Chihiro Sato

Nagoya University, Nagoya, Aichi, Japan

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Publications (72)225.29 Total impact

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    ABSTRACT: Interaction of Hsp70 with natural and artificial acidic glycans is demonstrated based on the native PAGE analysis. Hsp70 interacts with acidic glycopolymers that contain clustered sulfated and di-sialylated glycan moieties on a polyacrylamide backbone, but not with neutral or mono-sialylated glycopolymers. Hsp70 also interacts and forms a large complex with heparin, heparan sulfate, and dermatan sulfate that commonly contain 2-O-sulfated iduronic acid residues, but not with other types of glycosaminoglycans (GAGs). Hsp70 consists of the N-terminal ATPase domain and the C-terminal peptide-binding domain. The interaction analyses using the recombinant N- and C-terminal half domains show that the ATPase domain mediates the direct interaction with acidic glycans, while the peptide-binding domain stabilizes the large complexes with particular GAGs. To our knowledge, this is the first demonstration of direct binding of Hsp70 to the particular GAGs. This property may be involved in the physiological functions of Hsp70 at the plasma membrane and extracellular environments.
    Biochemical and Biophysical Research Communications 06/2014; · 2.41 Impact Factor
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    ABSTRACT: Polysialic acid is a linear homopolymer of α2-8 linked sialic acids attached mainly onto glycoproteins. Cell surface polysialic acid plays roles in cell adhesion and differentiation events in a manner that is often dependent on the degree of polymerization (DP). Anti-oligo/polysialic acid antibodies have DP-dependent antigenic specificity, and such antibodies are widely utilized in biological studies for detecting and distinguishing between different oligo/polysialic acids. A murine monoclonal antibody mAb735 has a unique preference for longer polymers of polysialic acid (DP>10), yet the mechanism of recognition at atomic level remains unclear. Here we report the crystal structure of mAb735 single chain variable fragment (scFv735) in complex with octasialic acid at 1.8 Å resolution. In the asymmetric unit, two scFv735 molecules associate with one octasialic acid. In both complexes of the unit, all the complementarity-determining regions except for L3 interact with three consecutive sialic acid residues out of the eight. A striking feature of the complex is that 11 ordered water molecules bridge the gap between antibody and ligand while the direct antibody-ligand interaction is less extensive. The dihedral angles of the trisialic acid unit directly interacting with scFv735 are not uniform, indicating that mAb735 does not strictly favor the previously proposed helical conformation. Importantly, both reducing and non-reducing ends of the bound ligand are completely exposed to solvent. We suggest that mAb735 gains its apparent high affinity for a longer polysialic acid chain by recognizing every three sialic acid unit in a paired manner.
    Journal of Biological Chemistry 10/2013; · 4.65 Impact Factor
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    ABSTRACT: Oligo/polysialic acids consisting of consecutive α(2,8)-linkages on gangliosides and glycoproteins play a role in cell adhesion and differentiation events in a manner that is dependent on the degree of polymerization (DP). Anti-oligo/polysialic acid antibodies often have DP-dependent antigenic specificity, and such unique antibodies are often used in biological studies for the detection and differentiation of oligo/polysialic acids. However, molecular mechanisms remain unclear. We here use NMR techniques to analyze the binding epitopes of the anti-oligo/polysialic acid monoclonal antibodies (mAb) A2B5 and 12E3. The mAb A2B5, which has a preference for trisialic acid, recognizes sialic acid residues at the non-reducing terminus and those in nascent units. On the other hand, mAb 12E3, which prefers oligo/polysialic acids of more than six sugar units, recognizes inner sialic acid residues. In both structural complexes, the interresidue transferred NOE correlations are significantly different from those arising from analogs of the free states, indicating that the bound and free sugar conformations are distinct. The ability of the two mAbs to distinguish the chain lengths comes from different binding epitopes and possibly from the conformational differences in the oligo/polysialic acids. Information on the recognition modes is needed for the structural design of immunoreactive antigens for the development of high-affinity anti-polysialic acid antibodies and of related vaccines against pathogenic, polysialic acid-coated bacteria.
    Bioorganic & medicinal chemistry 07/2013; · 2.82 Impact Factor
  • Chihiro Sato, Ken Kitajima
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    ABSTRACT: Sialic acids (Sia) are involved in many biological activities and frequently exist as monosialyl residues at the non-reducing terminal end of glycoconjugates. Occasionally, polymerized structures in the form of disialic acid (diSia), oligosialic acid (oligoSia), and polysialic acid (polySia) are also found in glycoconjugates. In particular, polySia, which is an evolutionarily conserved epitope from sea urchin to humans, is one of the most biologically important glycotopes in vertebrates. The biological functions of polySia, especially on neural cell adhesion molecules (NCAMs), have been well studied and an in-depth body of knowledge concerning polySia has been accumulated. However, considerably less attention has been paid to glycoproteins containing di- and oligoSia groups. However, advances in analytical methods for detecting oligo/polymerized structures have allowed the identification and characterization of an increasing number of glycoproteins containing di/oligo/polySia chains in nature. In addition, sophisticated genetic techniques have also helped elucidate the underlying mechanisms of polySia-mediated activities. In this review, recent advances in the study of the chemical properties, distribution, and functions of di-, oligo-, and polySia residues on glycoproteins are described.
    Journal of Biochemistry 06/2013; · 2.72 Impact Factor
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    Chihiro Sato, Ken Kitajima
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    ABSTRACT: Psychiatric disorders are a group of human diseases that impair higher cognitive functions. Whole-genomic analyses have recently identified susceptibility genes for several psychiatric disorders, including schizophrenia. Among the genes reported to be involved in psychiatric disorders, a gene encoding a polysialyltransferase involved in the biosynthesis of polysialic acid (polySia or PSA) on cell surfaces has attracted attention for its potential role in emotion, learning, memory, circadian rhythm, and behaviors. PolySia is a unique polymer that spatio-temporally modifies neural cell adhesion molecule (NCAM) and is predominantly found in embryonic brains, although it persists in areas of the adult brain where neural plasticity, remodeling of neural connections, or neural generation is ongoing, such as the hippocampus, subventricular zone (SVZ), thalamus, prefrontal cortex, and amygdala. PolySia is thought to be involved in the regulation of cell-cell interactions; however, recent evidence suggests that it is also involved in the functional regulation of ion channels and neurologically active molecules, such as Brain-derived neurotrophic factor (BDNF), FGF2, and dopamine (DA) that are deeply involved in psychiatric disorders. In this review, the possible involvement of polysialyltransferase (ST8SIA2/ST8SiaII/STX/Siat8B) and its enzymatic product, polySia, in schizophrenia is discussed.
    Frontiers in Cellular Neuroscience 01/2013; 7:61. · 4.47 Impact Factor
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    ABSTRACT: We previously reported on the accumulation of a substantial amount of free N-acetylneuraminic acid (Neu5Ac)-containing complex-type N-glycans in human pancreatic cancer cells (Yabu M, Korekane H, Takahashi H, Ohigashi H, Ishikawa O, Miyamoto Y. 2012. Accumulation of free Neu5Ac-containing complex-type N-glycans in human pancreatic cancers. Glycoconjugate J Epub ahead of print). In the present paper, we further extend our cancer glycomic study of human prostate cancer. Specifically, we demonstrate that, in addition to the free Neu5Ac-containing N-glycans, significant amounts of free deaminoneuraminic acid (KDN)-containing N-glycans had accumulated in the prostate cancer tissues from four out of five patients. Indeed in one of the four cases, the free KDN-glycans accumulated as major components in prostate cancer tissue. The structures of the KDN-containing free oligosaccharides were analyzed by a variety of methods. Specifically we used fluorescent labeling with aminopyridine combined with two dimensional mapping, KDNase digestion and mass spectrometry to facilitate identification. The analysis also utilized newly synthesized KDN-linked oligosaccharides as standards. The prostate-specific glycans were composed of five species having the following sequence, KDN-Gal-GlcNAc-Man-Man-GlcNAc (α2,6-KDN-linked glycans being the dominant form). The most abundant free KDN-containing N-glycan was KDNα2-6Galβ1-4GlcNAcβ1-2Manα1-3Manβ1-4GlcNAc followed by KDNα2-6Galβ1-4GlcNAcβ1-2Manα1-6Manβ1-4GlcNAc. This is the first study to show unequivocal chemical evidence for the occurrence of KDN-glycoconjugates in human tissues together with their detailed structures. These oligosaccharides might be developed as tumor markers, especially for prostate cancer.
    Glycobiology 09/2012; · 3.54 Impact Factor
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    ABSTRACT: A highly glycosylated protein, which has unique, novel features in localization, structure, and potential function, is found in pig sperm, and named WGA-gp due to its high binding property with wheat germ agglutinin (WGA). WGA-gp is localized mainly in flagella and enriched in membrane microdomains or lipid rafts. It is not detected by ordinary protein staining methods due to a high content of both N- and O-glycans consisting of neutral monosaccharides. Interestingly, WGA-gp may be involved in intracellular Ca(2+) regulation. Treatment of sperm with anti-WGA-gp antibody enhances the amplitude of Ca(2+) oscillation without changing the basal intracellular Ca(2+) concentrations. All these features of WGA-gp, except for different carbohydrate structures occupying most part of the molecules, are similar to those of flagellasialin in sea urchin sperm, which regulates the intracellular Ca(2+) concentration. Presence of carbohydrate-enriched flagellar proteins involved in intracellular Ca(2+) regulation may be a common feature among animal sperm.
    Biochemical and Biophysical Research Communications 08/2012; 426(3):356-62. · 2.41 Impact Factor
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    ABSTRACT: The sialic acid (Sia) N-acetylneuraminic acid (Neu5Ac) and its hydroxylated derivative N-glycolylneuraminic acid (Neu5Gc) differ by one oxygen atom. CMP-Neu5Gc is synthesized from CMP-Neu5Ac, with Neu5Gc representing a highly variable fraction of total Sias in various tissues and among different species. The exception may be the brain, where Neu5Ac is abundant and Neu5Gc is reported to be rare. Here, we confirm this unusual pattern and its evolutionary conservation in additional samples from various species, concluding that brain Neu5Gc expression has been maintained at extremely low levels over hundreds of millions of years of vertebrate evolution. Most explanations for this pattern do not require maintaining neural Neu5Gc at such low levels. We hypothesized that resistance of α2-8-linked Neu5Gc to vertebrate sialidases is the detrimental effect requiring the relative absence of Neu5Gc from brain. This linkage is prominent in polysialic acid (polySia), a molecule with critical roles in vertebrate neural development. We show that Neu5Gc is incorporated into neural polySia and does not cause in vitro toxicity. Synthetic polymers of Neu5Ac and Neu5Gc showed that mammalian and bacterial sialidases are much less able to hydrolyze α2-8-linked Neu5Gc at the nonreducing terminus. Notably, this difference was not seen with acid-catalyzed hydrolysis of polySias. Molecular dynamics modeling indicates that differences in the three-dimensional conformation of terminal saccharides may partly explain reduced enzymatic activity. In keeping with this, polymers of N-propionylneuraminic acid are sensitive to sialidases. Resistance of Neu5Gc-containing polySia to sialidases provides a potential explanation for the rarity of Neu5Gc in the vertebrate brain.
    Journal of Biological Chemistry 06/2012; 287(34):28917-31. · 4.65 Impact Factor
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    ABSTRACT: Modulation of levels of polysialic acid (polySia), a sialic acid polymer, predominantly associated with the neural cell adhesion molecule (NCAM), influences neural functions, including synaptic plasticity, neurite growth, and cell migration. Biosynthesis of polySia depends on two polysialyltransferases ST8SiaII and ST8SiaIV in vertebrate. However, the enzyme involved in degradation of polySia in its physiological turnover remains uncertain. In the present study, we identified and characterized a murine sialidase NEU4 that catalytically degrades polySia. Murine NEU4, dominantly expressed in the brain, was found to efficiently hydrolyze oligoSia and polySia chains as substrates in sialidase in vitro assays, and also NCAM-Fc chimera as well as endogenous NCAM in tissue homogenates of postnatal mouse brain as assessed by immunoblotting with anti-polySia antibodies. Degradation of polySia by NEU4 was also evident in neuroblastoma Neuro2a cells that were co-transfected with Neu4 and ST8SiaIV genes. Furthermore, in mouse embryonic hippocampal primary neurons, the endogenously expressed NEU4 was found to decrease during the neuronal differentiation. Interestingly, GFP- or FLAG-tagged NEU4 was partially co-localized with polySia in neurites and significantly suppressed their outgrowth, whereas silencing of NEU4 showed the acceleration together with an increase in polySia expression. These results suggest that NEU4 is involved in regulation of neuronal function by polySia degradation in mammals.
    Journal of Biological Chemistry 03/2012; 287(18):14816-26. · 4.65 Impact Factor
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    ABSTRACT: Polysialic acid (polySia) is a unique polysaccharide that modifies neural cell adhesion molecule (NCAM) spatiotemporally. Recently, we demonstrated that polySia functions as a reservoir for several neurotrophic factors and neurotransmitters. Here, we showed the direct interaction between polySia and fibroblast growth factor-2 (FGF2) by native-PAGE, gel filtration, and surface plasmon resonance. The minimum chain length of polySia required for the interaction with FGF2 was 17. Compared with heparan sulfate, a well known glycosaminoglycan capable of forming a complex with FGF2, polySia formed a larger complex with distinct properties in facilitating oligomerization of FGF2, as well as in binding to FGF receptors. In polySia-NCAM-expressing NIH-3T3 cells, which were established by transfecting cells with either of the plasmids for the expression of the polysialyltransferases ST8SiaII/STX and ST8SiaIV/PST that can polysialylate NCAM, FGF2-stimulated cell growth, but not cell survival, was inhibited. Taken together, these results suggest that polySia-NCAM might be involved in the regulation of FGF2-FGF receptor signaling through the direct binding of FGF2 in a manner distinct from heparan sulfate.
    Journal of Biological Chemistry 12/2011; 287(6):3710-22. · 4.65 Impact Factor
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    ABSTRACT: Sialic acid, a common terminal substitution of glycoconjugates, has been so far consistently identified in all vertebrates as well as in a growing number of bacterial species. It is assumed to be widely distributed among animal species of the deuterostome phylum, based on its identification in few echinoderm and all vertebrate species. However, whole sections of deuterostome, especially those intermediate species between invertebrates and vertebrates including cephalochordates, urochordates and hemichordates, are still unexplored in term of sialylation capacities. The discovery of functional sialic acid machinery in some of these species may shed new light onto the evolution of glycosylation capacities in deuterostome lineage. In a first approach, we investigated the sialylation pattern of a cephalocordate species, Branchiostoma belcheri, which occupies a strategic phylogenetic position to understand the transition of invertebrates toward vertebrates. Structural analysis of B. belcheri glycoconjugates established that this organism synthesizes large quantities of various sialic acids, some of which present rare or novel structures such as methylated sialic acids. These sialic acids were shown to be mainly associated with mono- and disialylated core 1-type O-glycans. Moreover, screening of the animal organs revealed the existence of exquisite tissue specificity in the distribution of sialic acids. Description of sialylation profiles was then correlated with the expression patterns of key enzymes involved in the biosynthesis of major forms of sialic acids, which provides the first complete overview of the sialylation patterns in cephalochordates.
    Glycobiology 11/2011; 22(4):479-91. · 3.54 Impact Factor
  • Ken Kitajima, Chihiro Sato
    07/2011: pages 33 - 63; , ISBN: 9781118017586
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    ABSTRACT: Naturally occurring polysialic acid (polySia) structures have a large diversity, primarily arising from the diversity in the sialic acid components as well as in the intersialyl linkages. In 2004, we demonstrated the presence of a new type of polySia, 8-O-sulfated N-acetylneuraminic acid (Neu5Ac) capped α2,9-linked polyNeu5Ac, on the O-glycans of a major 40-80 kDa sialoglycoprotein, flagellasialin, in sea urchin sperm. In this study, we demonstrated that another type of polySia, the α2,8-linked polyNeu5Ac, exclusively occurs on O-glycans of a 190 kDa glycoprotein (190 kDa-gp), whereas the α2,9-linked polyNeu5Ac is exclusively present on flagellasialin. The 190 kDa-gp is localized in both flagellum and head of sperm. We also demonstrated that polysialogangliosides containing the α2,8-linked polyNeu5Ac are present in sperm head. Thus, this study shows two novel features of the occurrence of polySia in nature, the co-localization of polySia with different intersialyl linkages, the α2,8- and α2,9-linkages, in a single cell and the occurrence of α2,8-linked polyNeu5Ac in glycolipids. Anti-α2,8-linked polyNeu5Ac antibody had no effect on fertilization, which contrasted with the previous results that anti-α2,9-linked polyNeu5Ac antibody inhibited sperm motility and fertilization. Based on these properties, distinct functions of α2,8- and α2,9-polySia structures are implicated in fertilization.
    Glycobiology 07/2011; 21(12):1596-605. · 3.54 Impact Factor
  • Ryo Isomura, Ken Kitajima, Chihiro Sato
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    ABSTRACT: Polysialic acid (polySia), a unique acidic glycan modifying neural cell adhesion molecule (NCAM), is known to regulate embryonic neural development and adult brain functions. Polysialyltransferase STX is responsible for the synthesis of polySia, and two single nucleotide polymorphisms (SNPs) of the coding region of STX are reported from schizophrenic patients: SNP7 and SNP9, respectively, giving STX(G421A) with E141K and STX(C621G) with silent mutations. In this study, we focused on these mutations and a binding activity of polySia to neural materials, such as brain-derived neurotrophic factor (BDNF). Here we describe three new findings. First, STX(G421A) shows a dramatic decrease in polySia synthetic activity on NCAM, whereas STX(C621G) does not. The STX(G421A)-derived polySia-NCAM contains a lower amount of polySia with a shorter chain length. Second, polySia shows a dopamine (DA) binding activity, which is a new function of polySia as revealed by frontal affinity chromatography for measuring the polySia-neurotransmitter interactions. Interestingly, the STX(G421A)-derived polySia-NCAM completely loses the DA binding activity, whereas it greatly diminishes but does not lose the BDNF binding activity. Third, an impairment of the polySia structure with an endosialidase modulates the DA-mediated Akt signaling. Taken together, impairment of the amount and quality of polySia may be involved in psychiatric disorders through impaired binding to BDNF and DA, which are deeply involved in schizophrenia and other psychiatric disorders, such as depression and bipolar disorder.
    Journal of Biological Chemistry 04/2011; 286(24):21535-45. · 4.65 Impact Factor
  • Chihiro Sato, Ken Kitajima
    Seikagaku. The Journal of Japanese Biochemical Society 03/2011; 83(3):189-96. · 0.04 Impact Factor
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    ABSTRACT: Flagellar movement of the sea urchin sperm is regulated by intracellular Ca(2+). Flagellasialin, a polysialic acid-containing glycoprotein, as well as other membrane proteins seems responsible for the Ca(2+) control. To elucidate the mechanism of Ca(2+) dynamics underlying flagellar movement, we analysed the sperm's mechanosensory behavioural responses by using microtechniques. In sea water containing 10 mM Ca(2+), the sperm swim in circular paths. When a mechanical stimulus was applied to the sperm head with a glass microstylus, the sperm showed a series of flagellar responses, consisting of a stoppage of beating (quiescence) and a recovery of swimming in a straight path, followed by swimming in a circular path again; as the result the sperm avoided the obstacle. Ca(2+)-imaging with Fluo-4 showed that the intracellular Ca(2+) was high in the quiescence and gradually decreased after that. The effects of blockers and antibodies against candidate components revealed that the Ca(2+) influx was induced by Ca(2+) channels and the Ca(2+) efflux was induced by a flagellasialin-related Ca(2+)-efflux system, plasma membrane Ca(2+)-ATPases and the K(+)-dependent Na(+)/Ca(2+) exchanger. The results show that the Ca(2+)-dependent mechanosensory behaviour of the sea urchin sperm is regulated by organized functioning of the membrane environment including the plasma membrane proteins and flagellasialin.
    Cell Structure and Function 02/2011; 36(1):69-82. · 1.65 Impact Factor
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    ABSTRACT: Sialic acids are a family of nine-carbon carboxylated sugars having a nonulosonate skeletal structure (Fig. 35.1). This structure is uniquely different from that of other sugar units of animal glycans. The most popular sialic acid is N-acetylneuraminic acid (Neu5Ac), which is universally found on cell surface glycocalyx and in secreted glycoproteins of vertebrates and some invertebrates. Sialic acids have low acid–base dissociation constants and give a negative charge on the cell surface under a wide range of physiological pH. In nature, more than 50 kinds of sialic acids are known. Nearly all of them are derived from Neu5Ac by a substitution on the hydroxyl groups (e.g., O-acetyl-Neu5Ac) and/or a hydroxylation of the N-acetyl group (e.g., N-glycolylneuraminic acid, Neu5Gc). Each modified sialic acid has properties different from those of Neu5Ac and is believed to contribute to specific physiological functions. In animal cells, sialic acids are most frequently the terminal sugars of cell surface glycolipids and glycoproteins, and any change that occurs on sialic acids can considerably influence the biological properties of a cell.
    Advances in experimental medicine and biology 01/2011; 705:669-78. · 1.83 Impact Factor
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    ABSTRACT: Few common carbohydrate epitopes consisting of terminal beta-(1,2)-xylose and/or alpha-(1,3)-fucose residues are shared by a variety of glycoproteins from plants, insects and parasitic worms, termed cross-reactive carbohydrate determinant (CCD), and frequently recognized by IgE antibodies of patients with food and/or respiratory allergy, though clinical relevancy of such CCD-specific IgE is still controversial. Attention has also been focused on CCDs from the undesired post-translational modification of recombinant therapeutic proteins produced by transgenic plants and insects. In the present study, to clarify immunogenic potentials of CCD-bearing glycoproteins, the antibody response to a model plant glycoprotein, horseradish peroxidase (HRP) was investigated in a mouse model. C3H/He mice were immunized with HRP plus Al(OH)(3) or Freund's adjuvant, and IgG and IgE responses to CCDs in addition to HRP were analyzed by ELISA using some distinct glycoproteins with known N-glycan structures. IgE response to HRP was induced remarkably, whereas that to CCD was weaker and delayed. Moreover, apparent ratio of the CCD-specific antibodies to HRP-specific ones tended to be higher in IgG2a and IgG2b isotypes than IgG1, IgG3 and IgE. In contrast to rabbit antibodies, the CCD-specific antibodies from the mice gave poor reactivity with bromelain and honeybee phospholipase A2, suggesting the critical role of both beta-(1,2)-xylose and alpha-(1,3)-mannose in the CCD-recognition by the mouse antibodies. Moreover, the mouse antibodies showed weaker cross-reactivity to pollen- and insect-derived glycoproteins than the rabbit ones. Thus, in this mouse model, not only IgE but also IgG2 antibody responses to CCDs were induced by immunizing with a CCD-bearing glycoprotein, suggesting that CCDs affected not only Th2-type but also Th1-type antibody response at least in C3H/He mice.
    Immunology letters 09/2010; 133(1):28-34. · 2.91 Impact Factor
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    ABSTRACT: The monoclonal antibody mAb.A2B5 is a marker for the detection of oligodendrocyte progenitor cells that differentiate into type-2 astrocytes and oligodendrocytes. It is also a useful antibody for separating these cells from other lineage populations. The epitope of this antibody is considered to be the gangliosides GT3 and GQ1c. In this study, we sought to define more precisely the structure of the epitope. Accordingly, we chemically synthesized defined oligosialic acid structures linked to phosphatidylethanolamine and bovine serum albumin and used these to determine the antigenic specificity. mAb.A2B5 recognized the Neu5Acalpha2-->8Neu5Acalpha2-->8Neu5Acalpha--> structure on both glycolipids and glycoproteins. We then examined whether the mAb.A2B5 epitope exists on glycoproteins in developing mouse brains. Western blot analyses revealed the expression of four glycoproteins reactive with the mAb.A2B5, and their expression was dependent on the stage of neural development. All the immunoreactivity in these glycoproteins with mAb.A2B5 disappeared after sialidase treatment and were resistant to chloroform/methanol extraction. These epitopes were also detected in brain homogenates from both GD3 synthetase-null and GD3/GD2 synthetase double null mice. These findings show that the alpha2,8-trisialic acid (triSia) unit recognized by mAb.A2B5 resides not only on gangliosides but also on glycoproteins in developing mouse brain. We postulate that the triSia structure on glycoproteins may be involved in oligodendrocyte differentiation, similar to the case with the alpha2,8-triSia structure on gangliosides. Real time polymerase chain reaction analysis of the developmental expression of all known ST8Sia genes, which are responsible for the biosynthesis of alpha2,8-linked Sia residues, showed that ST8Sia III gene expression correlated with expression of the triSia epitope. We suggest that ST8Sia III is the principal sialyltransferase responsible for synthesis of the alpha2,8-triSia units on glycoproteins.
    Glycobiology 04/2010; 20(7):916-28. · 3.54 Impact Factor
  • Sadako Inoue, Chihiro Sato, Ken Kitajima
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    ABSTRACT: N-Glycolylneuraminic acid (Neu5Gc) is the second most populous sialic acid (Sia). The only known biosynthetic pathway of Neu5Gc is the hydroxylation of cytidine-5'-monophosphate-N-acetylneuraminic acid (CMP-Neu5Ac), catalyzed by CMP-Neu5Ac hydroxylase (CMAH). Neu5Gc is abundantly found in mammals except for human, in which CMAH is inactivated due to mutation in the CMAH gene. Evidence has accumulated to show occurrence of Neu5Gc-containing glycoconjugates in sera of cancer patients, human cancerous tissues and cultured human cell lines. Recently, occurrence of natural antibodies against Neu5Gc was shown in healthy humans and is a serious problem for clinical xenotransplantation and stem cell therapies. Studying human occurrence of Neu5Gc is of importance and interest in a broad area of medical sciences. In this study, using a fluorometric high performance liquid chromatography method, we performed quantitative analyses of Sias both inside and in the external environment of the cell and found that (i) incorporation of Neu5Gc was most prominent in soluble glycoproteins found both in the extracellular space and inside the cell as the major Sia compounds. (ii) Of the total Neu5Gc in the Sia compounds that the cells synthesized, 90% was found in the secreted sialoglycoproteins, whereas for Neu5Ac, 70% was found in the secreted sialoglycoproteins. (iii) The Neu5Gc ratio was higher in the secreted sialoglycoproteins (as high as 40% of total Sias) than in intracellular sialoglycoproteins. (iv) The majority of the secreted sialoglycoproteins was anchored on the culture dishes and solubilized by brief trypsin treatment. Based on these findings, a new idea on the mechanism of accumulation of Neu5Gc in cancer cells was proposed.
    Glycobiology 03/2010; 20(6):752-62. · 3.54 Impact Factor

Publication Stats

879 Citations
225.29 Total Impact Points

Institutions

  • 1998–2014
    • Nagoya University
      • • Graduate School of Bio-Agricultural Sciences
      • • Department of Applied Molecular Biosciences
      • • Department of Bioengineering Sciences
      Nagoya, Aichi, Japan
  • 2011
    • Université des Sciences et Technologies de Lille 1
      • Unité de Glycobiologie Structurale et Fonctionnelle (UGSF)
      Lille, Nord-Pas-de-Calais, France
    • Georgia State University
      Atlanta, Georgia, United States
  • 2006
    • Mie University
      Tu, Mie, Japan
  • 1993–1998
    • The University of Tokyo
      • • Faculty of Science and Graduate School of Science
      • • Department of Biophysics and Biochemistry
      Tokyo, Tokyo-to, Japan