Faculty Profiles - SATO, Chihiro

特集腫瘍と糖鎖--糖鎖の基礎研究から腫瘍の分子標的同定に向けてがんとシアル酸重合体

Sulfation of sialic acid is ubiquitous and essential for vertebrate development. Open Access

Ertunc N, Phitak T, Wu D, Fujita H, Hane M, Sato C, Kitajima K

Scientific reports Vol. 12 ( 1 ) page： 12496 2022.7

Language：English Publishing type：Research paper (scientific journal) Publisher：Scientific Reports

Glycosylation of proteins and lipids occurs in vertebrates, usually terminating with sialylation, which regulates the physicochemical and biological properties of these glycoconjugates. Although less commonly known, sialic acid residues also undergo various modifications, such as acetylation, methylation, and sulfation. However, except for acetylation, the enzymes or functions of the other modification processes are unknown. To the best of our knowledge, this study is the first to demonstrate the ubiquitous occurrence of sulfated sialic acids and two genes encoding the sialate: O-sulfotransferases 1 and 2 in vertebrates. These two enzymes showed about 50% amino acid sequence identity, and appeared to be complementary to each other in acceptor substrate preferences. Gene targeting experiments showed that the deficiency of these genes was lethal for medaka fish during young fry development and accompanied by different phenotypes. Thus, the sulfation of sialic acids is essential for the vertebrate development.

DOI： 10.1038/s41598-022-15143-4

佐藤ちひろ

医学のあゆみ Vol. 281 ( 9 ) page： 870 - 875 2022.5

Shrimp thrombospondin (TSP): presence of O-β1,4 N-acetylglucosamine polymers and its function in TSP chain association in egg extracellular matrix. Open Access

Magerd S, Senarai T, Thongsum O, Chawiwithaya C, Sato C, Kitajima K, Weerachatyanukul W, Asuvapongpatana S, Surinlert P

Scientific reports Vol. 12 ( 1 ) page： 7925 2022.5

Language：English Publishing type：Research paper (scientific journal) Publisher：Scientific Reports

We characterized the existence of O-β(1,4)-GlcNAc polymers (β1,4GNP) that were anchored on the O-linked glycosylation sites of shrimp thrombospondin (pmTSP-II). There were five putative β1,4GNP linkages on the epithelial growth factor-like domain of pmTSP-II. Antibody against O-β-GlcNAc (CTD110.6) was used to prove the existence of linear and complex β1,4GNP. The antibody well reacted with linear chito-triose, -tetraose and -pentaose conjugated with phosphatidylethanolamine lipid. The immunoreactivity could also be detected with a complex β1,4GNP within pmTSP-II (at MW > 250 kDa). Upon denaturing the protein with SDS-PAGE buffer, the size of pmTSP-II was shifted to be 250 kDa, approximately 2.5 folds larger than the deduced molecular mass of pmTSP-II (110 kDa), suggesting additional association of pmTSP-II apart from its known disulfide bridging. This was confirmed by chitinase digestion on pmTSP-II protein leading to the subsequent smaller protein bands at 110–170 kDa in time- and concentration-dependent manners. These bands well reacted with CTD110.6 antibody and disappeared after extensive chitinase hydrolysis. Together, we believe that β1,4GNP on pmTSP-II serve the function in an inter-chain association to provide structural architecture of egg extracellular matrix, a novel function of pmTSP-II in reproductive biology.

DOI： 10.1038/s41598-022-11873-7

Analysis of biochemical features of ST8 α-N-acetyl-neuraminide α2,8-sialyltransferase (St8sia) 5 isoforms.

Araki E, Hane M, Hatanaka R, Kimura R, Tsuda K, Konishi M, Komura N, Ando H, Kitajima K, Sato C

Glycoconjugate journal Vol. 39 ( 2 ) page： 291 - 302 2022.4

Language：English Publishing type：Research paper (scientific journal) Publisher：Glycoconjugate Journal

Gangliosides are important components of the membrane and are involved in many biological activities. St8sia5 is an α2,8-sialyltransferase involved in ganglioside synthesis, and has three isoforms. In this study, we analyzed the features of three isoforms, St8sia5-S, -M, and -L that had not been analyzed, and found that only St8sia5-L was localized in the Golgi, while the majority of St8sia5-M and -S were localized in the ER. The localization of Golgi of St8sia5 depended on the stem region. In addition, the incorporation of exogenous GD3 was upregulated only in St8sia5-L expressing cells. Taken together, the localization of St8sia5 is important for the activity of the enzyme.

DOI： 10.1007/s10719-021-10034-8

A point-mutation in the C-domain of CMP-sialic acid synthetase leads to lethality of medaka due to protein insolubility.

がんとシアル酸重合体 Reviewed

佐藤ちひろ

医学の歩み Vol. 281 page： 870 - 875 2022

Wu D, Arakawa H, Fujita A, Hashimoto H, Hibi M, Naruse K, Kamei Y, Sato C, Kitajima K

Scientific reports Vol. 11 ( 1 ) page： 23211 2021.12

Language：English Publishing type：Research paper (scientific journal) Publisher：Scientific Reports

Vertebrate CMP-sialic acid synthetase (CSS), which catalyzes the synthesis of CMP-sialic acid (CMP-Sia), consists of a 28 kDa-N-domain and a 20 kDa-C-domain. The N-domain is known to be a catalytic domain; however, the significance of the C-domain still remains unknown. To elucidate the function of the C-domain at the organism level, we screened the medaka TILLING library and obtained medaka with non-synonymous mutations (t911a), or single amino acid substitutions of CSS, L304Q, in the C-domain. Prominently, most L304Q medaka was lethal within 19 days post-fertilization (dpf). L304Q young fry displayed free Sia accumulation, and impairment of sialylation, up to 8 dpf. At 8 dpf, a marked abnormality in ventricular contraction and skeletal myogenesis was observed. To gain insight into the mechanism of L304Q-induced abnormalities, L304Q was biochemically characterized. Although bacterially expressed soluble L304Q and WT showed the similar Vmax/Km values, very few soluble L304Q was detected when expressed in CHO cells in sharp contrast to the WT. Additionally, the thermostability of various mutations of L304 greatly decreased, except for WT and L304I. These results suggest that L304 is important for the stability of CSS, and that an appropriate level of expression of soluble CSS is significant for animal survival.

DOI： 10.1038/s41598-021-01715-3

Identification and functional characterization of a Siglec-7 counter-receptor on K562 cells. Reviewed International coauthorship

Yoshimura A, Asahina Y, Chang LY, Angata T, Tanaka H, Kitajima K, Sato C

The Journal of biological chemistry Vol. 296 page： 100477 2021.1

Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Biological Chemistry

Sialic acid (Sia)-binding immunoglobulin-like lectin 7 (Siglec-7) is an inhibitory receptor primarily expressed on natural killer (NK) cells and monocytes. Siglec-7 is known to negatively regulate the innate immune system through Sia binding to distinguish self and nonself; however, a counterreceptor bearing its natural ligand remains largely unclear. Here, we identified a counter-receptor of Siglec-7 using K562 hematopoietic carcinoma cells presenting cell surface ligands for Siglec-7. We affinity-purified the ligands using Fc-ligated recombinant Siglec-7 and diSia-dextran polymer, a strong inhibitor for Siglec-7. We then confirmed the counter-receptor for Siglec-7 as leukosialin (CD43) through mass spectrometry, immunoprecipitation, and proximity labeling. Additionally, we demonstrated that the cytotoxicity of NK cells toward K562 cells was suppressed by overexpression of leukosialin in a Siglec-7-dependent manner. Taken together, our data suggest that leukosialin on K562 is a counter-receptor for Siglec-7 on NK cells and that a cluster of the Sia-containing glycan epitope on leukosialin is key as trans-ligand for unmasking the cis-ligand.

DOI： 10.1016/j.jbc.2021.100477

Lower promoter activity of the ST8SIA2 gene has been favored in evolving human collective brains.

Hayakawa T, Terahara M, Fujito NT, Matsunaga T, Teshima KM, Hane M, Kitajima K, Sato C, Takahata N, Satta Y

PloS one Vol. 16 ( 12 ) page： e0259897 2021

Language：English Publishing type：Research paper (scientific journal) Publisher：PLoS ONE

ST8SIA2 is an important molecule regulating expression of the phenotype involved in schizophrenia. Lowered promoter activity of the ST8SIA2 gene is considered to be protective against schizophrenia by conferring tolerance to psychosocial stress. Here, we examined the promoter-type composition of anatomically modern humans (AMHs) and archaic humans (AHs; Neanderthals and Denisovans), and compared the promoter activity at the population level (population promoter activity; PPA) between them. In AMHs, the TCT-type, showing the second lowest promoter activity, was most prevalent in the ancestral population of non-Africans. However, the detection of only the CGT-type from AH samples and recombination tracts in AH sequences showed that the CGT- and TGT-types, exhibiting the two highest promoter activities, were common in AH populations. Furthermore, interspecies gene flow occurred into AMHs from AHs and into Denisovans from Neanderthals, influencing promoter-type compositions independently in both AMHs and AHs. The difference of promoter-type composition makes PPA unique in each population. East and Southeast Asian populations show the lowest PPA. This results from the selective increase of the CGC-type, showing the lowest promoter activity, in these populations. Every non-African population shows significantly lower PPA than African populations, resulting from the TCT-type having the highest prevalence in the ancestral population of non-Africans. In addition, PPA reduction is also found among subpopulations within Africa via a slight increase of the TCT-type. These findings indicate a trend toward lower PPA in the spread of AMHs, interpreted as a continuous adaptation to psychosocial stress arising in migration. This trend is considered as genetic tuning for the evolution of collective brains. The inferred promoter-type composition of AHs differed markedly from that of AMHs, resulting in higher PPA in AHs than in AMHs. This suggests that the trend toward lower PPA is a unique feature in AMH spread.

DOI： 10.1371/journal.pone.0259897

The expression of sulfated sialic acids (SiaS) is dynamically regulated in mammalian cells

Specific labeling and chemical analysis of sialic acid and sialyloligo/polymers.

Nishihara S, Angata K, Aoki-Kinoshita KF, Hirabayashi J, Sato C

2021

Kitajima, K; Ertunc, N; Sato, C

GLYCOBIOLOGY Vol. 30 ( 12 ) page： 1066 - 1067 2020.12

Sialic acid sulfation is induced by the antibiotic treatment in mammalian cells.

Ertunc N, Sato C, Kitajima K

Bioscience, biotechnology, and biochemistry Vol. 84 ( 11 ) page： 2311 - 2318 2020.11

Language：English Publishing type：Research paper (scientific journal) Publisher：Bioscience, Biotechnology and Biochemistry

Sialic acids (Sias) are an outermost-situated sugar of glycoproteins and glycolipids to play important roles in various biological phenomena. They are often modified by additional substituents, such as O-acetyl group, to display more than 50 different structures in nature. Of those modified Sia, nothing is known about the occurrence and biological functions of sulfated Sias (SiaSs) in mammals. To elucidate the significance of sialic acid sulfation, we investigated various mammalian-cultured cell lines for the expression of SiaS using the specific antibody 3G9. First, SiaS is expressed in a cell line-dependent and a cell density-dependent manner. Second, in CHO cells, the expression of SiaS is reversibly induced by treatment with the antibiotic G418. Taken together, the expression of SiaS is changed by intrinsic and extrinsic factors in mammalian cells. This is the first demonstration of regulated expression of SiaS.

DOI： 10.1080/09168451.2020.1792763

Systems glycomics of adult zebrafish

Kitajima Ken, Yamakawa Nao, Sato Chihiro, Yann Guérardel

Vol. 92 ( 3 ) page： 349 - 359 2020.6

Identification and characterization of a novel, versatile sialidase from a Sphingobacterium that can hydrolyze the glycosides of any sialic acid species at neutral pH. Reviewed

Iwaki Y, Matsunaga E, Takegawa K, Sato C, Kitajima K

Biochemical and biophysical research communications Vol. 523 ( 2 ) page： 487 - 492 2020.3

Language：English Publishing type：Research paper (scientific journal) Publisher：Biochemical and Biophysical Research Communications

Bacterial sialidases are widely used to remove sialic acid (Sia) residues from glycans. Most of them cleave the glycosides of N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) under acidic pHs; however, currently available bacterial sialidases had no activity to the glycosides of deaminoneuraminic acid (Kdn). In this study, we found a novel sialidase from Sphingobacterium sp. strain HMA12 that could cleave any of the glycosides of Neu5Ac, Neu5Gc, and Kdn. It also had a broad linkage specificity, i.e., α2,3-, α2,6-, α2,8-, and α2,9-linkages, and the optimal pH at neutral ranges, pH 6.5–7.0. These properties are particularly important when sialidases are applied for in vivo digestion of the cell surface sialosides under physiological conditions. Interestingly, 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (Neu5Ac2en), which is a transition state analog-based inhibitor, competitively inhibited the enzyme-catalyzed reaction for Kdn as well as for Neu5Ac, suggesting that the active site is common to the Neu5Ac and Kdn residues. Taken together, this sialidase is versatile and useful for the in vivo research on sialo-glycoconjugates.

DOI： 10.1016/j.bbrc.2019.12.079

Structure and Function of di/oligo/polysialic Acid (Polymerized Sialyl Structure) Invited

PSA-NCAM Colocalized with Cholecystokinin-Expressing Cells in the Hippocampus Is Involved in Mediating Antidepressant Efficacy. Reviewed Open Access

Yamada J, Sato C, Konno K, Watanabe M, Jinno S

The Journal of neuroscience : the official journal of the Society for Neuroscience Vol. 40 ( 4 ) page： 825 - 842 2020.1

Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Neuroscience

The extracellular glycan polysialic acid linked to neural cell adhesion molecule (PSA-NCAM) is principally expressed in the developing brain and the adult neurogenic regions. Although colocalization of PSA-NCAM with cholecystokinin (CCK) was found in the adult brain, the role of PSA-NCAM remains unclear. In this study, we aimed to elucidate the functional significance of PSA-NCAM in the CA1 region of the male mouse hippocampus. Combined fluorescence in situ hybridization and immunohistochemistry showed that few vesicular glutamate transporter 3-negative/CCK-positive (VGluT3 –/CCK +) cells were colocalized with PSA-NCAM, but most of the VGluT3 +/ CCK + cells were colocalized with PSA-NCAM. The somata of PSA-NCAM +/CCK + cells were highly innervated by serotonergic boutons than those of PSA-NCAM –/CCK + cells. The expression ratios of 5-HT3A receptors and p11, a serotonin receptor-interacting protein, were higher in PSA-NCAM +/CCK + cells than in PSA-NCAM –/CCK + cells. Pharmacological digestion of PSA-NCAM impaired the efficacy of antidepressant fluoxetine (FLX), a selective serotonin reuptake inhibitor, but not the efficacy of benzodiazepine anxiolytic diazepam. A Western blot showed that restraint stress decreased the expressions of p11 and mature brain-derived neurotrophic factor (BDNF), and FLX increased them. Interestingly, the FLX-induced elevation of expression of p11, but not mature BDNF, was impaired by the digestion of PSA-NCAM. Quantitative reverse transcription-polymerase chain reaction showed that restraint stress reduced the expression of polysialyltransferase ST8Sia IV and FLX elevated it. Collectively, PSA-NCAM colocalized with VGluT3 +/CCK + cells in the CA1 region of the hippocampus may play a unique role in the regulation of antidepressant efficacy via the serotonergic pathway.

DOI： 10.1523/JNEUROSCI.1779-19.2019

Sato Chihiro

Trends in Glycoscience and Glycotechnology Vol. 31 ( 181 ) page： SJ32 - SJ35 2019.7

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：FCCA(Forum: Carbohydrates Coming of Age)

<p>Sialic acid (Sia) is known to present as a monosialyl residue at a non-reducing terminal end of glycan in nature. In some cases, Sias occur as polymerized sialyl structures, di/oligo/polysialic acids (diSia/oligoSia/polySias). Here, we focus on the structure, function and biological significance of diSia/oligoSia/polySias. Following the definition of di/oligo/polySia structures and establishment of chemical and immunochemical detection methods, we analyzed these structures and their function based on a set of working hypotheses. So far, the biological meanings of degree of polymerization (DP) were shown and they lead to the change the concept of polySia. In addition, this research is applicable in the study of diseases such as mental disorder and cancer. However, research on the biological significance of the diversity in di/oligo/polySia structures is still ongoing.</p>

DOI： 10.4052/tigg.1915.2sj

CiNii Research

Structure and Function of di/oligo/polysialic Acid (Polymerized Sialyl Structure) Invited

Sato Chihiro

Trends in Glycoscience and Glycotechnology Vol. 31 ( 181 ) page： SE32 - SE35 2019.7

Language：English Publishing type：Research paper (scientific journal) Publisher：FCCA(Forum: Carbohydrates Coming of Age)

DOI： 10.4052/tigg.1915.2SE