth, we analyzed the structures of N-glycans expressed in O. sativa seedlings grown under light conditions and performed comparative analyses of the structures in the shoot and root. The analyses show that fundamental N-glycan structures are common to the shoot and root, whereas paucimannosidic-type N-glycans dramatically decreased in the root grown under light conditions. Further, to investigate the effects of light on N-glycan structures in O. sativa seedlings, we analyzed N-glycan structures in O. sativa seedlings grown in the dark. Understandably, N-glycan expression in the root was almost unaffected by light. However, despite a marked difference in phenotype, N-glycan expression in the shoot was also unaffected by light. This result suggests that the shoot and root of O. sativa have different glycoproteins and distinct N-glycan synthetic systems. Thus, we propose that the N-glycan synthetic system of the O. sativa shoot is almost unaffected by light conditions and that many photosynthesis-related proteins are not modified by N-glycans.

Comparative analysis of N-glycans in the ungerminated and germinated stages of Oryza sativa.

All fundamental information such as signal transduction, metabolic control, infection, cell-to-cell signaling, and cell differentiation related to the growth of plants are preserved in germs. In preserving these information, glycans have a key role and are involved in the development and differentiation of organisms. Glycans which exist in rice germ are expected to have an important role in germination. In this study, we performed structural and correlation analysis of the N-glycans in rice germ before and after germination. Our results confirmed that the N-glycans in the ungerminated stage of the rice germ had low number of N-glycans consisting only of six kinds especially with high-mannose and paucimannose type N-glycans being 16.0% and 76.7%, respectively. On the other hand, after 48 hours germinated germ stage, there was an increase in the complex type N-glycans with the appearance of Lewis a structure, the most complex type and a decrease in paucimannose types. These results suggest that at least six kinds of N-glycans are utilized for long time preservation of rice seed, while the diversification of most complex types of N-glycans is produced an environment dependent for shoot formation of rice.

Signal enhancement of protein binding by electrodeposited gold nanostructures for applications in Kretschmann-type SPR sensors.

We propose a novel Kretschmann-type surface plasmon resonance (SPR) sensor chip having a surface covered with electrodeposited gold nanostructures to enhance the sensitivity of SPR biosensing. The nanostructure is three-dimensional and has a larger surface area than a conventional flat surface chip, which increases the amount of protein binding and also induces a large change in the effective dielectric constant of the sensing area. The gold nanostructures were formed by electrodeposition under galvanostatic conditions, so their size could be controlled by manipulating the deposition time and current. The sensing characteristics, including the concentration dependence and noise level, indicated that the performance of the resulting chip (called a Au-black chip) was equivalent to that of a conventional sensor chip. We also determined the optimal electrodeposition conditions to obtain a sharp SPR curve for protein detection assay; the optimal thickness of the gold layer was 50-60 nm. Enhanced protein sensing was demonstrated by using a binding assay of anti-BSA antibody and BSA molecules. The protein binding signal was several times higher than that of the conventional assay. The insights into electrodeposition for SPR sensing presented here will enable improved sensitivity for detecting low-concentration and small proteins.

Hypersialylated type-I lactosamine-containing N-glycans found in Artiodactyla sera are potential xenoantigens.

There is increasing interest in biologics, i.e. human-originated biological pharmaceutics. Most of the protein drugs developed so far, such as immunoglobulins and erythropoietin, are secreted glycoproteins; as a result, any non-human-type glycans, such as αGal and NeuGc, derived from animal cells and sera must be removed to circumvent undesirable immunogenic reactions. In this study, we made an extensive search for potential xenoantigenic glycans among a panel of mammalian sera. As a result, sera belonging to the order Artiodactyla, i.e. bovine, lamb and goat sera, were found to contain substantial amounts of hypersialylated biantennary glycans closely associated with a type-I lactosamine structure containing a unique tetrasaccharide, Siaα2-3Galß1-3(Siaα2-6)GlcNAc. In all three Artiodactyla sera, the most abundant structure was Siaα2-3Galß1-3(Siaα2-6)GlcNAcß1-2Manα1-3[Siaα2-6Galß1-4GlcNAcß1-2Manα1-6]Manß1-4GlcNAcß1-4GlcNAc. A dually hypersialylated biantennary structure, Siaα2-3Galß1-3(Siaα2-6)GlcNAcß1-2Manα1-3[Siaα2-3Galß1-3(Siaα2-6)GlcNAcß1-2Manα1-6]Manß1-4GlcNAcß1-4GlcNAc, was also abundant (10%) in bovine serum. The amount of hypersialylated glycans among total sialylated glycans was 46, 26 and 23% in bovine, lamb and goat sera, respectively. On the other hand, such structures could not be detected in the sera of other animals including human. The biological functions and the immunogenicity of the hypersialylated glycans in these animals remain to be elucidated; however, it is worth noting that glycoproteins biosynthesized from Artiodactyla cells and those contaminated with bovine serum might enhance undesirable antigenicity in human patients.

Size-exclusion SPR sensor chip: application to detection of aggregation and disaggregation of biological particles.

We propose a novel surface plasmon resonance (SPR) sensor chip with a microfabricated slit array. The microslit excludes micrometre-size objects larger than its slit size from the SPR sensing area, so that it functions as an in situ filter. We demonstrated the sensing of microparticles of different diameters using the chip, and the results show a successful size-exclusion effect. As a demonstration of the biological application, we performed the detection of aggregation and disaggregation of biological particles using sugar-chain-immobilized gold nanoparticles as a test sample.

Evaluation of electrodeposited gold nanostructures for applications in QCM sensing.

The electrodeposition of gold nanostructures increases the surface area of a biosensor, which brings an enhancement of the sensitivity by increasing the amount of analyte binding to the surface. To evaluate the relationship among the surface structure, the area and the analyte binding, we quantitatively analyzed them for quartz crystal microbalance (QCM) sensing by scanning electron microscopy and cyclic voltammetry measurements. The results indicate a several-times increase of analyte bindings, and also the limitation of the sensing performance.

Development of a chemical strategy to produce rare aldohexoses from ketohexoses using 2-aminopyridine.

Rare sugars are monosaccharides that are found in relatively low abundance in nature. Herein, we describe a strategy for producing rare aldohexoses from ketohexoses using the classical Lobry de Bruyn-Alberda van Ekenstein transformation. Upon Schiff-base formation of keto sugars, a fluorescence-labeling reagent, 2-aminopyridine (2-AP), was used. While acting as a base catalyst, 2-AP efficiently promoted the ketose-to-aldose transformation, and acting as a Schiff-base reagent, it effectively froze the ketose-aldose equilibrium. We could also separate a mixture of Sor, Gul, and Ido in their Schiff-base forms using a normal-phase HPLC separation system. Although Gul and Ido represent the most unstable aldohexoses, our method provides a practical way to rapidly obtain these rare aldohexoses as needed.

Development of D-allose sensor on the basis of three strategic enzyme reactions.

Rare sugars are defined as monosaccharides and their derivatives that rarely exist in nature, according to the International Society of Rare Sugars. D-Allose (3-epi d-glucose) is one of the rare sugars, for which various physiological activities have recently been found, with increasing attention to its applications to bio-industry. Until now, however, there is no convenient method of measuring these sugars in a specific manner. For detecting D-allose, three consecutive enzyme reactions were devised by fabricating of a reaction batch chamber packed with L-rhamnose isomerase (LRI), D-tagatose 3-epimerase (DTE) and a screen-printed electrode, on which D-fructose dehydrogenase (DFDH) was immobilized. To obtain a substantial sensing system, extensive experimental parameters were optimized. These included the concentration of photo-crosslinkable poly (vinyl alcohol) bearing stilbazolium groups (PVA-SbQ), reaction ratios, and temperature of the batch chamber. By adopting the three consecutive enzyme reactions, an undesirable reverse reaction was minimized. As a result, the developed sensor system exhibited a good linear response on D-allose in the range from 0.1 to 50 mM (r(2)=0.998). The system has an apparent advantage over the previous chromatography approach in terms of simplicity and inexpensiveness.

Comprehensive analysis of N-linked oligosaccharides from eggs of the family Phasianidae.

We have reported a strategic procedure for the preparation of human-type N-linked oligosaccharides targeting hen egg white and yolk. To determine whether the technique is applicable to other avian species, we performed comparative analysis of N-linked oligosaccharides derived from eggs of other pheasant species. Our investigation of the principal oligosaccharides resulted in several major findings: (i) Glycan profiles as well as total yields were different between species and tissues (egg white and yolk). (ii) A common feature of egg white glycans is agalactosylated, hybrid-type, and complex-type oligosaccharides containing bisecting GlcNAc as major components. (iii) Egg yolk of pheasant species contained alpha2-6sialylated, biantennary complex-type oligosaccharides as major components. (iv) Egg yolk of Japanese pheasant and golden pheasant contained unusual persialylated oligosaccharides. Our results suggest that pheasant egg glycomes are significantly different from other avian species, although some common features are present.

A comprehensive HPLC analytical system for the identification and quantification of hexoses that employs 2-aminobenzamide coupling.

Rare sugars are defined as monosaccharides with extremely low natural abundances. Their natural distributions and biological functions remain to be clarified. To establish a robust analytical system that can separate, identify and quantify rare sugars, 12 d-hexoses-including five rare aldohexoses and three rare ketohexoses-were labelled with 2-aminobenzamide (2-AB), and the fluorescently tagged monosaccharides were separated by high-performance liquid chromatography (HPLC). Because the ketohexoses were much less reactive than were the aldohexoses, the reaction conditions were optimized to achieve the maximum yields (>75%) for both aldohexoses and ketohexoses. The calibration curve determined for the rare ketohexose, d-psicose (Psi), was linear between 1 pmol and 1 micromol and had a correlation coefficient of 0.9999. Using the developed method, the psicose content in a leaf of Itea virginica, in which the presence of psicose has previously been reported, was found to be 2.7 mg psicose/g leaf. The result proved feasibility of the method even for natural products. Because the system is a comprehensive tool, it should help answer questions concerning the biosyntheses and functions of rare hexose sugars.

Strategic glycan elution map for the production of human-type N-linked oligosaccharides: the case of hen egg yolk and white.

Glycans play important roles in various biological phenomena, but the lack of a systematic procedure for producing complex structures of glycans severely restricts their application in the medical and industrial fields. In this paper, we propose a basic strategy for the preparation of substantial amounts (>100 mg) of N-linked oligosaccharides, where the structure of each glycan is mapped with its elution position in liquid chromatography as well as the empirical yield. In model experiments using hen egg white and yolk as starting materials, the former provided a series of agalactosylated complex-type and hybrid-type N-linked oligosaccharides containing bisecting N-acetylglucosamine (GlcNAc) in addition to two high-mannose type glycans. In contrast, egg yolk gave predominantly alpha2-6sialylated biantennary glycans together with a high-mannose type one, reflecting the difference in the origins of the tissues. Due to the total identity of the glycans obtained to human ones, the present strategy should provide a practical scheme for the production of human-type N-linked oligosaccharides.

Development of an amperometric flow analysis sensor for specific detection of D-psicose.

The aim of this study was to develop a simple, rapid and highly sensitive sensor for measuring the rare sugar d-psicose. The proposed system adopts amperometric flow analysis and two consecutive enzyme reactions consisting of a reactor packed with d-tagatose 3-epimerase (DTE)-immobilized beads, which converts d-psicose to d-fructose, and a carbon-paste electrode containing d-fructose dehydrogenase (DFDH). In order to fabricate a robust sensor system, various experimental parameters were optimized including the buffer composition, flow rate for the two enzyme reactions and the size of micro-flow cell. The developed sensor responded linearly to d-psicose concentration in the range from 0.08 to 50mM (R(2)=0.988). The signal/noise ratio was 3.0 for the 0.08 mM d-psicose solution, and the relative standard deviations were 1.7 (n=20) and 2.6% (n=20) for the 10 and 20mM d-psicose solutions, respectively. One round of assay was completed within 8 min. Our results suggest that the sensor can be used not only for the detection of d-psicose in food samples but also for monitoring d-psicose within the environment. Moreover, the sensor system can be applied to the detection of many other rare sugars by using the same measurement principle.

A practical approach to N-glycan production by hydrazinolysis using hydrazine monohydrate.

Hydrazinolysis is a versatile method to liberate N-linked glycans from glycoproteins. However, the method is usually performed with anhydrous hydrazine, a highly toxic and explosive chemical used in rocket fuel. Thus despite the need to produce functionally important glyco-materials, hydrazinolysis is limited to small scale (e.g., 0.2-1 mL) reactions. In the present study, we report an alternative procedure for hydrazinolysis using hydrazine monohydrate in place of anhydrous hydrazine. The developed procedure was applied to both purified glycoproteins (Taka-amylase and transferrin) and hen egg yolk protein fraction with comparable yields to the traditional method using anhydrous hydrazine. The sialyl linkage of alpha2-6disialobiantennary oligosaccharides proved to be fully stable. The developed procedure facilitated the large-scale preparation of N-linked glycans. The new method should make a substantial contribution to both small- and large-scale production of functional glycans, including therapeutically relevant human-type glycans.

Gas-phase pyridylamination of saccharides: development and applications.

Pyridylamination is a versatile method for fluorescence labeling of oligosaccharides. The technique affords sensitive detection of saccharides with reducing termini and high-resolution separation by high-performance liquid chromatography. The conventional method, based on a liquid-phase reaction, has been extensively used in various aspects of glycobiology and glycotechnology. Unfortunately, the necessity for removing excess 2-aminopyridine makes the technique both laborious and time-consuming. Furthermore, removal of excess reagent can result in a significant loss of short saccharide components. In the present paper, we report an alternative methodology based on a "gas-phase" reaction, in which dried saccharides are reacted with vaporized 2-aminopyridine. The resultant Schiff base was also reduced in the gas phase within the same reaction microtube using a purpose-built device. The newly developed procedure was applied to both monosaccharide (GlcNAc) and oligosaccharides (isomalto-oligosaccharides) at quantitative yields with no requirement to remove excess reagent. The acid-labile sialyl linkages of alpha2-6-disialobiantennary oligosaccharides proved to be fully stable during the procedure. The developed method was also successfully applied to profiling N-linked oligosaccharides liberated from glycoproteins by hydrazinolysis and, thus, should contribute to various fields of glycomics.

Carbohydrate-recognition domains of galectin-9 are involved in intermolecular interaction with galectin-9 itself and other members of the galectin family.

Galectin-9 (Gal-9) is a tandem-repeat-type member of the galectin family associated with diverse biological processes, such as apoptosis, cell aggregation, and eosinophil chemoattraction. Although the detailed sugar-binding specificity of Gal-9 has been elucidated, molecular mechanisms that underlie these functions remain to be investigated. During the course of our binding study by affinity chromatography and surface plasmon resonance (SPR) analysis, we found that human Gal-9 interacts with immobilized Gal-9 in the protein-protein interaction mode. Interestingly, this intermolecular interaction strongly depended on the activity of the carbohydrate recognition domain (CRD), because the addition of potent saccharide inhibitors abolished the binding. The presence of multimers was also confirmed by Ferguson plot analysis of result of polyacrylamide gel electrophoresis and matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Moreover, this intermolecular interaction was observed between Gal-9 and other galectin members, such as Gal-3 and Gal-8, but not Gal-1. Because such properties have not been reported yet, they may explain an unidentified mechanism underlying the diverse functions of Gal-9.