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Polysialic acid is an important glyco-epitope in vertebrate brains, while altered expressions of polySia and biosynthetic enzyme have been reported in brain diseases such as schizophrenia and depression. Recently, the binding between polySia and dopamine and the involvement of this in Akt signaling has been demonstrated. However, the molecular mechanism underlying the binding of polySia and dopamine remains unknown. Therefore, here, we demonstrated the interaction between dopamine and polySia using frontal affinity chromatography alongside docking simulations. In addition, we prepared dopamine-lead compounds to understand the detailed molecular basis of polySia binding by frontal affinity chromatography, enzyme-linked immunosorbent assay, and docking simulations., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Formation of membrane microdomain is critical for cell migration (epiboly) during gastrulation of medaka fish [Adachi et al. (Biochem. Biophys. Res. Commun. 358:848-853, 2007)]. In this study, we characterized membrane microdomain from gastrula embryos to understand its roles in epiboly. A cell adhesion molecule (E-cadherin), its associated protein (beta-catenin), transducer proteins (PLCgamma, cSrc), and a cytoskeleton protein (beta-actin) were enriched in the membrane microdomain. Le(X)-containing glycolipids and glycoproteins (Le(X)-gp) were exclusively enriched in the membrane microdomain. Interestingly, the isolated membrane microdomain had the ability to bind to each other in the presence of Ca(2+). This membrane microdomain binding was achieved through the E-cadherin homophilic and the Le(X)-glycan-mediated interactions. E-cadherin and Le(X)-gp were co-localized on the same membrane microdomain, suggesting that these two interactions are operative at the same time. Thus, the membrane microdomain functions as a platform of the E-cadherin- and Le(X)-glycan-mediated cell adhesion and signal transduction.

Zebrafish appears as a relevant model for the functional study of glycoconjugates along vertebrate's development. Indeed, as a prelude to such studies, we have previously identified a vast array of potentially stage-specific glycoconjugates, which structures are reminiscent of glycosylation pathways common to all vertebrates. In the present study, we have focused on the identification and regulation of major protein and lipids associated alpha2-8-linked oligosialic acids motifs in the early development of zebrafish. By a combination of partial hydrolysis, anion exchange HPLC-FD and mass spectrometry, we demonstrated that glycoproteins and glycolipids differed by the extent and the nature of their substituting oligosialylated sequences. Furthermore, relative quantifications showed that alpha2-8-linked sialylation was differentially regulated in both families of glycoconjugates along development. Accordingly, we established that alpha2,8-sialyltransferase mRNA levels was directly correlated with changes of alpha2,8-sialylation status of glycolipids, but independent of those observed on major glycoproteins that appear to originate from the mother.

KDN is an abbreviation for 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid, and its natural occurrence was revealed in 1986 by a research group including the present authors. Since sialic acid was used as a synonym for N-acylneuraminic acid at that time, there was an argument if this deaminated neuraminic acid belongs to the family of sialic acids. In this review, we describe the 20 years history of studies on KDN (KDNology), through which KDN has established its position as a distinct member of the sialic acid family. These studies have clarified that: (1) KDN occurs widely among vertebrates and bacteria similar to the occurrence of the more common sialic acid, N-acetylneuraminic acid (Neu5Ac), but its abundant occurrence in animals is limited to lower vertebrates. (2) KDN is found in almost all types of glycoconjugates, including glycolipids, glycoproteins and capsular polysaccharides. (3) KDN residues are linked to almost all glycan structures in place of Neu5Ac. All linkage types known for Neu5Ac; alpha2,3-, alpha2,4-, alpha2,6-, and alpha2,8- are also found for KDN. (4) KDN is biosynthesized de novo using mannose as a precursor sugar, which is activated to CMP-KDN and transferred to acceptor sugar residues. These reactions are catalyzed by enzymes, some of which preferably recognize KDN, but many others prefer Neu5Ac to KDN. In addition to these basic findings, elevated expression of KDN was found in fetal human red blood cells compared with adult red blood cells, and ovarian tumor tissues compared with normal controls. KDNase, an enzyme which specifically cleaves KDN-linkages, was discovered in a bacterium and monoclonal antibodies that specifically recognize KDN residues in KDNalpha2,3-Gal- and KDNalpha2,8-KDN-linkages have been developed. These have been used for identification of KDN-containing molecules. Based on past basic studies and variety of findings, future perspective of KDNology is presented.

Deaminoneuraminic acid (KDN) is a unique member of the sialic acid family. We previously demonstrated that free KDN is synthesized de novo from mannose as its precursor sugar in trout testis, and that the amount of intracellular KDN increases in mouse B16 melanoma cells cultured in mannose-rich media [Angata et al. (1999) J. Biol. Chem. 274, 22949-56; Angata et al. (1999) Biochem. Biophys. Res. Commun. 261, 326-31]. In the present study, we first demonstrated a mannose-induced increase in intracellular KDN in various cultured mouse and human cell lines. These results led us to examine whether KDN expression in mouse organs is altered by exogenously administered mannose. Under normal feeding conditions, intracellular free KDN was present at very low levels (19-48 pmol/mg protein) in liver, spleen, and lung, and was not detected in kidney or brain. Oral ingestion of mannose, both short-term (90 min) and long-term (2 wk), resulted in an increase of intracellular KDN up to 60-81 pmol/mg protein in spleen and lung and 6.9-18 pmol/mg protein in kidney and brain; however, no change was observed in liver. The level of KDN in organs appears not to be determined only by the KDN 9-phosphate synthase activity, but might also be affected by other enzymes that utilize mannose 6-phosphate as a substrate as well as the enzymes that breakdown KDN, like KDN-pyruvate lyase. In blood, the detectable amount of free KDN did not change on oral ingestion of mannose. These findings indicate that mannose in the diet affects KDN metabolism in various organs, and provide clues to the mechanism of altered KDN expression in some tumor cells and aged organs.

The low density, detergent-insoluble membrane fraction (LD-DIM), where gangliosides are likely to be highly enriched, was prepared from sperm of two sea urchin species, Hemicentrotus pulcherrimus and Strongylocentrotus purpuratus. Immunoblotting showed the presence in the LD-DIM of two receptors for egg ligands, a glycosylphosphatidylinositol (GPI)-anchored protein, and four proteins which may be involved in signal transduction. Co-immunoprecipitation revealed that at least three proteins, the speract receptor, the 63kDa GPI-anchored protein and the alpha subunit of a heterotrimeric Gs protein, are localized in the LD-DIM. This suggests that the LD-DIM fraction may be a membrane microdomain for speract-speract receptor interaction, as well as the subsequent signal transduction pathway involved in induction of sperm respiration, motility and possibly the acrosome reaction.

Novel sulfated and nonsulfated oligosialylglycosphingolipids were isolated from sperm of the sea urchin, Hemicentrotus pulcherrimus, and their structures were established as follows: [formula: see text] This provides the first evidence for the natural occurrence of a tetrasialic acid structure in glycosphingolipids. The finding of sulfated oligosialyl chains is especially noteworthy in that the sulfate group exclusively resides on the C-8 of the nonreducing terminal residues of oligo/polysialyl chains and that sulfation appears to be a termination signal for elongation of oligosialyl chains. Sulfation at the nonreducing terminal Neu5Ac residues of oligosialyl chains was also found to facilitate the formation of an inter-residue lactone between the carboxyl group at the nonreducing terminal sulfated Neu5Ac and the hydroxyl group at C-9 of the penultimate Neu5Ac residue. The long chain base was 4-hydroxysphinganine (t18:0) and the major fatty acid species were identified as C20:1, C21:1, and C22:1.

Recently, we have reported purification and characterization of a de-N-glycosylating enzyme, peptide: N-glycanase (PNGase) found in C3H mouse fibroblast L-929 cells, and designated L-929 PNGase [Suzuki T, Seko A, Kitajima K, Inoue Y, Inoue S (1994) J Biol Chem 269, 17611-18]. The unique properties of L-929 PNGase are that the enzyme had a high affinity to the substrate glycopeptide (e.g. Km = 114 microM for fetuin derived glycopentapeptide) and that the PNGase-catalysed reaction is strongly inhibited by the released free oligosaccharides but not by the free peptides formed, suggesting that L-929 PNGase is able to bind to a certain type of carbohydrate chain. In this study, we report the new findings of the mannan-binding property of L-929 PNGase: the de-N-glycosylating enzyme activity of L-929 PNGase was inhibited by yeast mannan and triomannose, Man alpha 1-->3(Man alpha 1-->6)Man, but not by mannose and alpha-methyl-D-mannoside. Furthermore, L-929 PNGase was revealed to bind to the glycan moiety of yeast mannan by using mannan-conjugated Sepharose 4B gel as a ligand, suggesting that L-929 PNGase could serve not only as an enzyme but also as a carbohydrate recognition protein in vivo. Such 'dual' properties found for animal-derived L-929 PNGase are unique and are not shared with other previously characterized plant- and bacterial-origin PNGases--PNGase A and PNGase F, respectively.

A novel glycosyltransferase which catalyses transfer of deaminated neuraminic acid, KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) from CMP-KDN to the non-reducing termini of oligo-polysialyl chains of polysialoglycoprotein (PSGP), was discovered in the ovary of rainbow trout (Oncorhynchus mykiss). The KDN-transferase activity was optimal at neutral pH, and stimulated 2 to 2.5-fold by 2-5 mM Mg2+ or Mn2+. Expression of KDN-transferase was developmentally regulated in parallel with expression of the alpha 2-->8-polysialyltransferase, which catalyses synthesis of the oligo-polysialyl chains in PSGP. Incorporation of the KDN residues into the oligo-polysialyl chains prevented their further elongation, resulting in 'capping' of the oligo-polysialyl chains. This is the first example of a glycosyltransferase that catalyses termination of alpha 2-->8-polysialylation in glycoproteins.