Sialic Acids in Neurology

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Sialic Acids in Neurology Chihiro Sato, Ken Kitajima Bioscience and Biotechnology Center, Nagoya University, Nagoya, Japan

Contents 1. Introduction 2. History, Definition, and Occurrence 2.1 History 2.2 Definition of Oligo/PolySia 2.3 Occurrence 3. Analytical Methods 3.1 Biochemical Probes 3.2 Chemical Detection Method 4. Biosynthesis 4.1 Common Features 4.2 Oligo/PolySia-Biosynthesizing Enzymes: ST8Sia2, ST8Sia4, and ST8Sia3 4.3 Di/TriSia-Synthesizing Enzymes: ST8Sia1, ST8Sia5, and ST8Sia6 5. Phenotypes of PolySia-Impaired Animals 6. Biochemical Features of Di/Oligo/PolySia and Their Functions 6.1 Repulsive Field of PolySia 6.2 Attractive Field of PolySia 6.3 Regulatory Role for Receptors 7. Related Diseases 7.1 Mental Disorders and Neurodegenerative Diseases 7.2 Cancer 8. Perspectives Acknowledgments References

2 3 3 4 5 8 8 12 15 15 16 19 20 23 23 25 37 38 38 43 44 46 46

ABBREVIATIONS AMG ASD BD BDNF CX DA

amygdala autism spectrum disorder bipolar disorder brain-derived neurotrophic factor cerebral cortex dopamine

Advances in Carbohydrate Chemistry and Biochemistry ISSN 0065-2318 https://doi.org/10.1016/bs.accb.2018.09.003

#

2018 Elsevier Inc. All rights reserved.

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ECM FGF2 HIP NACM Neu5Ac OB PolySia PolyST PSA SGZ Sia SNP ST8Sia SVZ SZ UDP

extracellular matrix fibroblast growth factor-2 hippocampus neural cell-adhesion molecule N-acetylneuraminic acid olfactory bulb polysialic acid polysialyltransferase polysialic acid subgranular zone sialic acid single-nucleotide polymorphism ST8 α-N-acetylneuraminide α-2,8-sialyltransferase subventricular zone schizophrenia uridine diphosphate

1. INTRODUCTION Sialic acids (Sias) comprise a family of 9-carbon carboxylated sugars. The Sia family consists of nearly 50 members that are derivatives of N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), and 3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid (deaminoneuraminic acid; 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid; Kdn) (Fig. 1).1 The diversity of these structures is attributable to several modifications such as acetylation, sulfation, methylation, lactylation, and lactonization. In most cases, Sias occurring as monosialyl residues (monoSias) cap the nonreducing terminal ends of glycan chains of glycoconjugates (glycoproteins and glycolipids). MonoSias function as mediators for ligand receptors and regulate the cell–cell and/or cell–extracellular matrix (ECM) interactions.1,2 In some cases, Sias that are linked to each other form oligo/polymerized Sia structures (oligo/polySia), specifically, disialic acid (diSia), trisialic acid (triSia), oligosialic acid (oligoSia), and polysialic acid (polySia).3 The di/tri/oligo/polymerized Sia glycotope has been shown to exhibit more structural diversities in “Sia components,” “intersialyl linkages,” and at a “degree of polymerization (DP)” (Fig. 1).3 Sensitive chemical methods have been developed to detect di/tri/oligo/ polymerized Sia structures.4,5 The specificity of probes such as antibodies and enzymes has been estimated, and new probes have been developed,3,6,7 which indicate the diversity of the oligo/polymeric structures. All these methods demonstrate the frequent occurrence of di/tri/oligo/polySias in glycoproteins.3 Mono/di/triSia epitopes on glycolipids are known to

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Fig. 1 Diversity in oligo/polymerized sialic acids present in glycoproteins and glycolipids. Oligo/polymerized sialic acids (red diamond) have a diversity in components (Neu5Ac: purple diamond, Neu5Gc: blue diamond, Kdn: green diamond), intersialyllinkage (α2,4-, α2,8-, α2,8/9, and α2,11-; α2,7-linkage is not reported), degree of polymerization (DP) (2–400), and modifications (acetylation, methylation, sulfation, lactylation, and lactonization). Theoretically, the four factors, components, intersialyl-linkage, DP, and modifications, regulate the diversity of oligo/polymerized Sia structures.

occur in the gangliosides, and this topic has been focused upon in a different chapter of this book. The function of the monoSia epitope present in a specific glycoprotein has not been thoroughly understood, especially in the brain, probably because monoSias cap almost all sialoglycoconjugates; their functions could be understood using St3gal, St6gal1/2, and St6galnac knockout (KO) mice. Therefore, in this chapter, the history, definition, occurrence, methods for analysis, biosynthesis, and biochemical features of di/tri/oligo/polymerized Sia epitopes on glycoproteins, and their related diseases and biological functions, which were evaluated by phenotypes of gene-targeted mice, have been focused upon.

2. HISTORY, DEFINITION, AND OCCURRENCE 2.1 History The polySia structure was first reported in a filtrate of Escherichia coli K235L+ O, and it was named colominic acid.8,9 It was reported that colominic acid appeared to be derived from the acid hydrolysate of membrane-bound polySia (polyNeu5Ac)10; it exhibited the α2,8-interlinkage,11 and its DP was

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greater than 200.12 The glycocalyx of Neisseria meningitidis serogroup B was found to be structurally and immunologically identical to the α2,8polyNeu5Ac structure of E. coli K1.13 These two bacterial capsular polysaccharides were shown to be neuroinvasive determinants,14,15 and α2,8-polyNeu5Ac has been considered to be related to the brain system. Based on the structural analysis of capsular polysaccharides derived from the N. meningitidis serogroup C and E. coli K92, the α2,9-polyNeu5Ac13,16 and alternative α2,8- and α2,9-polyNeu5Ac (α2,8/9)17 structures were reported. There have been reports regarding linkage diversity since the discovery of the polySia structure. However, there has been no report to date regarding oligo/polyNeu5Gc and oligo/polyKdn in bacterial polysaccharides. In vertebrates, α2,8-polyNeu5Gc was found in O-linked glycans on the polysialoglycoproteins (PSGPs) of the cortical alveoli of eggs derived from Oncorhynchus mykiss (rainbow trout).18 The α2,8-polyNeu5Ac structure was also present in the N-linked glycans of neural cell-adhesion molecules (NCAMs) derived from developing rat brains; however, adult brains have amounts approaching 10% of this unique glycotope.19 The α2,8-polyNeu5Ac, α2,8-polyNeu5Gc, and α2,8-polyNeu5Ac/Neu5Gc structures were present in the α2,8-polySias of PSGPs derived from Salmonidae fish eggs. In addition, α2,8-oligoKdn was found in fish ovarian fluid. Diversity has been observed in α2,8-linked oligo/polySias since 1993.20 However, because the diversity and degree of polymerization (DP) of the polySia were not taken into consideration, the definition put forward earlier was inaccurate. Di/tri/oligo/polySias-containing glycoproteins other than PSGP and NCAM have now been shown to be present in several glycoproteins, and an understanding of the biological relevance of the DP is developing.

2.2 Definition of Oligo/PolySia Until 1990 polySia structures were considered to be present only in neuroinvasive bacterial polysaccharides, NCAMs, and PSGPs. The polySia structures from these glycoconjugates were analyzed by chemical methods such as periodate analysis,11,12 methylation–GC/MS analysis,16 and anion-exchange chromatography analysis.21 PolySia probes such as antipolyNeu5Ac antibodies were then developed, especially for producing vaccines against N. meningitidis; however, it was not easy to produce the probes, owing to the immunotolerance of the α2,8-polySia structure in hosts.22

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Anti-polySia antibodies have been widely used for the detection of polySia, because their sensitivity was higher than that of conventional chemical methods of detection. However, the specificity of the anti-polySia antibody was not well characterized until the mid-1990s, because concepts regarding the presence of shorter polySia structures or diversity of polySia structures with regard to the linkage and Sia components in vertebrate were yet to be established. The diversity of polySia structures was first described in a report in 1993.20 In addition, polySia was not well characterized, because NCAM was considered to be the only polySia carrier in the vertebrate brain, and the functional differences of polySias had not been completely investigated. Therefore, it was essential to understand the precise antigenicity of the antibody before its use, along with the components, linkages, and DP.3,23,24 We have been proposing to classify polymerized Sia structures according to their antibody specificity and conformation as follows: diSia (DP 2), triSia (DP 3), oligoSia (DP 2–7), and polySia (DP ≧ 8) (Fig. 1).3,23,24 PolySias could be identified with specific probes such as anti-polySia antibodies and monoclonal antibodies, with a specific enzyme such as endo-N-acylneuraminidase (Endo-N), which could cleave the oligo/polySia structure, or by using the newly developed chemical methods that are described later.

2.3 Occurrence As mentioned earlier, the polySia structure was first reported to be present in acid hydrolysates of E. coli K-235, and it was named colominic acid.8,9 The glycocalyx of neuroinvasive bacterial polysaccharides from E. coli K1 and N. meningitidis group B was then shown to possess the α2,8-linked polyNeu5Ac structure, in which DP ¼ 55–100.13–15 N. meningitidis group C and E. coli K-92 were shown to have α2,9-linked polyNeu5Ac and α2,8/α2,9-linked polyNeu5Ac structures. All these bacteria are neuroinvasive, and this feature greatly influences their immunotolerance, probably because of their ability to pass the blood–brain barrier.14,15 In vertebrates, polySia is enriched, especially in embryonic brains. The presence of polySia is restricted to the deuterostome lineage, and echinoderms exhibit a variety of polySia structures (Fig. 2A).3 Interestingly, the α2,8-linked polyNeu5Ac structure is the only epitope that occurs in embryonic and adult brains (Fig. 2B), perhaps because it has certain specific functions, especially in the brain. The major carrier of polySia in the brain is the

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Fig. 2 Occurrence of oligo/polySia in organism and brain. (A) Oligo/polymerized sialic acids have been reported to occur in Gram-negative bacteria and in the deuterostome lineage of the animal kingdom. Echinoderms are rich in the diversity in oligo/polySia. In vertebrates, α2,8-linked oligo/polySia have been reported. (B) α2,8-Linked oligo/polyNeu5Ac have been reported in vertebrate brains. Usually, in embryonic brain, polyNeu5Ac is enriched; however, in adult brain, polyNeu5Ac is present in restricted regions, especially at OB systems (OB, RMS, LV) and HIP (DG/SGL) where neurogenesis is ongoing. Other regions, such as AMG, Hypo, PFC, pons, and spinal code (SC), are also reported to have polySia-bearing cells.

neural cell-adhesion molecule, NCAM (NCAM/Ncam1). Almost the entire NCAM in the embryonic brain is modified by α2,8-linked polyNeu5Ac, and in the adult brain, α2,8-linked polyNeu5Ac-modified NCAM is present in restricted areas, such as the regions that are highly

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plastic or those undergoing neural remodeling (Fig. 2B).25,26 Based on the results obtained with NCAM-KO mice, 85% of α2,8-linked polyNeu5Ac was present in NCAM. Around 15% of α2,8-linked polyNeu5Ac is considered to be linked to other glycoconjugates.27 Another possible carrier protein in the brain is the synaptic cell-adhesion molecule (SynCAM/Syncam/ Cadm1), which was found to be polysialylated in NCAM-KO mice.28 Syncam1 was proven to be polysialylated by the polysialyltransferase (polyST) ST8Sia2.29 Another candidate protein is neuropilin (NRP) 2, which was shown to be present in the human T cell and mouse oligodendrocyte,30 and was also proven to be polysialylated by ST8Sia4.31 However, it should be noted that the loss of substrate might lead to the polysialylation of other proteins that are not polysialylated in the presence of NCAM. DiSia is shown to be linked to NCAM and other unidentified glycoproteins using the anti-diSiaGal antibody,32 although its function remains unknown.6 The presence of triSia on glycoproteins in the brain was also demonstrated with the use of the anti-triSia antibody.33 The presence of polySia/oligoSia/ diSia-containing glycoproteins in tissues other than the brain has also been reported.3,34 The expression of polysialylated NCAM (polySia–NCAM) in mice starts at E9.5 and increases until just before birth; the level of polySia decreases drastically after birth.35 In adults, almost the entire polySia content disappears, except for the restricted areas, in which neurogenesis and neural remodeling occur, such as the olfactory bulb (OB) system and hippocampus (HIP).25 The expression level of polySia is highly correlated with the mRNA expression of two polySTs, ST8Sia2 and ST8Sia4, although their expression mechanism has not been investigated yet. During development, the expression of mRNA of both polySTs starts from E8 to E9 at the time of neural tube closure, whereas their expression is downregulated in adults. The level of ST8Sia2 declines drastically after birth, while the level of ST8Sia4 declines gradually, as demonstrated by both Northern blot analysis and real-time PCR.35,36 In adults, ST8Sia4 is continuously expressed at lower levels in various tissues. In humans, a similar drastic decrease in the expression of ST8Sia2 was demonstrated by microarray analysis, and the ST8Sia2 mRNA level in neonates decreased drastically as compared to that in toddlers (30%), teenagers (20%), and adults (10%).37 In embryonic rodent brains, overall polySia staining is observed38; however, in adult brains, two major polySia-staining areas are well known.25,26,38–40 One is the area consisting of the precursor cells of interneurons (INs), which are generated from the subventricular zone (SVZ) of the lateral ventricle and rostral migratory stream (RMS); these INs migrate

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to the OB.41 The other is the subgranular zone (SGZ) of the dentate gyrus (DG) that includes the granule cell layer and the mossy fibers of the HIP.42 It is well known that neurogenesis and neural plasticity are observed in these two areas, even in the adult brain. The other areas that exhibit physiological plasticity, such as the hypothalamus (Hypo) (supraoptic nucleus and suprachiasmatic nucleus (SCN))43 and thalamus (periventricular, paratenial, and anteroventral–anterodorsal nuclei),44 also show polySia– NCAM staining. Interestingly, the polySia–NCAM in the SCN is regulated by photic stimulation, which influences circadian rhythm regulation.45 In the cortex, polySia staining is observed in the layer II of the piriform cortex.46 It is also known that polySia–NCAM is observed in the amygdala (AMG),47 substantia nigra (SNA),48 and pons (lateral part of parabranchial nucleus).44 In the spinal cord, polySia is expressed in some sensory neurons, but not in motor neurons.44 The expression of polySia is confined to laminae I and II of the dorsal horn, lateral spinal nucleus, and the region surrounding the central canal (lamina X).44 In the peripheral nervous system, polySia expression is observed in the visual system (axons of ganglion cells in the optic nerve and tectum, but not the somata in the retina).49 The expression of polySia is observed to be restricted in the sciatic nerves of adults, particularly in some unmyelinated axons and Schwann cells, though not in the myelinated axons.50 In addition, the reexpression of polySia reportedly occurs in the sciatic nerves of adults owing to injury and repair.51

3. ANALYTICAL METHODS 3.1 Biochemical Probes 3.1.1 Antibodies The first approach for developing probes for polySia involved the use of antibodies developed for producing vaccines against neuroinvasive N. meningitidis group B (MenB) bacteria. The horse 46 serum was developed for this purpose.52 Several attempts were made to develop anti-MenB antibodies using autoimmune mice, because the antigenicity of α2,8-linked polyNeu5Ac was low, owing to the presence of common epitopes in newborn infants. The first approach used for confirming the specificity of the antibody toward polySia was to assess its ability to inhibit the binding of MenB polysaccharides with colominic acid (α2,8-polyNeu5Ac).53 On performing precipitation assays, the radiolabeled oligo/polySia, H.46, and 735,

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which were used as polySia-specific probes, were shown to react with polyNeu5Ac structures with a DP 8 or 1054; however, the immunospecificity of a majority of other “anti-polySia antibodies” remained unknown. The comprehensive analyses of anti-oligo/polySia antibody specificities were first achieved using lipidated oligo/polySias (neooligo/polySia-glycoconjugate) and phosphatidylethanolamine dipalmitoyl (PE)-conjugated oligo/polySias as test antigens.23 This system was used to classify not only anti-“polySia” antibodies but also anti-di/oligo/polySia antibodies. Thus, a long list of characterized antibodies recognizing di/tri/oligo/polySia structures now appeared to be present (Table 1), although the precise immunospecificities of a few antibodies remained unknown. This was an epoch-making system, because it preceded the establishment of the array system, and antiganglioside antibodies that had been considered to react only with glycolipids appeared to be able to react with oligomerized Sia epitopes on glycoproteins. Anti-di/oligo/polySia antibodies could be classified into three groups, based on their immunospecificity for the DP and recognition of nonreducing termini of antibodies. In addition, these antibodies could also be subdivided based on the different Sia components, such as anti-Neu5Ac, anti-Neu5Gc, and anti-Kdn antibodies. Furthermore, depending on the types of linkages present, the antibody could be classified into at least two subgroups, groups I and II, consisting of α2,8-linked or α2,9-linked Sia-specific antibodies, respectively. Group I consists of antibodies that recognize α2,8-linked polySia structures and fully extended helical polySia chains with a DP  8. Group I antibodies are thought to recognize the extended helical conformation attributable to α2,8-linked polySia residues within the internal region of polySia chains, but they do not require the nonreducing terminal residues for recognition, because the destruction of the nonreducing terminal end did not influence antibody activity. These antibodies do not recognize α2,9-linked polySias, but recognize the α2,8-polyNeu5Ac structure; therefore, this group is named as α2,8-Group IA. The horse IgM polyclonal antibody (H.46)52 and mouse monoclonal IgG2a antibody 73555 are included in this group. The crystal structures of the 735 without56 and with57 polySia (DP ¼ 8) were analyzed, and it was proven that the presence of a minimum of three sialic acid residues was required for the recognition of the 735-scFv fragment. However, stronger and more stable binding requires at least 11 residues to be recognized, indicating that both extended and flexible epitopes were required for this purpose.57 The anti-polySia antibody IgMNOV was

III

A

II

Neu5Ac

2-2B

Kdn8kdn

K

Neu5Ac

FS1

Kdn

Neu5Gc

Neu5Ac

S2-566

AC1

Neu5Ac

A2B5

Neu5Gc

Neu5Ac

5A5

2-4B

Neu5Ac

OL28

Neu5Ac

4F7

Neu5Ac

Neu5Ac

735

12E3

Neu5Ac 13 NR n.d. n.d. 6 5 4 3 2 3 2 2–3 R 2–3 R 2–3 R

α2,8α2,9α2,8α2,8α2,8α2,8α2,8α2,8α2,8α2,8α2,8α2,8-

R

R

R

R

R

R

R

10 NR

Requirement of NR

α2,8-

Components Linkage DP

H.46

G

A

G

A

I

Name of Group Category Antibody

n.d.







+/

+/

+/

+/

+/

+



+

+



























α2,3,6Endo-N Sialidase

+

+

+

+

+

+

+

+

+

+



+

+

α2,3,6,8Sialidase

+

+

+

+

+

+

+

+

+

+

+

+

+

α2,3,6,8,9Sialidase

Table 1 Specificity of Anti-di/oligo/polySia Antibodies and Epitope Sensitivity Using Various Types of Sialidases Specificity of Di/Oligo/PolySia Epitope Sensitivity Using Sialidase Enzyme

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identified in the serum of a patient with IgM gammopathy, and this antibody reacted with both polyNeu5Ac and DNA/polynucleotides.58 This property is important because of the similarity between the helical conformation of polySia and DNA in negatively charged arrays. Other interesting antibodies include the anti-polySia antibodies specific to N-substituted polySia, which are used as immunizing antigens.59 Anti-polyNeu5Pro showed a high affinity toward polyNeu5Ac. These studies demonstrated that the antigenic specificity of anti-polySia antibodies is closely related to the conformational state of the negatively charged array. As for the α2,9-Group IA antibodies, antibodies specific to N. meningitidis Group C and sea urchin sperm (mouse monoclonal IgG3) were developed, but the DP of these antibodies has not been determined. Group II antibodies, designated as “anti-oligo + polySia antibodies,” consist of antibodies that recognize polySia chains and di/tri/oligoSias with DP 2–7. In addition, these antibodies are considered to recognize the distal portion of oligo/polySia chains, including the nonreducing termini, because the destruction of the nonreducing terminal end greatly influenced the activity toward antigens. These antibodies (12E3,46 OL28,60 2-2B,61 5A562) were first developed as anti-polySia antibodies because they could recognize polySia–NCAM; however, after determining their specificity based on the lipidated oligo/polySia content, it was found that these antibodies could recognize not only polyNeu5Ac but also di/tri/oligoNeu5Ac, indicating that these antibodies could recognize the nonreducing terminal end of the polyNeu5Ac structure, and a variety of DPs (2–6). Therefore, these antibodies could be included in the α2,8-Group IIA. In α2,8-Group II, there are two other types of antibodies, 2-4B23 and kdn8kdn,63 which are included in α2,8-Group IIG and α2,8-Group IIK because of their reactivity toward oligo/polyNeu5Gc and oligoKdn, respectively. Group III, designated as “anti-di/oligoSia antibodies,” recognize specific conformations of di/triSia with DP 2–3, but do not bind polySia. These antibodies were developed for ganglioside detection, but were confirmed to bind di/triSia epitopes on glycoproteins. The S2-566,64 A2B5,65 and FS166 antibodies are included in α2,8-Group IIIA. Group II and III antibodies are useful for detecting and determining diSia and oligoSia structures in combination with exo- and endo-sialidase treatment, as described in Table 1. 3.1.2 Enzymes endo-Sialidase can serve as a specific probe to detect and selectively modify α2,8-linked polySia chains.7,67,68 A soluble enzyme derived from

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bacteriophage K1F, designated Endo-N, catalyzes the cleavage of α2,8polySia chains as follows: ð! 8Neu5Acylα2 !Þn  X ðn  5Þ ! ð! 8Neu5Acylα2 !Þ24 + ð! 8Neu5Acylα2 !Þ2  X:7 Two other types of endo-sialidases with substrate specificities that differ from that of Endo-N of bacteriophage K1F have been isolated; these include Endo-NF68 and a bacteriophage endosialidase,67 and they require minimum DP  11 and DP  3, respectively, for cleavage. Based on the analysis of the crystal structures of Endo-NF, the oligoSia, pentaSia, and tetraSia bind to the enzymes, and the size of the oligomers is different, i.e., 12.4 and ˚ , respectively, indicating that the oligoSia structure binds flexibly to 14.7 A Endo-NF.69 There is no report to date regarding an endo-sialidase specific toward α2,9-polySia. Recently, it was found that a recombinant protein derived from Endo-NE that did not exhibit enzymatic activity was a new probe for α2,8-linked polySia.70 exo-Sialidases that cleave specific Sia linkages in enzymes such as α2,3sialidase (NANase I, Sialidase A), α2,3- and α2,6-sialidase (NANase II, Sialidase B), α2,3-, α2,6-, α2,8-sialidase (NANase III, Sialidase C), and α2,3-, α2,6-, α2,8-, α2,9-sialidase have also been used to theoretically confirm the presence of the α2,8/9-polySia structure and α2,8/9-di/tri/ oligoSia (DP 2–5) on glycoproteins. Notably, di/tri/oligoSia (DP  5) structures are not cleaved by Endo-N,7,20 but are cleaved by exo-sialidases (Table 1). It is important to think about the presence of sialidase-resistant structures in nature.71 In addition, cleavage of the oligo/polySia structure also occurs in acidic conditions where the pH is 4.0–6.5, without sialidases. The control experiments in which chemical treatments such as acid or periodate treatments are performed sometimes positively influence the results.

3.2 Chemical Detection Method 3.2.1 Fluorometric C7/C9 Analysis When a di/oligo/polymer of α2,8-linked N-acylneuraminic acid (Neu5Acyl) residues is subjected to periodate oxidation, the nonreducing terminal residue is oxidized to the C7 analogue of N-acylneuraminic acid, C7(Neu5Ac) (5-acetamido-3,5-dideoxy-L-arabino-hept-2-ulosonic acid), or C7(Neu5Gc) (5-hydroxyacetamido-3,5-dideoxy-L-arabino-hept-2-ulosonic acid), from monoNeu5Ac or monoNeu5Gc residues, while the internal residues of Neu5Ac (C9(Neu5Ac)) or Neu5Gc (C9(Neu5Gc)) still remain

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unchanged because the α2,8-linkage inhibits the cleavage of diol residues.4,12 Accordingly, the detection of C9-compounds among the products of periodate oxidation indicates the presence of internal sialyl residues or a polymeric structure composed of α2,8-linked N-acylneuraminic acid. C7- and C9-compounds can be quantitated by fluorometric high-performance liquid chromatography (HPLC) after treatment with the α-keto acid-specific fluorescent labeling reagent, 1,2-diamino-4,5-methylenedioxybenzene (DMB)4,72,73 (Fig. 3A). This chemical method has been successfully used to confirm the ubiquitousness of di/oligo/polySia in a wide variety of glycoproteins at femtomole (fmol) levels. These improved detection methods have

Fig. 3 Analytical methods for the detection of oligo/polySia. (A) Fluorometric C7/C9 analysis. α2,8-Linked oligo/polyNeu5Acyl structures are oxidized using NaIO4 under mild conditions. Under these conditions, nonreducing terminal sialic acid is changed from C9 to C7, while α2,8-linked sialic acids remain unchanged (C9). After hydrolysis using strong acid solution, the released monoNeu5Acyl structures are labeled with the α-keto acid-specific labeling reagent DMB. After separation with HPLC using an ODS column, the C9 amounts are considered to be the amounts of α2,8-linked polySia. (B) Mild acid hydrolysis–fluorometric anion-exchange HPLC analysis. It was first demonstrated that the polySia structure can be directly labeled with DMB, and labeled oligo/ polySia structures can be separated using anion-exchange HPLC. Using colominic acid, a peak around DP 40 was observed.

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led to the identification of polymerized Sia-modified carrier proteins and have helped to identify the specific functions of polySia. However, this method has several limitations. First, this method is only applicable for the detection of α2,8-linked di/tri/oligo/polymers of N-acylneuraminic acid and cannot be used to determine the DP of polymers with α2,9, α2,8/α2,9 linkages, or α2,11 (or α2,5Oglycolyl)-linkages. Second, the detected C9-derivatives do not always arise from α2,8-linked Neu5Ac, because 8-O-substituted Neu5Acyl residues might also yield indistinguishable C9-derivatives. Because of this reason, samples are typically treated with a mild alkali solution prior to periodate oxidation, although a few substituents are not released under these conditions, such as those of sulfated molecules. Third, the molar proportion of C9- to C7derivatives is not directly representative of the DP, unless linear oligo/polySia chains are being analyzed. Thus, this method does not allow the determination of the DP for samples containing multiple sialylated glycan chains. Fourth, the chemical reagents for the treatment of glycolipids are ineffective at times because of micelle formation. 3.2.2 Mild Acid Hydrolysis-Fluorescent Anion-Exchange HPLC Analysis Our group was the first to report that di/oligo/polymers produced by the mild acid hydrolysis of di/oligo/polySia chains could be directly labeled with DMB5 and analyzed by anion-exchange HPLC5 (Fig. 3B). Several anion-exchange chromatography columns can be used to analyze DMBlabeled Sia polymers, such as MonoQ or Mini Q HR5/5 (0.5  5 cm, GE, Uppsala, Sweden), Resource Q (1 mL, GE, Uppsala, Sweden), CarbopacPA100 (4  250 mm, Dionex), and DNApac PA100 (4  250 mm, Dionex) columns. DMB labeling can be used for the detection of various types of oligo/polymers of Sia found in glycoconjugates, which can differ in component Sia species, interresidual linkages, and DP. This analysis can be applied to glycoproteins blotted on PVDF membranes. However, because oligo/polySia is easily degraded under mild acidic conditions, it is difficult to accurately determine the DP of oligo/polySia in glycans. This method can be used to determine the maximum DP on the oligo/polySia chains and the ratio of the DPs. 3.2.3 Conventional Chemical Methods For the analysis of samples containing relatively higher amounts of di/tri/ oligo/polySia structures, a number of conventional methods, including methylation analysis,74 nuclear magnetic resonance (NMR) spectroscopy,75 and mild acid hydrolysis followed by thin-layer chromatography (TLC)76 can be used.

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3.2.4 Chemical Biological Approaches The in vivo modification of Sia, achieved by the treatment of samples with precursors of Sia biosynthesis, is useful for detection, imaging, and targeting and is a useful and widely available technique. Reutter et al. first demonstrated that the addition of N-substituted mannosamine changed Sia on the cell surface to N-substituted forms, such as Man2NPro, Man2NBut, and Man2NPent.77 N-Levulinoyl mannosamine (Man2NLev)78 and N-azido-acetylmannosamine (Man2NAz)79 were used as precursors to modify Sia in a highly selective manner, and it was observed that the incorporation of these unnatural Sias occurred on glycoconjugates containing Sia in cells in vivo. Using unnatural substrates such as ManPro and ManBut, polySTs were shown to utilize CMP-Sia. But less efficiently than CMPSiaPro, and both brought about slower polymerization than CMP-Sia. Furthermore, the activity of ST8Sia2 was diminished by the addition of ManBut within the priming Sia residues.80 ST8Sia2, ST8Sia3, and ST8Sia4 were also shown to be inhibited by 20 -OMe-CMP, CMP, and 5-Me-CMP, respectively, and the addition of these compounds to the cell culture medium leads to a decrease in polySia expression.81

4. BIOSYNTHESIS 4.1 Common Features The biosynthetic reaction for the synthesis of α2,8-linked oligo/polymerized Sia catalyzed by α2,8-sialyltransferase/ST8Sia is as follows: ðSiaÞn -glycoconjugates + CMP-Sia ! ðSiaÞn + 1 -glycoconjugates + CMP The enzymes for this reaction were cloned and named CMP-Sia:ST8 α-sialide α-2,8-sialyltransferase (ST8Sia).82,83 In humans, ST8SIA is used, and in the case of other animals, St8sia is used. In this chapter, ST8Sia is used for both cases. ST8Sia1–ST8Sia6 have been known as mammalian ST8Sias. In fish, ST8Sia7 and other STSias with unknown linkage specificities have been cloned from the sea urchin (Strongylocentrotus purpuratus) and lancelet (Branchiostoma floridae).84 From the evolutionary perspective, the existing ST8Sias in vertebrates are important because of their involvement in biological functions, although in the future, the number of ST8Sia genes might decrease because some of them synthesize common structural epitopes. Although the overall sequence identity among sialyltransferases (SiaT) (ST3Gal, ST6Gal, and ST6GalNAc) is low, all SiaTs catalyzing the transfer of Sia to glycoconjugates using CMP-Sia as a donor substrate belong to

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the CAZy (carbohydrate-active enzymes) glycosyltransferase family.84,85 ST8Sias have type II transmembrane topology and a majority of them are localized in the Golgi apparatus.82,83 ST8Sias have a short cytoplasmic region connected to the transmembrane (TM) region, followed by an intraluminal region consisting of a stem region and a catalytic domain. In the catalytic domains of ST8Sias and other SiaTs, four conserved consensus sequences, namely the sialyl motif large (SM-L), SM-small (SM-S), motif III, and SM-very short (SM-VS) sequences, are observed82,83; these four motifs are important for the maintenance of the 3D structure for substrate binding and catalysis. SM-L sequences are characterized by a 55 amino acid region in the center of the enzyme that is known as a donor substrate (CMPSia) binding site.86 SM-S sequences are located at the C-terminal region of the enzyme and consist of 28 amino acids that are involved in binding with both donor and acceptor substrates.87 SM-VS (HXXXXEX) sequences are located at the C-terminal region of the enzyme; His (H) and Glu (E) residues are highly conserved between all SiaTs. This motif is reported to be involved in catalytic activity. Motif III ((Y/H)HYYD) is also reportedly involved in the catalytic activity of SM-S and SM-VS sequences.88 ST8Sias possess characteristic Cys (C) residues, of which one occurs at the COOH-terminal end, three occur in sialyl motif L, and one occurs in sialyl motif S. Disulfide bonds between these residues (Cys in SM-L and COOH-terminal domain, Cys in SM-L and SM-S) are important for enzymatic folding and activity.89 Based on the similarity of the gene structure and amino acid sequences, ST8Sias are categorized into two groups. One group is comprised of di/triSia-synthesizing enzymes ST8Sia1, ST8Sia5, and ST8Sia6. Another is the group comprised of oligo/polySia-synthesizing enzymes ST8Sia2, ST8Sia4, and ST8Sia3,84 although ST8Sia3 never synthesizes the polySia structure.90

4.2 Oligo/PolySia-Biosynthesizing Enzymes: ST8Sia2, ST8Sia4, and ST8Sia3 The elucidation of the biosynthetic pathway of polySia on NCAM was achieved by the identification and cloning of two types of α2,8-polySTs, i.e., ST8Sia2/STX/ST8SiaII/siat8b and ST8Sia4/PST/ST8SiaIV/siat8d from rodents and humans.91–94 The maximum DP of polySias occurring in nature is reported to be in the range of 8–400,95,96 although for in vitro synthesizing assays, the DP should be within the range of 40–60.39 The approximate DP value was 60 when the cooperative synthesis of ST8Sia2 and ST8Sia4 was demonstrated.

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The ST8Sia2 gene was first cloned from rodents97 and humans,94 and it could transfer Sia residues from CMP-Sia to Siaα2,3GalNAc and Siaα2, 6Gal/GalNAc on both N-linked and O-linked glycans in vitro, because it could synthesize polySias on the N-glycans of fetuin and α1 acids (tetraantennary N-glycans), human chorionic gonadotropin (CG) β-subunits (biantennary N-glycans), glycoproteins, or O-linked glycans in ovomucoid, but their activity toward these proteins was very low as compared with the natural substrate, NCAM (1400-fold).98 In addition, this enzyme could utilize oligosaccharides such as Siaα2 ! 3/6Galβ1 ! 4GlcNAcβ1 ! 6Manα1 ! 6Manβ1 ! octyl, mono/diSia-LacNAc99 or triNeu5Ac-DMB.100 It has been proven that ST8Sia2 does not require a second Sia on its monoSia chain (considered as an initiase activity) in vitro,39 although this has still not been observed in nature because biochemical evidence was reported using fish PSGP.101 The Lec 4 and Lec 13 mutant cells, which have defective GnT-V and GDP-fucose synthetase, respectively, were used to show that GlcNAcβ1,6Manα1,6R and α1,6-linked fucose are not required for polysialylation,102 although α1,6-linked fucose is reportedly involved in conferring stability to polySia-carrying glycoproteins.98 As a naturally occurring glycoprotein acceptor substrate, two N-linked glycans out of three in the Ig5 domain of NCAM (Fig. 4A) are shown to be the most preferred substrate of ST8Sia2.104 ST8Sia2 also synthesizes polySia on N-linked glycans of the

Fig. 4 Biosynthesis of oligo/polySia by ST8Sia2/ST8Sia4. (A) NCAM is the best substrate for ST8Sia2 and ST8Sia4. NCAM has five N-linked glycans and ST8Sia2/4 can synthesize polySia on the fifth and sixth N-linked glycans on Ig5. The Ig5 domain and the first FN type III domains are important for polysialylation. (B) Molecular modeling of ST8Sia2 using the crystal structure of ST8Sia3 (5BO6)103. SM-L: sialyl motif L (green), SM-S: sialyl motif S (red), SMIII: sialyl motif III (yellow), VS: sialyl motif VS (pink): These regions are important for enzymatic activity. PBR: polybasic region (blue), PSTD: polysialyltransferase domain (sky blue). These two areas are important for protein-specific polysialylation.

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Ig1 domain of synaptic cell-adhesion molecules (SynCAM1/CADM1) and ST8Sia2, which is called autopolysialylation.105 ST8Sia4 is the first enzyme that was cloned as a polysialyltransferase from chinse hamster cells.92 ST8Sia4 also synthesizes both N-linked and O-linked glycans and oligosaccharides having both α2,3- and α2,6-linked monoSia epitopes.39 Similar to ST8Sia2, ST8Sia4 also synthesizes polySia preferentially on NCAM. It prefers the sixth N-linked glycan on the Ig5 domain of NCAM over the fifth glycan.98 It also utilizes the O-linked glycan on PSGP as a substrate in fish.106 The PSGP family is composed of polySiacontaining glycoproteins that ubiquitously occur in Salmonidae fish eggs.107 PSGPs are also the major glycoprotein components of cortical alveoli, which are Golgi-derived secretory organelles found in the peripheral cytoplasm of mature eggs of almost all animal species, including humans. L-PSGP is a species-specific single, trideca-, or dodecapeptide, which has the structure (D)DAT*S*XAAT*GPSX (X ¼ E or A, Z ¼ D or S or G, *indicates the position of the O-linked polySia chain). Interestingly, ST8Sia2 derived from the trout ovary and embryo did not utilize monosialylated PSGP via the α2,6GalNAcT structure.106 ST8Sia4 also synthesized polySia that showed an activity toward O-linked glycans from NRP2.108 The ST8Sia3 gene was first cloned from rodents, and it synthesized the oligoSia epitope not only on glycoproteins but also on glycolipids.109 Interestingly, ST8Sia3 never synthesizes polySias because it can synthesize up to seven oligoSias.99 Recent glycan array data have suggested that ST8Sia3 prefers Neu5Acα2,3Gal(6SO3)β1,4GlcNAc-(keratan sulfate) terminal ST8Sia3.103 Among the ST8Sias, ST8Sia3 is the only enzyme for which the structure could be determined using X-ray crystallography. In addition to the commonly observed structure of ST8Sias, novel domains among polySTs have also been revealed. One of these is the polybasic polysialyltransferase domain (PSTD) (32 amino acids) that occurs prior to the SM-S sequence, and influences the polysialylation activity of ST8Sia2 and ST8SIA4 and a polybasic motif named the polybasic region (PBR) (pI 12) localized just before the SM-L in ST8Sia4 and ST8Sia2110 (Fig. 4B). These regions are equidistant from the region containing SM-L sequences. The PBR consists of 35 amino acids, of which seven are the basic amino acids Arg (R) and Lys (K), and this region is involved in NCAMspecific polysialylation via binding to the acidic patch of the first fibronectin type III domain.110 The second docking site in the Ig5 domain of ST8Sia4 has also been demonstrated. The PBR of ST8Sia4 also binds to the MeprinA5 protein-μ tyrosine phosphatase (MAM) domain to synthesize polySia on

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O-linked glycans in the adjacent linker region of NRP-2. ST8SIA2 and ST8SIA4 have six and five N-glycosylation sites, and autopolysialylation occurs on both polySTs on several N-linked glycans.111,112 Recently, it was revealed that this autopolysialylation enhanced substrate-specific polysialylation.108 Based on docking models of the Ig5-FN crystal and the ST8Sia4 structure prepared using the ST8Sia3 crystal as a template, PBR and PSTD form an extended basic groove, probably to enable substrate binding and product synthesis.

4.3 Di/TriSia-Synthesizing Enzymes: ST8Sia1, ST8Sia5, and ST8Sia6 The key enzymes responsible for the synthesis of diSia and triSia on glycolipids are ST8Sia1 and ST8Sia5, and these two enzymes have been investigated in detail.113 ST8Sia1 synthesizes GD3, and it prefers to use GM3 as a substrate. It could also synthesize GT3. On the other hand, ST8Sia5 synthesizes GD1c, GT1a, GT1b, and GT3. ST8Sia6 is different from the ST8Sia1 and ST8Sia5 enzymes because it exhibits a low activity toward glycolipids (however, ST8Sia6 could utilize GM1b, GD1a, and GD1b) and it mainly synthesizes the di/triSia structure on O-linked glycan chains of glycoproteins and sialyloligosaccharides,113 although the specific acceptor substrate remains unknown. To date, ST8Sia6 KO mice have not been developed. There is very little knowledge regarding the enzymes that are responsible for the synthesis of α2,8-linkages of Kdn,23,114 the α2,5Oglycolyl-linkage of Neu5Gc,23,115 and the α2,9-linkage of Neu5Ac on glycoproteins,116 although the enzymatic activity of the oligomerized Kdn has been measured in fish eggs.117 The stop signal for elongation is also unknown. The enzymes that catalyze sialyl residue modifications such as lactylation, sulfation, and methylation2,118 have not been isolated or cloned. Recently, O-acetyltransferase that was specific to sialic acid was cloned.119 However, the stop signal of polysialylation is well documented in PSGP polysialylation. After polymerization, polysialyltransferase transfers the Kdn from the CMP-Kdn donor to the nonreducing terminal end of polySia. In rainbow trout, this effectively stops polysialylation, as polysialyltransferase is unable to transfer Neu5Ac or Neu5Gc onto the Kdn residue.117 In mammals, polysialylation might be capped by O-acetylation, as indicated by the presence of O-acetylated Sia on chick NCAM.120 The α2,9-linked polySia derived from flagellasialin of sea urchin is also capped with sulfated Sia at the nonreducing terminal residue, indicating that sulfation might act as the stop signal for polysialylation in this organism.116

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5. PHENOTYPES OF PolySia-IMPAIRED ANIMALS To understand the function of polySia or polySTs at an animal level, techniques based on polySia probes or gene targeting have been used. Out of ST8Sia2, 3, 4, and 6, ST8Sia2- and ST8Sia4-gene-targeted mice were developed to understand the phenotypes of polyST-impaired mice. To understand the effects of polySia and NCAM on the structure and function of the brain, NCAM/ mice were developed.27 These mice were healthy and fertile. An almost total loss of polySia staining clearly indicates that NCAM is a major carrier protein for polySia in the brain (85%). In the brain, 10% of the overall brain size and 36% of the OB size were reduced because of the loss of the granule cell layer, which is usually composed of polySia-positive cells derived from the SVZ in the LV. The mutant mice can smell different odors. The cytoarchitecture of regions of the brain such as the cerebellum was normal. The behavioral phenotypes were deficits of special learning tested by the Morris water maze that was associated with hippocampus function, and exhibited different exploratory activities. In the HIP, polySia immunostaining of DG and MF disappeared. Later, NCAM/ mice showed some behavioral changes, locomotion, and social interactions.121 Cognitive functions of NCAM/ mice and conditional NCAM-deficient mice (forebrain specific), such as contextual fear conditioning and cued fear conditioning, are also impaired, especially under stress.122 Interestingly, the expression of the D2-receptor and sensitivity of dopamine are upregulated in cells derived from NCAM/ mice.123 It should be noted that NCAM is not the only substrate of polyST, as described earlier. Next, ST8Sia4/ mice were established.124 The amounts of polySia in the brain until P4 was almost the same as that in control mice. After 4–6 weeks, the amounts of polySia in the OB, medulla oblongata (MO), HIP, neocortex, and Hypo were decreased, although the polySia expression in nonneural tissues such as the kidney, heart, spleen, and thymus was the same. In 4-month-old brains, polySia expression was low in more than five brain regions, as seen using the anti-polySia antibody for detection; however, the results of immunostaining were different when an anti-NCAM antibody was used for detection. Anatomically, the size of the OB, precursor migration from SVZ, chain organization of migrating cells, and mossy fiber laminated organization were normal. However, the expression of polySia in the CA1 region of Ammon’s horn was drastically

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downregulated. The LTP and LTD in CA1 were impaired, but the LTP in CA3 was not impaired. ST8Sia4/ mice display a decreased motivation for social interactions.125 Next, ST8Sia2/ mice were developed and characterized.126 PolySia staining was decreased at the OB and cerebral cortex (CX), but remained almost unchanged in the HIP, cerebellum, and Hypo. In experiments related to the HIP, an abnormal deficit of polySia-expressing cells was observed in the DG (inner rim of the granular layer, from which newborn precursors from the subgranular layer first acquire the polySia stain). Interestingly, ST8Sia2/ mice exhibit the misguidance of infrapyramidal MF and the formation of ectopic synapses in the hippocampus CA3 region, although the LTP levels in CA1 and CA3 were normal. ST8Sia2/ mice exhibit a higher exploratory drive, similar to the NCAM-KO mice. ST8Sia2-KO mice reduce behavioral responses to fear, as measured by the cue test (AMG dependent) and context test (AMG and HIP dependent). In addition, ST8Sia2/ mice show impaired social interactions.125 Interestingly, the abnormal phenotypes are not totally compensated by either ST8Sia2/ or ST8Sia4/ mice, but many phenotypes of NCAM1/ mice were observed in either ST8Sia2- or ST8Sia4-KO mice. These results clearly indicate that each enzyme is able to synthesize the polySia chain via unexpected biosynthetic pathways and plays different roles in the brain. To remove polySia completely and to understand polySia-specific functions, ST8Sia2 and ST8Sia4 double KO mice were established.127,128 ST8Sia2//ST8Sia4/ mice are polySia-impaired mice that show severe phenotypes and die within 8 weeks. They are small and show a slow, weak, and uncoordinated movement. The major phenotypic characteristics that lead to postnatal death are the anatomical disorganization of the forebrain, smaller OB, CX, CB, thin cortex, reduced size of the internal capsule, large lateral, and third ventricles that lead to hydrocephalus, malformation of the anterior commissure, corticospinal tract, and mammillothalamic tract. The migration of cells from SVZ in lateral ventricles was inhibited, which lead to a reduced OB size. The chain tangential migration of olfactory interneurons in the RMS and that of GABAergic interneurons from the ganglionic eminence to the dorsal cerebral cortex were impaired. In addition, the number of GABAergic neurons was decreased. In case of pyramidal cells in layer II/III and IV of the neocortex, the number of cells stained was decreased and they were aberrantly localized, indicating the impairment of the cell number and radial migration of pyramidal cells. These lead to an impairment in the excitatory neural network system. Analysis of DNA

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fragmentation and BrdU experiments clearly showed that neural cells and precursor cells in the cortex and SVZ were undergoing apoptosis, and that polySia is not involved in the proliferation of BrdU-positive cells. On the other hand, in the HIP, except for the cells facing the lateral ventricles, cells did not appear to undergo apoptosis. All these data showed that polySia is necessary to form and maintain forebrain structures during mouse brain development. PolySia was shown to be involved not only in the migration but also in the differentiation of both neural precursor cells and glial precursor cells via inhibition of the onset of neural cell or glial cell development. Interestingly, a deficit of polySia leads to a decrease in the level of mRNA expression for Pax6, which is important for tangential and radial cell migration, and for the differentiation of neural precursors during cortex development, although the mechanism is still unknown. PolySia is also shown to be involved in the myelination, and the delamination of MF is a polySia-related function. Interestingly, in NCAM, ST8Sia2, and ST8Sia4 triple-KO mice, the severe phenotype of the DKO mice is rescued, suggesting that an uncontrolled type of NCAM-mediated cell adhesion was followed by increased signal transduction events.40,129 In NCAM//ST8Sia2//ST8Sia4/ mice, improved signaling via increased cell–cell interactions in the polySia-deficient brain is likely to result from the reduced levels of celladhesion molecules, resulting from the deficiency of NCAM1. Thus, the reduction of NCAM leads to the recovery of normal physiological interactions and to the rescue of the severe phenotype of polySiadeleted mice. The tissue- and stage-specific expression of ST8Sia3 has been reported, while diSia- and triSia-containing glycoproteins were present in various tissues.6,33 The phenotype of a morpholino-knockdown of ST8Sia3 in zebrafish appeared to lead to anomalous somite morphologies,130 indicating that di/oligosialylation is involved in the somite development in zebrafish. In mice, the injection of RNAi for ST8Sia3 into the ventricular brain cavity using 1-day-old mice leads to a reduction in ST8Sia3 mRNA expression in the cerebellum, and mice recovered from this effect during adulthood. However, mice showed a poor performance in the T-maze experiment at all times, indicating that the ST8Sia3 or a diSia epitope is associated with working and procedural memory in the cerebellum.130a Interestingly, ST8Sia3-KD mice showed an irregular spongiform in most parts of the brain, especially in the cerebellum. The phenotypes of ST8Sia3-KO mice are yet to be developed.

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6. BIOCHEMICAL FEATURES OF Di/Oligo/PolySia AND THEIR FUNCTIONS At an animal level, it is proven that an impairment in the polySia leads to impairments in neurological phenomena, such as the impaired LTP and LTD in the HIP, ectopic synapse formation in CA3, and several behaviors such as fearfulness and aggressiveness. Therefore, polySia is involved in normal brain development and functioning. To understand all the features of polySia, it is very important to conduct research in a pure system. In animal- or cell-based systems, many undetected molecule-based interactions occur. The final output obtained is a summation of actions of all molecules. Therefore, people tend to misunderstand that drastic or sustained phenomena are the only important points to be considered. We need to consider the timescale during polySia-impaired periods as well. However, the properties of polySia that are discovered are reliable if the experiments are carried out in a pure system. If a feature or an effect is not observed in other experimental systems, although it is observed in a pure system, it is because of the undetected interactions occurring in other systems.

6.1 Repulsive Field of PolySia PolySia on NCAM has an antiadhesive effect on molecules bound by homophilic (NCAM–NCAM) and heterophilic (NCAM–CAMs/extracellular matrix (ECM)) interactions. Therefore, polySia interferes in cell–cell adhesions by inhibiting NCAM–NCAM, NCAM–other CAM/ECM, CAM–CAM, and CAM–ECM binding.26 This feature is attributable to the hydration effects of the long, negatively charged linear polymer of Sias.131 The effect on the cell surface was measured by estimating the intercellular distance by electron microscopy before and after Endo-N treatment. Based on the experiment, there was a distance of 10–15 nm among cells owing to the presence of polySia.132 To uncover information about the steric effects of polySia on polySia–NCAM, direct measurement was performed. Based on light scattering studies, fluorescence correlation spectroscopy, and surface force apparatus-based measurement, it was demonstrated that in polySia–NCAM, polySia doubles the hydrodynamic radius of NCAM, and that the magnitude of repulsion also depended on the amount of polySia on the membrane.26 Therefore, a repulsive field is observed around polySia, which causes the distance between cell–cell and cell–ECM to be maintained (Fig. 5A).

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Fig. 5 Repulsive field of polysialic acid. (A) Repulsive field of polySia on polySia–NCAM. The large exclusion volume of polySia–NCAM creates repulsive fields on the cell surface to negatively regulate cell–cell/molecule interactions, as shown in gray shadow. Therefore, polySia changes a cell-adhesion molecule to an antiadhesion molecule and regulates the space between cells. PolySia shields both cis- and trans-areas of the cell. (B) Schematic drawings of the surfaces of immobilized sensor chips and measurement of repulsive field of polySia–NCAM by SPR. Purified polySia–NCAM–Fc (Continued)

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Recently, the repulsive feature of polySia on polySia–NCAM was measured using the surface plasmon resonance (SPR) instrument.133 The sensor chip was coated with a self-assembly membrane (SAM), and protein A was immobilized onto the surface. The polySia–NCAM–Fc, sialidase-treated asialo–NCAM, and endo-N-treated di/triSia–NCAM were immobilized via protein A–Fc interactions. After immobilization, polySia–NCAM, di/triSia–NCAM, and asialo–NCAM were used as analytes to measure interactions between ligands and analytes. The positive RU value indicates that binding occurs, and the negative RU value indicates that the repulsive interactions are occurring because of the decrease in the nonspecific binding to the molecule. Based on the results under this system, novel and interesting features of polySia–NCAM were revealed (Fig. 5B). One of these is that the repulsive field was only observed in polySia synthesized by ST8Sia2, but not by ST8Sia4. Second, polySia–NCAM binds to polySia–NCAM. Third, di/triSia–NCAM can efficiently bind to the di/triSia–NCAM. The fact that this system is a flow system and the conventional system is a static system should be taken into account. In the second and third points, the observation of the bundle formation oligo/polySia,134 which was achieved under air experimental conditions, might have occurred because of the use of SPR-proven feature.

6.2 Attractive Field of PolySia As described earlier, polySia is a linear homopolymer of Sia with several negatively charged carboxyl groups that contribute not only to its polyanionic feature but also to the extremely large hydration effect. These physical properties provide the polySia with a large exclusion volume and inhibit interactions mediated by polySia-containing molecules around their area for localization. Therefore, polySias have been considered only to function as antiadhesive molecules, i.e., polySias, act as slippery nonreceptor Fig. 5—cont’d synthesized by ST8Sias is injected into the system to allow immobilization onto the proteinA-immobilized Au surface. NCAM–Fc is used as a negative control. The analytes such as polySia–NCAM–Fc derived from a variety of polySTs, oligoSia–NCAM–Fc, and asialo–NCAM–Fc can be used to understand the homophilic interactions. The sensor gram of RU values shows the weight of the binding molecules toward a sensor chip; therefore, subtraction of the sensor gram obtained using the NCAM–Fc surface as a negative control is required. Zero or minus RU values show no interaction or repulsive contribution of the polySia on polySia–NCAM, respectively, because the NCAM itself can bind to significant amount of analytes.

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molecules.26 It has long been thought that the only unique function of polySia is to impart a repulsive field for the trans-ligands or trans-cell interactions. Therefore, if polySia expression is observed in the context of many biological activities, it is considered that the polySia functions as an antiadhesive molecule. However, in 2008, polySia was clearly proven to specifically bind to a relatively smaller and neurologically/biologically active molecule (100 Da to 40 kDa).135 Since then, polySias have been demonstrated to bind to various molecules and display an attractive field around polySia-expressing regions (Fig. 6A)3,34,136,137 based on the SPR measurements (Fig. 6B). In addition, since then, the paradigm of functioning of polySia has changed; therefore, based on a biochemical perspective, the molecular mechanism of action of polySia in polySia-specific biological functions such as cell migration, neurogenesis, axon guidance, fasciculation, cell proliferation, learning and memory, social interaction, and diseases needs to be considered. 6.2.1 Neurotrophic Factors Neurotrophic factors (NTs) are factors that influence the neural cell survival, development, and functions via binding to the specific receptors in the central nervous system (CNS) and peripheral nervous system (PNS). NTs are classified into three groups based on their structural and functional properties as follows: the neurotrophin family, transforming growth factor (TGF) family (glial cell line-derived neurotrophic factor (GDNF) family), and ciliary neurotrophic factor (CNTF) family.138 Among these, the relationship between the neurotrophin family and polySia was well studied. The neurotrophin family consists of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin (NT)-3, and NT4, which are involved in various brain functions, including neural cell survival, differentiation, axon targeting, synapse maturation, and plasticity by binding to the specific receptors, tropomyosin-related kinase (Trk) receptor, and pan neurotrophin receptor of the tumor necrosis factor receptor family p75NTR. BDNF is enriched in adult brains and was first isolated from pig brains. It was biochemically shown to have a survival-promoting activity in dorsal root ganglion cells.139 BDNF is synthesized as a precursor protein, preproBDNF, which consists of 247 amino acid (aa) residues. Pre-proBDNF is proteolytically cleaved to proBDNF (229 aa, 32 kDa). ProBDNF is further

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Fig. 6 Attractive field of polysialic acid. (A) Attractive field of polySia on polySia–NCAM. PolySia on NCAM directly binds to bioactive molecules involved in neural functions, such as neurotrophins (BDNF and proBDNF), neurotransmitters (DA), and growth factors (FGF2), and thereby regulates their extracellular concentrations and signaling modes. (B) Schematic drawings of the surfaces of immobilized sensor chips and measurement of attractive field of polySia–NCAM by SPR. Purified polySia–NCAM–Fc synthesized by ST8Sias is injected into the system to allow immobilization onto the proteinAimmobilized Au surface. OligoSia–NCAM–Fc prepared using Endo-N (negative control 1), asialo–NCAM–Fc prepared using exo-sialidase (negative control 2) and NCAM–Fcderived Mock cells (negative control 3) can be used as the negative control. When the sensor gram of negative control 1 is subtracted from that of polySia–NCAM–Fcimmobilized chip, the contribution is considered to be oligo/polySia (DP  4). When the sensor gram of negative control 2 is subtracted from that of polySia–NCAM–Fcimmobilized chip, the contribution is considered to be oligo/polySia (DP  1). When the sensor gram of negative control 3 is subtracted from that of polySia–NCAM–Fcimmobilized chip, the contribution is considered to be glycan chains that changed by transfection with ST8Sia genes.

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processed in secretory vesicles by proteases such as furin in many tissue types, and seven-membered proprotein convertase (PC7) in the hippocampus or amygdala of the brain.140–142 The enzymes generate mature BDNF (mBDNF) (119 aa, 13.5 kDa) and prodomain (proD) (110 aa, 18 kDa) sequences. The aa sequence of mBDNF is conserved among mammalian animals and the C-terminal end contains basic amino acids.143 PreproBDNF has a highly conserved N-terminal signal peptide; it is cleaved in the endoplasmic reticulum (ER). The resulting proBDNF forms noncovalent dimers that are transferred to the Golgi apparatus via intermediate nonclathrin-coated transport vesicles. In the Golgi apparatus, proBDNF is further cleaved by enzymes localized in the trans-Golgi network (TGN) by furin or PC7, generating mBDNF, which typically exists as a stable noncovalent dimer. While proBDNF is processed inside the cells, it is secreted in an activity-dependent manner.144 ProBDNF functions as a p75NTR ligand. Recently, it has been found that proBDNF was extracellularly cleaved by the tissue plasminogen activator (tPA)/plasmin system145 or metalloproteinases (MMP)146,147 to yield functional mBDNF, which is involved in memory-related functions. The first important indication between polySia and mBDNF was biochemically confirmed using several methods, such as horizontal nativePAGE, gel filtration,135 and SPR-based measurement.148 The evidence obtained from these analyses was used for the first time to demonstrate that BDNF dimers bind to polySia directly. The minimum DP required for BDNF binding is 12,135 and this is the first demonstration of the biological significance of DP in polySia, although the concept had been reported previously.149 Based on the gel filtration analysis results, it was inferred that mBDNF and polySia (colominic acid, DPav 43) formed a large complex with a weight of approximately 2500 kDa. Stoichiometrical analyses showed that 14 mol of mBDNF dimers and 28 mol of polySia chains were present in the complex.135 The same affinity is also observed in interactions between BDNF and glycosaminoglycans (GAGs), other acidic glycoconjugates localized in brains, and other tissues such as hyaluronic acid (HA, (GlcAβ1, 3GlclNAcβ1,4)n), chondroitin (CH, (GlcAβ1,3GalNAcβ1,4)n), heparan sulfate (HS), chondroitin sulfate (C4/6S, (GlcAβ1,3GalNAc4/6Sβ1,4)n), keratan sulfate (KS, (GlclNAc6Sβ1,4Galβ1,3)n), and dermatan sulfate (DS, (IdoUAβ1, 3GalNAc4Sβ1,4)n).150 This study is the first demonstration of the comprehensive features of GAGs and polySias specific to neurotrophins.150 Based on the horizontal native-PAGE results, it was revealed that HS, CS, KS, and DS bind to BDNF, but not to HA and CH,150 and that BDNF

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(pI 10.5) binds to polySia but not to trypsin (pI 10.5).135 This indicates that BDNF–polySia binding is specific and does not occur because the protein is a basic protein or an acidic polymer, but because it has a specific array of negative charges. Though BDNF can bind to both polySia and GAGs such as HS and CS, the binding process is completely different with regard to several aspects. First, the binding sites of polySia and HS on the BDNF molecule are different. When glycan polymers, polySia, and HS are immobilized via reducing terminal end of biotin to the streptavidin sensor chip of an SPR-based instrument and BDNF is used as an analyte, the KD values for the glycan polymers are almost similar, i.e., 6.4  109 M (polySia–BDNF) and 1.4  109 M (HS–BDNF). However, when BDNF is immobilized onto the sensor chip (CM5) and the glycan polymers are used as analytes, the KD value of BDNF toward HS is almost the same as 1.5  109 M; however, the KD value toward polySia increases by two or three orders of magnitude (9.1  106 M).148 These results suggest that the polySia binding site on BDNF is preferentially utilized for immobilization on the sensor chip, while the HS binding sites are left unbound during immobilization. The second fact is that the complex size is different. Based on gel filtration, the size of the HS–BDNF complex (670 kDa) is lower than that of the polySia–BDNF complex (2400 kDa), and BDNF and polySia do not form a ternary complex with TrkB and p75NTR. BDNF in the BDNF–polySia complex is shown to migrate easily toward receptors, probably owing to the difference in KD values among molecules.135 The KD of BDNF toward polySia is confirmed to be approximately 109 M by using SPR.151 In contrast, the KDs of BDNF toward TrkB and p75NTR are 1012 and 1010 M, respectively (Fig. 7A). Therefore, polySia serves as a reservoir for BDNF molecules, for enabling the efficient supply of required molecules to p75NTR and TrkB, which are nearing the polySia–NCAM complex. Recently, it was observed that polySia binds not only to mBDNF but also to proBDNF (KD ¼ 1.3  109 M). However, it does not bind to the proD at all, and proBDNF in the complex with polySia but not in the complex with HS is inhibited by the conversion of proBDNF to mBDNF by plasmin,154 strongly suggesting that the reservoir or scaffold function of polySia is involved in the regulation of BDNF/proBDNF activities in the hippocampal cell. Interestingly, proBDNF can bind to oligoSia– NCAM with the same affinity that it shows toward polySia–NCAM. Notably, mice that cannot convert proBDNF to BDNF effectively showed “depression” phenotypes, and a low concentration of BDNF was observed in the hippocampus as a result.155

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Fig. 7 The binding between polySia on polySia–oligoSia and BDNF. (A) Features of polySia on polySia–NCAM as a BDNF reservoir. PolySia on NCAM can directly capture BDNF with the KD value 6.4  109 M. The polySia can easily pass the BDNF in polySia chains toward trkB and p75NTR, probably due to the higher affinities of BDNF toward receptors. PolySia also captures proBDNF (KD ¼ 1.3  109 M) and the proBDNF complexed with polySia cannot be cleaved with plasmin protease as compared with that with HS. (B) Sialidase-related releasing mechanism of polySia–BDNF in polySia can be released by the sialidase (Neu1) localized on the exosomes that are secreted from microglia cells after inflammatory stimulation. (C) Features of polySia on polySia–NCAM as a FGF2 reservoir. PolySia on NCAM can directly capture FGF2 with the KD value 8.8  1010 M. The polySia never passes the FGF2 in polySia chains toward FGFR, while it can easily pass FGF2 toward HS (the migration of FGF2 in HS chain cannot migrate to polySia chains). After passing FGF2 to the HS, HS on HSPG forms an FGF2/HS complex, which then binds to FGFR to form a functional ternary complex. *, Ref. 152. **, Ref. 153.

Recently, another striking mechanism of release of BDNF from the microglia cells was demonstrated.156 The microglial cell line contains polySia that disappears rapidly in response to inflammatory stimulation with lipopolysaccharides (LPSs). Furthermore, the rapid clearance of the surface polySia of microglia cells is shown to accompany the release of BDNF within the culture supernatant because of the action of mammalian sialidase, Neu1, on the exosome (Fig. 7B).156 These results suggest that microglia cells release prereserved BDNF immediately after LPS stimulation in order to supply it to the surrounding cells. The sialidase-mediated degradation of

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polySia and concomitant release of trophic factors after 15 min might occur when the surrounding cells need the trophic factors to protect them from the toxic effect of LPS-activated microglia cells. The BDNF mRNA has been detected all over the tissues throughout the CNS (OB, CX, HIP, AMG, THA, Hypo, and spinal cord) and in other nonneuronal tissues (salivary gland, skeletal muscle, spleen, vascular endothelial cells, aorta, kidney, ovary, heart, lung, retina, and immune cells).138 BDNF proteins have been also detected throughout the brain using immunohistochemical approaches, using the anti-BDNF antibody; the HIP and CX are particularly enriched in BDNF. The HIP is a critical region of the brain, responsible for learning and memory, for which BDNF plays a critical role. In the HIP, CA1 pyramidal cells have been shown to contain BDNF mRNA157,158; however, there are conflicting reports regarding the detection of BDNF proteins in the HIP.159 There might have been differences in the conditions used for the immunohistochemical analyses, particularly during the fixation and permeabilization steps,160 and in the cell states, as BDNF expression is dependent on several factors, including cellular electrical activity, environmental conditions, stress, and circadian rhythm.161 In addition, because BDNF binds to extracellular glycans such as polySia3,135,136 and other GAG chains, which are frequently remodeled in response to the cellular state,156 the staining patterns with anti-BDNF antibodies are potentially altered, based on the binding of glycans. The underlying molecular mechanisms of several polySia-related functions remain unknown, for example, learning and memory through LTP regulation in CA1 (observed using NCAM-KO and ST8Sia4-KO mice), progenitor cell migration from SVZ to OB (observed using NCAM-KO, ST8Sia2-KO, and ST8Sia4-KO mice), mossy fiber formation in HIP (observed using NCAM-KO and ST8Sia2-KO mice), and behavioral abnormalities (observed using NCAM-KO, ST8Sia2-KO, and ST8Sia4KO mice). These phenomena are influenced by neurologically active molecules such as BDNF. An interesting example is the process of regulation of LTP in CA1. In 2000, BDNF was shown to rescue the impaired LTP in the CA1 region of the HIP of NCAM-KO mice,162 and it was also shown to inhibit the LTP in the CA1 of wild-type mice after the addition of colominic acid or polySia–NCAM, although the mechanism of action remained unknown. It was proved by biochemical analyses135 that direct binding between BDNF and polySia can be involved in neurological functions related to memory and learning. In addition, the time-course of stimulated BDNF release is important for understanding the neurological

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functions of BDNF, as seen with LTP in the HIP during memory consolidation. The analyses of the HIP, cortical, and dorsal horn slices clearly showed that the activity-dependent release of BDNF occurred 5–20 min after stimulation.163 BDNF release is regulated by intravesicular pH, with the neutralization of the normally acidic conditions in vesicles, thereby increasing the rate of release.164,165 In the case of microglia, peaks in the amount of BDNF release were observed 5 and 60 min after stimulation.166 The second release of BDNF is associated with increased BDNF protein expression. This second release was also regulated by the concentration of Ca2+ outside the cell and is considered to be important for the consolidation of BDNF-induced phenomena, such as fear learning in the amygdala.167 Interestingly, the first unknown peak of the BDNF from microglia might have resulted from the Neu1-related release from microglia, as described earlier.156 In case of RMS in OB, polySia greatly influences migration; BDNF is also shown to be related to RMS migration,168 indicating that the direct polySia–BDNF interaction135 is also involved in this migration system. An in vitro study confirmed that polySia strongly binds to BDNF, especially if BDNF is present in the brain; the functions of polySia need to be reexamined, not because of their repulsive field but because of their attractive field. Interestingly, the impairment of the molecule binding properties of the polySia structure biosynthesized by ST8Sia2 from a schizophrenia patient has been demonstrated137 and discussed later.

6.2.2 Growth Factors Growth factors are protein molecules that cause the proliferation of specific cells. FGF is a family of growth factors, and FGF2 is a member of the FGF family that stimulates the growth of various cell types, ranging from fibroblast to tumor cells.169 FGF2 is expressed in the brain during the early stages of development and is involved in brain formation, i.e., FGF2 promotes the cell growth and survival of fetal and postnatal cells from various regions of the brain, including the HIP,170 the entorhinal, frontal, parietal, and occipital cortex,171 and the striatum and thalamus.172 In addition, FGF2 is essential for the differentiation of embryonic hippocampal cells173 and for the acceleration of axonal branching.174 FGF2 is also shown to be a potent modulator of the proliferation and differentiation of multipotent neural progenitor cells isolated from the adult SVZ.175 Thus, apart from BDNF, FGF2 also plays a pivotal role in adult neurogenesis. Interestingly, FGF2 is related to fear memory, social interaction,176 and psychiatric

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disorders.177–182 These biological functions of FGF2 are mediated via their binding to FGF receptors (FGFRs). Through FGF2–FGFR signaling, it has been analyzed that the HS present in the HS–proteoglycan (HSPG) complex could lead to conformational changes in FGF2, and that the formation of the HS–FGF2–FGFR ternary complex enhanced the proliferative signals in the cells. Therefore, FGF2-driven cell proliferation via HS has been well characterized. However, there has been no report regarding the involvement of polySia in FGF2 signaling until recently. In 2012, it was clearly demonstrated that FGF2 physically binds to polySia to regulate the FGF signaling, using biochemical and biophysical methods.148 In addition, polySia influences the cell growth, but not cell survival via FGF2 signaling. It is interesting to note that localization and deficient phenotypes of FGF2 are well overlapped with those of polySia–NCAM.176 It was shown in several biochemical studies that FGF2 monomers directly bind to polySia with the minimum DP required for binding, i.e., 17.148 In the case of HS, which is a much shorter chain, the minimum DP is approximately 6.183 Therefore, it is suggested that an unusually long polySia chain is required for normal FGF2 binding. Based on the elution positions observed during gel filtration, it is determined that the size of the complex formed by FGF2 and polySia (colominic acid, DPav 43) is around 5000 kDa, while the size of the complex formed by HS and FGF2 is 440 kDa. SPR-based measurements showed that the KD of FGF2 for polySia (1.5  108 M) is smaller than that for HS (2.8  108 M), and that FGF2 does not migrate toward FGFR from the complex containing polySia and FGF2 but migrates to HS (Fig. 7C). The latter phenomenon was confirmed by gel filtration and SPR analyses. FGF2 does not migrate when complexed with polySia to FGFR, even if the FGFRs are closely located next to the complex. The affinity of polySia toward FGF2 is greater than that of HS. In addition, the SPR experiment clearly showed that the FGF2–polySia complex binds to HS- or polySia-coated surfaces, whereas the HS–FGF2 complex cannot bind to either of these surfaces, indicating that the regions for the binding of FGF2 to polySia and HS are different from each other. Notably, FGF2–polySia binding largely depends on whether the conformation of FGF2 is correct, because FGF2 that is incubated for a longer duration no longer binds to polySia; however, even FGF2 that has been incubated for a longer duration can bind to HS, indicating that polySia determines the lifetime of FGF2, which is a property that is useful for the reservoir. It is thus suggested that FGF2 is captured by polySia and then transferred from polySia to HS, followed by the formation of the ternary complex with FGF2–HS and

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FGFR. Confirmation of the effect of polySia on FGF2-driven cell survival and cell proliferation in HS-dependent cell signaling was demonstrated using polySia-expressing NIH-3T3 fibroblast cells, by the transfection of the ST8Sia2 or ST8Sia4 gene.148 Although not involved in the survival of NIH-3T3 cells, polySia was involved in FGF2-dependent cell growth. To confirm the involvement of HS, HS was downregulated using the siRNA technique for reduction an HS synthetic enzyme, EXT1. Using four cell lines, Mock (HS+/polySia), HS–KD/apolySia (HS-less/polySia), HS–KD/polySia (HS-less/polySia+), HS/polySia (HS+/polySia+), and FGF2/HS-dependent cell proliferation was strongly inhibited in the presence of polySia at the cell surface. Although polySia-negative cells showed a sustained signal of p42/44 (Erk) phosphorylation, the polySia-positive cells showed transient and strong peak signals for ST8Sia2- and ST8Sia4transfected cells. These results showed that polySia affects the FGFdependent Erk signaling pathway. This unique feature may be explained by a low-path filter effect of signaling, in which a sustained signal enhances the downstream signal, while a transient one reduces the downstream signal.184 The transient signal, probably produced by a combination of polySia and HS, downregulates the proliferation. It should be noted that the dissociation constants of the FGL peptide and several Ig domains of NCAM specific toward FGFR are reported to be approximately 1–10 μM.185 The NCAM–FGFR association would greatly affect the FGF/FGFR signaling after NCAM polysialylation. The regulation of cell proliferation through NCAM–FGFR interactions was also demonstrated using NIH-3T3 cells.186 However, these studies have focused on nonpolysialylated NCAMs, and the possibility that the posttranslational modification of NCAM with polySia or other moieties is directly involved in FGF2 signaling has not been taken into account. It is important to consider the microenvironment in which polySia– NCAM, HS, and FGFR are expressed on the cell surface, because neural stem cells that cause proliferation in OB and HIP have polySia on their cell surface. Furthermore, it was shown that the KD between polySia and FGF2 is 1.5  108 M, while that between polySia–NCAM and FGF2 is 8.8  1010 M; the dissociation rate of FGF from polySia or polySia– NCAM was completely different. It strongly indicates that the polySia moiety in polySia–NCAM shows a much higher affinity to FGF2 than free polySia. It was thus observed that the inhibitory effect of polySia–NCAM on FGF2/HS-dependent cell signaling was much larger than that expected from polySia and HS, which had similar KD values. It is important to understand the FGF signaling comprehensively; the signaling in

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nonpolySia–NCAM (homophilic and heterophilic interaction in cis and trans modes), polySia–NCAM (antiadhesive and FGF2 binding properties), FGFR (NCAM, HS, and FGF2 binding properties), and FGF2 (polySia, FGF2, FGFR binding properties) should be carefully considered. The impairment in the binding of the polySia structure biosynthesized by ST8Sia2 in a schizophrenia patient is discussed later. 6.2.3 Neurotransmitters and Ions It is somehow difficult to understand the interaction between polySia and small molecules such as neurotransmitters; the interaction was first measured using the frontal affinity chromatography (FAC) analyses.187 Of numerous small molecules in neural systems, including histamine, acetylcholine, serotonin, catecholamines (dopamine, epinephrine, and norepinephrine), and their precursors, polySia binds directly to catecholamines with high affinity. No binding is observed with the diSia immobilized column (DP 2), indicating that the binding does not result because of ionic interactions. The KD of dopamine for polySia is 105(M) and KD changes sensitively, depending on the extracellular pH of the solution, indicating that specific interactions between these molecules might be fine-tuned through conformational changes induced by subtle changes in the extracellular pH.151 The binding mode remains unknown; however, these interactions might occur between specific structures of polySia and the catechol backbone, because it does not bind to positively charged molecules such as histamine and did not bind to the diSia structure. Using human neural cells, polySia was shown to be involved in Akt signaling via the dopamine receptor D2 (DRD2) and dopamine.187 In animals, it is known that polySia is present in dopaminergic neurons such as mesencephalic dopaminergic cells, and that polySia–NCAM is involved in target-induced morphological differentiation of arcuate dopaminergic neurons.188 It is also reported that polySia is required for DRD2mediated plasticity involving inhibitory circuits of the rat medial prefrontal cortex.189 Thus, it is evident that a strong relationship exists between polySia–NCAM and dopamine/DR. Actually, profiles of NCAM and polyST match those for dopaminergic marker gene expression in mouse.190 On the other hand, polySia is essential for the development of the midbrain dopamine system.190 Rather, polySia appears to regulate the normal functioning of various dopaminergic cells, and the impaired or unusual expression of polySia causes diseases. For example, a large number of polySia-positive cells are detected in the SNA, in which dopaminergic neurons are enriched in some patients suffering from Parkinson’s disease.191 In this case, highly

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expressed polySia–NCAM might trap dopamine and reduce the concentration of dopamine that can effectively bind to its receptor. To understand the regulation of dopamine signaling in the dopaminergic and/or other catecholamine-related neurons and other cell types, it is important to consider the presence and absence of polySia on the cells, because of the polySia molecules function as a reservoir for these small molecules. This is interesting because polySia is found in the heart, which is rich in catecholamines. Ca2+ ions are one of the most important ions for the regulation of neural and biological activities outside and inside the cells. Sia and polySia are both able to bind to Ca2+. At first, the binding between Neu5Ac and Ca2+ was demonstrated by 1H and 13C NMR spectroscopy.192 The affinity of Neu5Ac for Ca2+ was estimated to be 1.21  102 M1 and the stoichiometry was 1:1. It was also demonstrated that the glycerol side chain of Neu5Ac is deeply involved in the binding of Ca2+. Colominic acid (DPav 24 and 15) and oligoSia (DP 5) were shown to be able to bind to Ca2+ with an affinity of 1.39  104, 1.49  104, and 0.065  104 M1 with n (number of binding site) ¼ 0.3, 0.3, and 0.2, respectively, as estimated by equilibrium dialysis.193 Binding to Ca2+ was inhibited by the same amount of Mn2+ but not by Na+, although in the presence of 0.11 M NaCl (physiological conditions), binding to Ca2+ was not observed. It is noteworthy that a neurotransmitter binding to polySia was observed under physiological conditions. 6.2.4 Cytokines The affinity of binding of polySia to CCL21 was demonstrated by ELISA.194,195 However, the monoSia epitopes also have the ability to bind to CCL21. Notably, the affinity for binding of polySia to CCL21 was greatly lower than that of HS, based on the SPR experiments (S. Ono et al., unpublished results). Although the NRP-2-mediated chemotaxis of mature dendritic cells was driven by the C-terminal region of CCL21 through CCR7,194,195 the contribution of HS or other GAGs should also be considered, as described in the case of FGF2 and polySia. CCR7 was polysialylated in the immune system.196 6.2.5 Transcription Factors The binding of polySia to transcription factors was first shown by the use of a polySia-mimicking scFv antibody; this was based on the anti-idiotypic approach, and a direct binding was examined by ELISA, the results of which revealed that only the histone H1 specifically binds to polySia.197 Histone H1 is a nuclear protein and binds a linker DNA between nucleosomes

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mediating DNA packing, although in some cases, H1 is observed extracellularly near the cell surface of several types of cells.198 Extracellular histone is also present as a component of the neutrophil extracellular trap (NET) produced by neutrophils, and polySia was shown to be involved in NET-mediated cytotoxicity.199 Recently, polySia–NCAM was shown to be present inside the nucleus and was involved in the circadian rhythm regulation.200 The polySia–NCAM that was present extracellularly and intracellularly for regulating molecules via an attractive field would expand.

6.3 Regulatory Role for Receptors 6.3.1 Ion and Ion Channel PolySia was first shown to present in voltage-sensitive-Na+ channels in the electric eel (Electrophorus electricus)201 and in the α-subunit of Na+ channels in the adult rat brain.202 Although the function of polySia on Na+ channels is still unknown, the polyanionic nature of polySia may be involved in the retention of Na+ as a cation near the Na+ channel for the efficient provision of ions. PolySia was present in the ion channel and was able to bind to Ca2+.193 Although this property was not observed under physiological conditions, it has been considered that polySias play important roles in the regulation of channels involved in learning and memory. The direct relationship between polySia–NCAM and memory using in vitro electrophysiological methods was shown, and polySia was shown to directly modulate the activity of AMPA-R in immature pyramidal neurons isolated from the CA1 region of the hippocampus203 that is closely related to the impairment of LTP in CA1 using NCAM- and ST8Sia4-KO mice, as described earlier. Specifically, polySia prolongs the open channel time of AMPA-R-mediated currents and alters the bursting pattern of the receptor channels, but does not modify AMPA-R single-channel conductance.203 Although no direct evidence has been provided yet, polySia might directly interact with AMPA-R to regulate the AMPA-R level. Later, it was demonstrated that treatment with polySia alone or polySia–NCAM inhibits the activation of GluN2B-containing NMDA-Rs at low micromolar concentrations of glutamate,204 indicating that polySia regulates GluN2Bcontaining NMDA-Rs. 6.3.2 Siglecs The most familiar Sia-recognizing molecules that are present in vertebrate cells consist of a family of lectins, known as Sia binding immunoglobulinlike lectins (Siglecs). 205 Siglecs-1 to -16 are mainly present in blood and

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neuronal cells. Siglecs-1, -5, -7, -10, -11, and -16 are reported to have affinity toward α2,8-linkages.206 In particular, Siglecs-7, -11, and -16 bind to diSia and oligoSia with a high affinity.207–209 GD3 or the epitope synthesized by Campylobacter jejuni are shown to be a ligand for Siglec-7; however, a majority of natural ligands remain unknown. In the brain, Siglecs-11 and -16 were shown to present in microglia. The V-set domain that is related to sialic acid binding is the same between Siglecs-11 and -16. In mouse and human coculture systems, polySia (DPav 20) was shown to bind to Siglec-11 and inhibit immune responses such as the reduction of inflammatory neurotoxicity of phagocytosis.210 Using humanized Siglec-11 transgenic mice, a laser-damaged eye was rescued by the addition of polySia (DPav 20), because of the vascular leakage induced by laser coagulation. In addition, polySia prevented the deposition of the membrane attack complex in Siglec-11transgenic and wild-type animals. This system is closely related to agerelated macular degeneration (AMD), which is a major cause of blindness in elderly individuals.211 6.3.3 Other Molecules Oligo/polysialyltransferases, exo/endo-sialidases, and anti-di/oligo/polySia antibodies described earlier are considered to be molecules that bind to the di/oligo/polySia epitope. In addition, it is well known that several bacteria and viruses contain hemagglutinin or receptors that are capable of binding to Sia residues on host cells. Some of these hemagglutinins, such as those of the Sendai virus, specifically bind to α2,8-linkages.212

7. RELATED DISEASES PolySia is associated with several diseases, including mental disorder and cancer. Because the occurrence of polySias is restricted to the embryonic brain and other organs, the plastic area of adult brain, and cancers, it is named as an oncodevelopmental antigen.213 Therefore, the abnormal expression of polySia in these areas and expression of polySia in new (usually previously unidentified areas) areas lead to the diseases. The major carrier protein of polySia is NCAM; therefore, almost all studies focus on polySia–NCAM or the biosynthetic enzymes ST8Sia2 or ST8Sia4.

7.1 Mental Disorders and Neurodegenerative Diseases Schizophrenia (SZ) is a mental disorder that affects 1% of the world’s population, with multiple factors contributing to pathogenesis. Recently, it is

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considered that the disease is caused by both genetic and environmental factors. Based on the genome-related studies, dysbindin (DTNBP1), which forms a part of the protein complex related to lysosome-related organelles complex 1; dopaminergic genes such as Akt-1, catechol-O-methyltransferase (COMT), and DA receptor D2 (DRD2); disrupted in schizophrenia 1 (DISC1) gene, which was first identified in Scottish families and is considered to encode a scaffold protein for nuclear distribution element-like 1 (NEDL1) and 14-3-3; neuregulin (NRG); GAD1, which encodes glutamic acid decarboxylase (GAD67), a key enzyme involved in the synthesis of gamma-amino butyric acid (GABA); and BDNF were identified as related genes but none of them was shown to be a causative gene.137 Since 1996, some reports have suggested that the polySia or ST8Sia2 gene is associated with SZ. For example, the number of polySia–NCAM immunostained cells derived from the HIP of SZ brains is decreased as compared with that of normal brains,214 and this phenomenon was observed in Ncam1- and ST8Sia2/4-KO mice. Lowered polySia–NCAM expression was shown in layers IV and V of the dorsolateral PFC of SZ brains215; however, no such difference was observed in AMG,216 indicating that regionspecific polySia impairment was the main feature of the SZ-brain. As for the genome, it was first shown that chromosome 15q26, which is the genomic region where the gene encoding ST8Sia2 is localized, was related to SZ and bipolar disorders (BD) in the Eastern Quebec population.217 Then, it was shown from genome-wide studies of Japanese218 and Chinese–Han219 populations that a relationship exists between the single-nucleotide polymorphisms (SNPs) in the promoter region of the ST8Sia2 gene and SZ. Biochemical studies using SNP-7 (Glu141Lys) in the coding region of ST8Sia2 obtained from an SZ patient have clearly demonstrated that the in vitro and in vivo enzymatic activity of ST8Sia2 with SNP-7 drastically decreases, and that the polySia products are also impaired with respect to quantity (amounts) and quality (DP and negative charge of polySia– NCAM).133,154,187,220 In addition, polySia functions not only in repulsive fields (antiadhesive properties) but also in attractive fields (molecule binding properties); both functions were completely impaired in SZ patients.133 Interestingly, SNP-9 was first reported from Japanese populations,218 and although the levels were insignificant, they were shown to be significant among Spanish male SZ populations221; it was a silent SNP that leads Pro to Pro. Surprisingly, this SNP also leads to the impairment of the quality and quantity of polySia, probably because of the change (slow) in the ST8Sia2 translation (shorter active period) process, based on the codon usage

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ratio, because the ratio was changed from the highest to lowest. Considering that polySia functions as a regulator of biologically active molecules such as BDNF, FGF2, and dopamine, which are intimately involved in neural and brain functions,135 polySia–NCAM synthesized by the mutated ST8Sia2 gene likely plays a role in causing SZ.187,220 Based on the effects of the rSNP reported from Japanese populations,218 the functions of polySia such as embryonic cell migration and cell proliferation of adult neural cells were also impaired, probably because of the change in the promoter activity, in a celltype specific manner (M. Hane et al., unpublished results). Anatomically, the volume of OB derived from SZ brains is reduced,222 leading to a phenotype that is similar to that of NCAM-KO mice.27 In rodents, this phenotype is believed to result from a requirement for polySia during the migration of neural precursors from the SVZ through the RMS to the OB, and this polySia structure was compensated for by ST8Sia2 or ST8Sia4. In addition, the functional impairment and disturbed organization of the HIP are also involved in the etiology of SZ.223 In this regard, it is interesting that the loss of ST8Sia2, NCAM, or Endo-N injection results in the misguidance of infrapyramidal mossy fibers and formation of ectopic synapses in the HIP.126,224 The impairment of learning and memory is closely involved in the impairment of LTP in the HIP, and cognitive deficits are commonly observed in SZ patients. These deficits are suggested to be associated with impaired LTP.225 The impairment of LTP at CA1 in the HIP observed in NCAM-KO162,226 or ST8Sia4-KO mice124 is considered to be because of the polySia-dependent function of NCAM. Although LTP impairment in CA3 was not observed in ST8Sia2 and ST8Sia4, it was observed in NCAM-KO mice. Interestingly, the spatial learning,27 circadian rhythms,227,228 and social interactions125 of SZ patients were frequently observed to be disturbed, which is a characteristic of polySia-impaired mice. Recently, ST8Sia2-KO mice125,229 were shown to be able to be used as SZ-model mice, because these mice showed an impaired working memory, deficits in prepulse inhibition, anhedonic behavior, and increased sensitivity to amphetamine-induced hyperlocomotion. NCAM-KO mice were also demonstrated to be useful for studying specific endophenotypes related to SZ, although these mice do not display typical SZ-like phenotypes.121 As for the therapeutic aspects, the effects of chlorpromazine (CPZ) on polySia expression were studied.230 CPZ is the medication prescribed for positive symptoms of SZ. The addition of CPZ to human neural cells leads to the upregulation in polySia expression. In addition, mice that were administered CPZ showed an upregulated expression of polySia only in prefrontal cortex, but not in AMG, HIP, OB, and SCN. This is an interesting

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phenomenon, because the upregulated expression in neurons of PFC, in which the downregulation of the expression of polySia has been reported, leads to ideal conditions for positive symptoms that are deeply related to the dopamine-DR systems to emerge, although the CPZ is known to be an antagonist of D2DR. Many biochemical studies that demonstrate the underlying molecular mechanism between behavior or anatomical phenotypes and polySia are required. Bipolar disorder (BD) is one of the mental disorders that affect people worldwide, and many susceptibility genes have been identified by genomewide association study (GWAS). For example, DISC1, NRG1, and BDNF231,232 were shown to be related to BD. A number of susceptibility genes for BD partly overlap with those of SZ. Interestingly, several symptoms are common between SZ and BD,233,234 and environmental factors are also major components that cause BD.235,236 Expression analysis of the ST8Sia2 gene in adult postmortem DLPFC revealed that ST8Sia2 gene expression tends to be lower in the brains of BP and SZ patients,237 indicating that the DLPFC of BP and SZ patients might have lower polySia– NCAM expression levels, although no biochemical studies were performed to confirm this speculation. In one report, the increased expression of polySia–NCAM was observed in the AMG of a BD patient.216 Based on genome-related studies, several significant SNPs specific to BD have been reported, and the information was summarized in a review regarding mental disorders.137 One interesting intronic SNP (rs2168351) that is related to the BD patients was biochemically analyzed.37 Using mouse and human neural cells, an iSNP (rs2168351) regulates ST8Sia2 expression and therefore leads to the upregulation of the level of ST8Sia2 pre-mRNA, ST8Sia2, and polySia–NCAM, although the underlying mechanism of action remains unknown. Therefore, further studies of iSNP rs2168351, which is the only nucleotide changed in the 6-kbp long intron 4, need to be carried out, because this mutation clearly affects polySia expression. Autism spectrum disorder (ASD) is considered to be a heterogeneous neurodevelopmental condition characterized by early onset. ASD patients have difficulties in social communication, display little interest in others, and have restricted interests, receptive movement, and difficulties in communication through spoken language.238 Genetic factors are an important causative factor of ASD, and the accumulation of SNPs or mutations in the causative genes and/or impairment of epigenetic regulation results in altered brain structures.239 The insufficient development of PFC and Purkinje cells in the cerebellum has also been reported.240 In addition, the cortex, pons, and limbic area are impaired in affected individuals.241,242

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GWAS of 1558 ASD families (4712 subjects) from the database of the Autism Genome Project (AGP) consortium, which consists of more than 50 centers in North America and Europe, demonstrated that an SNP (rs3784730) of ST8Sia2 for verbal individuals was associated with ASD by exploratory analyses. However, the SNP was not found to be significant after correction for multiple tests of diagnostic groups and subphenotypes.243 Interestingly, a child diagnosed with ASD and epilepsy was found to have a heterozygous 520-kb deletion on chromosome 15q26.1 (chr15:90,517,962-91,039,825; NCBI36/hg18), where three genes, ST8Sia2, C15orf32, and FAM174B, are located.244 It is clear that C15orf32 is not expressed in the brain; however, the functions of C15orf32 and FAM174B are still unknown. These three genes may cause the ASD-like phenotypes to be generated. Later, the child’s father was also found to have the same deletion.245 However, based on his self-report, he was not diagnosed with ASD or epilepsy. Interestingly, he did have a history of attention deficit hyperactivity disorder (ADHD). It is noteworthy that the ST8Sia2-KO mice show hyperactivity and aggressiveness.125,126 The heterozygous deletion on chromosome 15q26.1 does not lead to the same phenotype as ASD; several factors other than a single causative gene, such as environmental factors, contribute to the development of ASD, for example, the child was in a chaotic home environment until the age of 18 months, which could have contributed to the development of ASD. Parkinson’s disease (PD) is known as a long-term degenerative disorder of the nervous system, especially motor systems. The number of dopamine neurons at the SNA, the region of midbrain, is reduced, which leads to shortage of the DA. In PD, the upregulated expression of polySia was reported in SNA.191 Notably, the side effect of CPZ that leads to the upregulated expression of polySia in PFC is Parkinsonism. Alzheimer’s disease (AZ) is another major neurodegenerative disease. In the brain of an individual with AZ, polySia was shown to increase at the SVZ of the lateral ventricle, and SGZ and the GL of DG with at high Braak stages.246 The injection of Aβ into the rat HIP leads to the impairment of memory and learning, and increased staining of polySia at CA1 and DG in HIP.247 On closer observation, polySia–NCAM staining in ED changed in AD patients.248 The staining at layer II/III and V of the entorhinal cortex that is severely affected by the disease was decreased; however, the staining of layer IV was increased,246 indicating the disorganization of the tissues, although the underlying mechanism of action of polySia toward these neurodegenerative disorders remains unknown.

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7.2 Cancer A majority of the polysialylated NCAMs are expressed in embryos, while normal cells in adult tissues do not typically display polySia on the cell surface, except for the restricted area of adult brain; however, some cancer cells have been known to reexpress polySia. Thus, polySia has been recognized as an oncodevelopmental antigen.213 For example, neuroblastomas,249,250 Wilms’ tumors,251 medulloblastomas,252 pheochromocytomas,253 small cell lung carcinomas,254 teratomas,255 malignant lymphomas,256 medullary thyroid carcinomas,257 non-small cell lung (NSCL) carcinomas,258,259 pituitary adenomas,252 childhood rhabdomyosarcomas,260 pancreatic cancer,261 and breast cancer60 reexpress polySia on their cell surface. The relationship between the expression of the polySia and cancer progression was first reported for small cell lung carcinoma NCI-H69-derived cell lines, E2, and F3 cells. While E2 cells express negligible amounts of polySia, F3 cells express a large amount of polySia, which is consistent with the high expression of ST8Sia2.262 E2 cells tend to aggregate easily, but F3 cells tend to disperse. After removal of polySia from the F3 cell, the cells begin to aggregate. F3 cells form much more colonies than E2 cells on soft agar or in the methylcellulose test. Even in vivo using nude mice, F3 cells show a high metastatic feature, as compared with E2 cells.263 Histochemically, highgrade tumors resulting because of lung carcinomas exhibit a significantly higher extent of polySia–NCAM immunostaining as compared to lowgrade tumors and are correlated with nodal spread and metastasis across histologic classes, but not in individual types of tumors.264 In NSCL carcinoma cells, tumor progression is related to the expression levels of polySia and its biosynthesizing enzyme, ST8Sia2.259,265,266 Because of the highly invasive, and cell proliferative features of polySia-expressing tumors, polySia– NCAM is used as a diagnostic marker for not only lung carcinoma but also other cancers such as neuroblastoma,267–269 glioblastoma,270,271 and pituitary tumor.272 The control of polySia–NCAM expression and targeting of the drug toward polySia–NCAM-expressing cancers are important therapeutic approaches. Therefore, inhibitors and antibodies specific for polySia– NCAM have been used as powerful tools for the control and diagnosis of cancer and cancer grades.273 Unnatural materials described earlier were used for the purpose.274 RBL and RMA cells were used as model cancer cells, and these cells were incubated with Man2NPro to convert cell surface polyNeu5Ac to polyNeu5NPro, which is an unnatural polySia that is

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highly immunogenic for humans. Once polyNeu5Ac is converted to polyNeu5NPro by incubating the cancer cells with Man2NPro, the antipolyNeu5NPro antibody could be used as a targeted anticancer drug. Antibody-dependent cytotoxicity was observed in vitro and in vivo.275,276 PolySia is safely used for drug development, because it shows immune tolerance in humans.53 In addition, polySia is an effective glycotope, and the lifetime of polySia-containing proteins in serum can be increased. Polysialylation appears to prevent the proteins from being removed by asialoglycoprotein receptors on the liver.277 Another therapeutic approach for regulating the polySia structure is to use ammonia (NH3). NH3 is shown to regulate the polySia expression by changing the nucleotide sugar pools,278 and expressions of polySias using CHO and SCLC were actually inhibited by NH3.279 Valproic acid (2-propylpentanoic acid, VPA) is one of the candidates showing anticancer activity, because VPA is an antiepileptic drug that downregulates the ST8Sia2 mRNA expression in the neuroblastoma cell lines UKF-NB-3, UKF-NB-4, BE(2)-C, and MHH-NB-11, but upregulates mRNA expression for ST8Sia4. The polySia expression on the cell surface unexpectedly increased because of the down regulation of ST8Sia2 and upregulation of ST8Sia4.280 Notably, VPA was the reagent used to establish the model animal for autism by prenatal exposure, which led to abnormal polySia expression in the HIP.281 Another material that regulates polySTs is CMP and its derivatives, as described earlier.81 The addition of CMP to neuroblastoma (SH-SY5Y) and ST8Sia2-overexpressed C6 glioma cells inhibited migration.282

8. PERSPECTIVES Based on the status of di/oligo/polySia research, the following would be interesting to study in the future. First, polySia has long been recognized as a negative regulator of cell–cell adhesion; therefore, its only remarkable physiological feature is its ability to generate a repulsive field toward cells or the ECM. This feature of polySia has an important and unique function in neurogenesis during embryogenesis, as well as in neuroplasticity. Recently, the attractive field of polySia has been clearly demonstrated, and polySia is shown to serve as a reservoir or a scaffold for components involved in the maintenance of neural activity and growth of brain cells, particularly, groups of neurotrophic factors, growth factors, and neurotransmitters. Information regarding the oligo/polySia binding molecules

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and the molecular mechanisms for their binding and release need to be understood in detail. Second, the reservoir function of polySias for growth factors, morphogens, and cytokines is also observed largely in GAGs, such as HS, KS, CS, and DS, and these acidic polymers sometimes coexist in a polySia environment. Based on the in vitro and in cell analyses, the specificity of FGF2 toward NIH-3T3 cells is differently regulated by HS and polySia. Therefore, coordinated systems are present between polySia and GAGs; such systems are present not only with the HS–polySia complex but also with other GAG–polySia complexes. Therefore, it is important to understand their function under these conditions. As described earlier, antibodies against polySia occasionally cross-react with polynucleotides, which are a different group of polyanions than polySia or GAGs. Thus, polySias may share mimetic conformations with these polyanionic compounds, such as those observed during the steric distribution of carboxyl anions along the helical chain. This might explain why anti-polySia antibodies sometimes detect the polySia epitope in organisms that would not be expected to express polySia, because of the lack of polySTs.283,284 The relationship between the mucin-type sialic acid array on O-linked glycans and polysialic acid should be considered. An alternative possibility is that the polySia epitope is synthesized by unknown mechanisms in those organisms. However, we have observed the molecular mimicry of diSia by carbonic anhydrase-lacking carbohydrates.285 The detection of polySia by methods other than immunochemical detection, such as chemical assays, needs to be confirmed before forming conclusions. The molecular mimicry of the di/oligo/polySia structure in various cell types is an interesting phenomenon that should be investigated further. Third, many important questions concerning the biosynthesis of di/oligo/ polySia remain unresolved. For example, how are the expression and disappearance of polySia regulated at the transcriptional, translational, and protein levels? Which STs are responsible for the synthesis of glycosidic linkages other than the α2,8-linkage? Although the chain length is known to be biologically important, it is not understood why a DP of at least 12 is required for polySia to act as a reservoir for BDNF,135 while a DP of 17 is needed to bind FGF2148; the regulation of diSia, oligoSia, and polySia chain length is also not well understood. Why could enzymes synthesize an epitope on both glycoproteins and glycolipids? The answers to these, and many other questions, would supply the necessary insights for understanding polySia biosynthesis.

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Fourth, although polySia has been relatively well studied, a greater focus on di- and oligoSia glycoproteins is expected to provide in-depth knowledge regarding the functions of these interesting glycoproteins. After we identified a large group of diSia/oligoSia-containing glycoproteins, a growing number of studies on diSia and oligoSia structures have demonstrated that the biological functions of di- and oligoSia are clearly distinct from those of polySia, although many details remain unknown. Fifth, it is important to understand the molecular mechanism of di/oligo/ polySias involved in the biological phenomena observed in gene-targeted mice, because the features of the molecule are revealed in simple systems. In particular, in SZ, BD, ASD, PD, AZ, and cancer, it is important to understand how the diseases are caused by the expression of polySia and understand the di/oligo/polySia structure based on the chemical and welldefined immunochemical probes for these diseases. In conclusion, di/oligo/polymerized Sia is a distinct, unusual carbohydrate structure with respect to its size, properties, and functions, when compared with carbohydrates that are commonly present on cell surfaces. This epitope has never been abandoned in the mammalian brain during the course of evolution, although structural diversities have become smaller and smaller. On the other hand, it is rather utilized in a sophisticated way to fine-tune various aspects of brain functions. Therefore, the study of diSia/oligoSia/ polySia-containing glycoproteins, through a distant and a close view, is expected to deepen the understanding of this interactive epitope.

ACKNOWLEDGMENTS This review was supported in part by Grants-in-Aid for Scientific Research (C) (15K06995) from the Ministry of Education, Science, Sports and Culture (C.S.) and the DAIKO foundation (C.S.).

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