Faculty Profiles - SATO, Chihiro

Identification of oligo-N-glycolylneuraminic acid residues in mammal- derived glycoproteins by a newly developed immunochemical reagent and biochemical methods

Sato C.

Journal of Biological Chemistry Vol. 273 ( 5 ) page： 2575 - 2582 1998.1

Occurrence of terminal α2 → 8-linked disialylated poly-N-acetyllactosamine chains with Le(X) and I antigenic glycotopes in tetraantennary arms of an N-linked glycoprotein isolated from rainbow trout ovarian fluid

Funakoshi Y., Taguchi T., Sato C., Kitajima K., Inoue S., Morris H.R., Dell A., Inoue Y.

Glycobiology Vol. 7 ( 2 ) page： 195 - 205 1997.3

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Glycobiology

The Pronase digestion of a 54K glycoprotein present in ovarian fluid of rainbow trout yielded a major glycopeptide. Carbohydrate compositional analysis revealed that this glycopeptide was likely to possess a single large N-glycan chain having low molecular weight oligomers of N-acetylneuraminic acid (oligoNeu5Ac). Structural studies of this glycopeptide revealed novel α2 → 8-linked disialylated poly-N-acetyllactosamine chains with Le(X) and I antigenic determinants on the N-linked tetraantennary core glycan. In our recent studies (Kitazume, S., Kitajima, K., Inoue, S., Inoue, Y. and Troy, F.A. (1994) J. Biol. Chem. 269, 10330-10340) we presented evidence that synthesis of α2 → 8-linked polysialic acid (polySia) chains is a two-step process in which chain initiation is catalyzed by an α2 → 8-sialyltransferase (α2 → 8-ST; initiase) that catalyzes synthesis of the first Sia α2 → 8-linkage, forming the disialic acid (diSia) unit, Siaα2 → 8-Siaα2 → 6-Gal-.Chain polymerization is then postulated to be catalyzed by a second enzyme, an α2 → 8-polyST ('polymerase') that converts the diSia units to polySia chains. The present structural studies leading to the discovery of α2 → 8-linked disialylated units that terminate poly-N-acetyllactosamine chains in an N-linked glycoprotein is further evidence in support of our hypothesis that more than one sialyltransferase activity is required for polySia chain synthesis and polymerization.

DOI： 10.1093/glycob/7.2.195

Assembly of neuron- and radial glial-cell-derived extracellular matrix molecules promotes radial migration of developing cortical neurons Reviewed

Mubuchi A, Takechi M, Nishio S, Matsuda T, Itoh Y, Sato C, Kitajima K, Kitagawa H, Miyata S.

eLife Vol. 12 page： RP92342 2024.3

Rat hepatocytes secrete free oligosaccharides. Reviewed International coauthorship

Huang C, Seino J, Honda A, Fujihira H, Wu D, Okahara K, Kitazume S, Nakaya S, Kitajima K, Sato C, Suzuki T.

J Biol Chem. Vol. 300 page： 105712 2024.3

Forced expression of α2,3-sialyltransferase IV rescues impaired heart development in α2,6-sialyltransferase I-deficient medaka Reviewed International coauthorship

Omoto T, Wu D, Maruyama E, Tajima K, Hane M, Sato C, Kitajima K.

Biochem Biophys Res Commun . Vol. 649 page： 62 2023.3

Fu B, Wu D, Yasumasu S, Hane M, Sato C, Kitajima K. Reviewed International coauthorship

Fu B, Wu D, Yasumasu S, Hane M, Sato C, Kitajima K.

Biomolecules. Vol. 13 page： 146 2023.1

Implication of N-glycolylneuraminic acid in regulation of cell adhesiveness of C2C12 myoblast cells during differentiation into myotube cells. International journal

Shiori Go, Chihiro Sato, Masaya Hane, Shinji Go, Ken Kitajima

Glycoconjugate journal Vol. 39 ( 5 ) page： 619 - 631 2022.10

Language：English Publishing type：Research paper (scientific journal)

A transition of sialic acid (Sia) species on GM3 ganglioside from N-acetylneuraminic acid (Neu5Ac) to N-glycolylneuraminic acid (Neu5Gc) takes place in mouse C2C12 myoblast cells during their differentiation into myotube cells. However, the meaning of this Sia transition remains unclear. This study thus aims to gain a functional insight into this phenomenon. The following lines of evidence show that the increased de novo synthesis of Neu5Gc residues in differentiating myoblast cells promotes adhesiveness of the cells, which is beneficial for promotion of differentiation. First, the Sia transition occurred even in the C2C12 cells cultured in serum-free medium, indicating that it happens through de novo synthesis of Neu5Gc. Second, GM3(Neu5Gc) was localized in myoblast cells, but not in myotube cells, and related to expression of the CMP-Neu5Ac hydroxylase (CMAH) gene. Notably, expression of CMAH precedes myotube formation not only in differentiating C2C12 cells, but also in mouse developing embryos. Since the myoblast cells were attached on the dish surface more strongly than the myotube cells, expression of GM3(Neu5Gc) may be related to the surface attachment of the myoblast cells. Third, exogenous Neu5Gc, but not Neu5Ac, promoted differentiation of C2C12 cells, thus increasing the number of cells committed to fuse with each other. Fourth, the CMAH-transfected C2C12 cells were attached on the gelatin-coated surface much more rapidly than the mock-cells, suggesting that the expression of CMAH promotes cell adhesiveness through the expression of Neu5Gc.

DOI： 10.1007/s10719-022-10049-9

The α2,8-sialyltransferase 6 (St8sia6) localizes in the ER and enhances the anchorage-independent cell growth in cancer. International journal Open Access

Rina Hatanaka, Erino Araki, Masaya Hane, Shiori Go, Di Wu, Ken Kitajima, Chihiro Sato

Biochemical and biophysical research communications Vol. 608 page： 52 - 58 2022.6

Language：English Publishing type：Research paper (scientific journal)

Sialylation, the final stage of post-translational modification of proteins, is achieved in the Golgi apparatus and is related to the malignant phenotype of cancer. Disialylation of ganglioside (GD3) by St8sia1 and polysialylation by St8sia2 and 4 have been shown to be related to malignant phenotypes; however, di/oligosialylation by St8sia6 is still unknown. In this study, we analyzed the malignant phenotype of St8sia6 and found that upregulation of St8sia6 in melanoma B16 cells increased anchorage-independent cell growth, which was not due to sialic acid cleavage by a sialidase. Moreover, unlike other sialyltransferases, St8sia6 localized to the endoplasmic reticulum (ER). We found that the localization to the Golgi apparatus could be regulated by swapping experiments using St8sia2; however, the malignant phenotype did not change. These data demonstrate that the enhancement of anchorage-independent cell growth by St8sia6 is not due to its localization of ER, but is due to the expression of the protein itself.

DOI： 10.1016/j.bbrc.2022.03.146

Comprehensive Analysis of Oligo/Polysialylglycoconjugates in Cancer Cell Lines. International journal

Masaya Hane, Ken Kitajima, Chihiro Sato

International journal of molecular sciences Vol. 23 ( 10 ) 2022.5

Language：English Publishing type：Research paper (scientific journal)

In cancer cells, cell-surface sialylation is altered, including a change in oligo/polysialic acid (oligo/polySia) structures. Since they are unique and rarely expressed in normal cells, oligo/polySia structures may serve as promising novel biomarkers and targets for therapies. For the diagnosis and treatment of the disease, a precise understanding of the oligo/polySia structures in cancer cells is necessary. In this study, flow cytometric analysis and gene expression datasets were obtained from sixteen different cancer cell lines. These datasets demonstrated the ability to predict glycan structures and their sialylation status. Our results also revealed that sialylation patterns are unique to each cancer cell line. Thus, we can suggest promising combinations of antibody and cancer cell for glycan prediction. However, the precise prediction of minor glycans need to be further explored.

DOI： 10.3390/ijms23105569

Role of Polysialic Acid in Schizophrenia

Polysialylation in a DISC1 Mutant Mouse. International journal Open Access

Yuka Takahashi, Chikara Abe, Masaya Hane, Di Wu, Ken Kitajima, Chihiro Sato

International journal of molecular sciences Vol. 23 ( 9 ) page： 5207 2022.5

Language：English Publishing type：Research paper (scientific journal)

Schizophrenia is a serious psychiatric disorder that affects the social life of patients. Psychiatric disorders are caused by a complex combination of genetic (G) and environmental (E) factors. Polysialylation represents a unique posttranslational modification of a protein, and such changes in neural cell adhesion molecules (NCAMs) have been reported in postmortem brains from patients with psychiatric disorders. To understand the G × E effect on polysialylated NCAM expression, in this study, we performed precise measurements of polySia and NCAM using a disrupted-in-schizophrenia 1 (DISC1)-mutant mouse (G), a mouse model of schizophrenia, under acute stress conditions (E). This is the first study to reveal a lower number and smaller length of polySia in the suprachiasmatic nucleus of DISC1 mutants relative to those in wild-type (WT) mice. In addition, an analysis of polySia and NCAM responses to acute stress in five brain regions (olfactory bulb, prefrontal cortex, suprachiasmatic nucleus, amygdala, and hippocampus) revealed that the pattern of changes in these responses in WT mice and DISC1 mutants differed by region. These differences could indicate the vulnerability of DISC1 mutants to stress.

DOI： 10.3390/ijms23095207

Chihiro Sato, Masaya Hane, Ken Kitajima

Comprehensive Glycoscience: Second Edition page： 276 - 286 2021.6

Polysialylation and disease. Reviewed International journal Open Access

Chihiro Sato, Ken Kitajima

Molecular aspects of medicine Vol. 79 page： 100892 - 100892 2021.6

Language：English Publishing type：Research paper (scientific journal)

Polysialic acid (polySia, PSA) is a unique constituent of the glycocalyx on the surface of bacterial and vertebrate cells. In vertebrates, its biosynthesis is highly regulated, not only in quantity and quality, but also in time and location, which allows polySia to be involved in various important biological phenomena. Therefore, impairments in the expression and structure of polySia sometimes relate to diseases, such as schizophrenia, bipolar disorder, and cancer. Some bacteria express polySia as a tool for protecting themselves from the host immune system during invasion. PolySia is proven to be a biosafe material; polySia, as well as polySia-recognizing molecules, are key therapeutic agents. This review first comprehensive outlines the occurrence, features, biosynthesis, and functions of polySia and subsequently focuses on the related diseases.

DOI： 10.1016/j.mam.2020.100892

Evolutionary conservation of human ketodeoxynonulosonic acid production is independent of sialoglycan biosynthesis. Reviewed International coauthorship International journal

Kunio Kawanishi, Sudeshna Saha, Sandra Diaz, Michael Vaill, Aniruddha Sasmal, Shoib S Siddiqui, Biswa Choudhury, Kumar Sharma, Xi Chen, Ian C Schoenhofen, Chihiro Sato, Ken Kitajima, Hudson H Freeze, Anja Münster-Kühnel, Ajit Varki

The Journal of clinical investigation Vol. 131 ( 5 ) 2021.3

Language：English Publishing type：Research paper (scientific journal)

Human metabolic incorporation of nonhuman sialic acid (Sia) N-glycolylneuraminic acid into endogenous glycans generates inflammation via preexisting antibodies, which likely contributes to red meat-induced atherosclerosis acceleration. Exploring whether this mechanism affects atherosclerosis in end-stage renal disease (ESRD), we instead found serum accumulation of 2-keto-3-deoxy-d-glycero-d-galacto-2-nonulosonic acid (Kdn), a Sia prominently expressed in cold-blooded vertebrates. In patients with ESRD, levels of the Kdn precursor mannose also increased, but within a normal range. Mannose ingestion by healthy volunteers raised the levels of urinary mannose and Kdn. Kdn production pathways remained conserved in mammals but were diminished by an M42T substitution in a key biosynthetic enzyme, N-acetylneuraminate synthase. Remarkably, reversion to the ancestral methionine then occurred independently in 2 lineages, including humans. However, mammalian glycan databases contain no Kdn-glycans. We hypothesize that the potential toxicity of excess mannose in mammals is partly buffered by conversion to free Kdn. Thus, mammals probably conserve Kdn biosynthesis and modulate it in a lineage-specific manner, not for glycosylation, but to control physiological mannose intermediates and metabolites. However, human cells can be forced to express Kdn-glycans via genetic mutations enhancing Kdn utilization, or by transfection with fish enzymes producing cytidine monophosphate-Kdn (CMP-Kdn). Antibodies against Kdn-glycans occur in pooled human immunoglobulins. Pathological conditions that elevate Kdn levels could therefore result in antibody-mediated inflammatory pathologies.

DOI： 10.1172/JCI137681

Exploring the Impact of Ketodeoxynonulosonic Acid in Host-Pathogen Interactions Using Uptake and Surface Display by Nontypeable Haemophilus influenzae. International coauthorship International journal

Sudeshna Saha, Alison Coady, Aniruddha Sasmal, Kunio Kawanishi, Biswa Choudhury, Hai Yu, Ricardo U Sorensen, Jaime Inostroza, Ian C Schoenhofen, Xi Chen, Anja Münster-Kühnel, Chihiro Sato, Ken Kitajima, Sanjay Ram, Victor Nizet, Ajit Varki

mBio Vol. 12 ( 1 ) page： 1 - 24 2021.1

Language：English Publishing type：Research paper (scientific journal)

Surface expression of the common vertebrate sialic acid (Sia) N-acetylneuraminic acid (Neu5Ac) by commensal and pathogenic microbes appears structurally to represent "molecular mimicry" of host sialoglycans, facilitating multiple mechanisms of host immune evasion. In contrast, ketodeoxynonulosonic acid (Kdn) is a more ancestral Sia also present in prokaryotic glycoconjugates that are structurally quite distinct from vertebrate sialoglycans. We detected human antibodies against Kdn-terminated glycans, and sialoglycan microarray studies found these anti-Kdn antibodies to be directed against Kdn-sialoglycans structurally similar to those on human cell surface Neu5Ac-sialoglycans. Anti-Kdn-glycan antibodies appear during infancy in a pattern similar to those generated following incorporation of the nonhuman Sia N-glycolylneuraminic acid (Neu5Gc) onto the surface of nontypeable Haemophilus influenzae (NTHi), a human commensal and opportunistic pathogen. NTHi grown in the presence of free Kdn took up and incorporated the Sia into its lipooligosaccharide (LOS). Surface display of the Kdn within NTHi LOS blunted several virulence attributes of the pathogen, including Neu5Ac-mediated resistance to complement and whole blood killing, complement C3 deposition, IgM binding, and engagement of Siglec-9. Upper airway administration of Kdn reduced NTHi infection in human-like Cmah null (Neu5Gc-deficient) mice that express a Neu5Ac-rich sialome. We propose a mechanism for the induction of anti-Kdn antibodies in humans, suggesting that Kdn could be a natural and/or therapeutic "Trojan horse" that impairs colonization and virulence phenotypes of free Neu5Ac-assimilating human pathogens.IMPORTANCE All cells in vertebrates are coated with a dense array of glycans often capped with sugars called sialic acids. Sialic acids have many functions, including serving as a signal for recognition of "self" cells by the immune system, thereby guiding an appropriate immune response against foreign "nonself" and/or damaged cells. Several pathogenic bacteria have evolved mechanisms to cloak themselves with sialic acids and evade immune responses. Here we explore a type of sialic acid called "Kdn" (ketodeoxynonulosonic acid) that has not received much attention in the past and compare and contrast how it interacts with the immune system. Our results show potential for the use of Kdn as a natural intervention against pathogenic bacteria that take up and coat themselves with external sialic acid from the environment.

DOI： 10.1128/mBio.03226-20

The conserved arginine residue in all siglecs is essential for Siglec-7 binding to sialic acid. Reviewed International journal

Atsushi Yoshimura, Rina Hatanaka, Hiroshi Tanaka, Ken Kitajima, Chihiro Sato

Biochemical and biophysical research communications Vol. 534 page： 1069 - 1075 2021.1

Language：English Publishing type：Research paper (scientific journal)

Siglecs are sialic acid (Sia)-binding immunoglobulin-like lectins; the majority of Siglecs functions as transmembrane receptors on the immune cells via Sia residues. Recently, a new Sia binding site in Siglec-7, termed site 2, where arginine (R) 67 was critical, was identified by computational modeling and biochemical analyses, relative to the primary Sia binding site, termed site 1, containing critical R124. Here, the presence of a new essential R94 residue, which is completely conserved among all identified Siglecs, was demonstrated. A mutation of R94 residue in Siglec-7 led to the disappearance of the Sia binding property, similar to a site 1 mutation (R124A). R94 is close to R67 in site 2, and site 2 mutations at either of them abolished the ligand-binding properties to both gangliosides and glycoproteins. These data suggest that, in addition to site 1, the conserved R residue among Siglecs in site 2 is another functional site.

DOI： 10.1016/j.bbrc.2020.10.023

Comparative Studies of Polysialic Acids Derived from Five Different Vertebrate Brains. Reviewed International journal

Yi Yang, Ryo Murai, Yuka Takahashi, Airi Mori, Masaya Hane, Ken Kitajima, Chihiro Sato

International journal of molecular sciences Vol. 21 ( 22 ) page： 1 - 22 2020.11

Language：English Publishing type：Research paper (scientific journal)

Polysialic acid (polySia/PSA) is a linear homopolymer of sialic acid (Sia) that primarily modifies the neural cell adhesion molecule (NCAM) in mammalian brains. PolySia-NCAM not only displays an anti-adhesive function due to the hydration effect, but also possesses a molecule-retaining function via a direct binding to neurologically active molecules. The quality and quantity of polySia determine the function of polySia-NCAM and are considered to be profoundly related to the maintenance of normal brain functions. In this study, to compare the structures of polySia-NCAM in brains of five different vertebrates (mammals, birds, reptiles, amphibians, and fish), we adopted newly developed combinational methods for the analyses. The results revealed that the structural features of polySia considerably varied among different species. Interestingly, mice, as a mammal, possess eminently distinct types of polySia, in both quality and quantity, compared with those possessed by other animals. Thus, the mouse polySia is of larger quantities, of longer and more diverse chain lengths, and of a larger molecular size with higher negative charge, compared with polySia of other species. These properties might enable more advanced brain function. Additionally, it is suggested that the polySia/Sia ratio, which likely reflects the complexity of brain function, can be used as a new promising index to evaluate the intelligence of different vertebrate brains.

DOI： 10.3390/ijms21228593

Combinational Analyses with Multiple Methods Reveal the Existence of Several Forms of Polysialylated Neural Cell Adhesion Molecule in Mouse Developing Brains. Reviewed International journal

Airi Mori, Yi Yang, Yuka Takahashi, Masaya Hane, Ken Kitajima, Chihiro Sato

International journal of molecular sciences Vol. 21 ( 16 ) page： 1 - 20 2020.8

Language：English Publishing type：Research paper (scientific journal)

Polysialic acid (polySia/PSA) is an anionic glycan polymer of sialic acid, and it mostly modifies the neural cell adhesion molecule (NCAM) in mammalian brains. Quality and quantity of the polySia of the polySia-NCAM is spatio-temporally regulated in normal brain development and functions, and their impairments are reported to be related to diseases, such as psychiatric disorders and cancers. Therefore, precise understanding of the state of polySia-NCAM structure would lead to the diagnosis of diseases for which their suitable evaluation methods are necessary. In this study, to develop these evaluation methods, structures of polySia-NCAM from mouse brains at six different developmental stages were analyzed by several conventional and newly developed methods. Integrated results of these experiments clearly demonstrated the existence of different types of polySia-NCAMs in developing brains. In addition, combinational analyses were shown to be useful for precise understanding of the quantity and quality of polySia, which can provide criteria for the diagnosis of diseases.

DOI： 10.3390/ijms21165892

Discovery of a new sialic acid binding region that regulates Siglec-7. Reviewed International coauthorship International journal

Nao Yamakawa, Yu Yasuda, Atsushi Yoshimura, Ami Goshima, Paul R Crocker, Gérard Vergoten, Yuji Nishiura, Takashi Takahashi, Shinya Hanashima, Kana Matsumoto, Yoshiki Yamaguchi, Hiroshi Tanaka, Ken Kitajima, Chihiro Sato

Scientific reports Vol. 10 ( 1 ) page： 8647 - 8647 2020.5

Language：English Publishing type：Research paper (scientific journal)

Siglec-7 is a human CD33-like siglec, and is localised predominantly on human natural killer (NK) cells and monocytes. Siglec-7 is considered to function as an immunoreceptor in a sialic acid-dependent manner. However, the underlying mechanisms linking sialic acid-binding and function remain unknown. Here, to gain new insights into the ligand-binding properties of Siglec-7, we carried out in silico analysis and site-directed mutagenesis, and found a new sialic acid-binding region (site 2 containing R67) in addition to the well-known primary ligand-binding region (site 1 containing R124). This was supported by equilibrium dialysis, STD-NMR experiments, and inhibition analysis of GD3-binding toward Siglec-7 using synthetic sialoglycoconjugates and a comprehensive set of ganglioside-based glycoconjugates. Our results suggest that the two ligand-binding sites are potentially controlled by each other due to the flexible conformation of the C-C' loop of Siglec-7.

DOI： 10.1038/s41598-020-64887-4

Effect of expression alteration in flanking genes on phenotypes of St8sia2-deficient mice. Reviewed International journal

Keisuke Ikegami, Kazumasa Saigoh, Atsuko Fujioka, Mamoru Nagano, Ken Kitajima, Chihiro Sato, Satoru Masubuchi, Susumu Kusunoki, Yasufumi Shigeyoshi

Scientific reports Vol. 9 ( 1 ) page： 13634 - 13634 2019.9

Language：English Publishing type：Research paper (scientific journal)

ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 2 (ST8SIA2) synthesizes polysialic acid (PSA), which is essential for brain development. Although previous studies reported that St8sia2-deficient mice that have a mixed 129 and C57BL/6 (B6) genetic background showed mild and variable phenotypes, the reasons for this remain unknown. We hypothesized that this phenotypic difference is caused by diversity in the expression or function of flanking genes of St8sia2. A genomic polymorphism and gene expression analysis in the flanking region revealed reduced expression of insulin-like growth factor 1 receptor (Igf1r) on the B6 background than on that of the 129 strain. This observation, along with the finding that administration of an IGF1R agonist during pregnancy increased litter size, suggests that the decreased expression of Igf1r associated with ST8SIA2 deficiency caused lethality. This study demonstrates the importance of gene expression level in the flanking regions of a targeted null allele having an effect on phenotype.

DOI： 10.1038/s41598-019-50006-5