Polysialic Acid Synthase (ST8Sia II/STX) mRNA Expression in the Developing Mouse Central Nervous System

Abstract: A comparative study was undertaken to correlate the immunohistochemical localization of polysialic acid (PSA) and the in situ localization of ST8Sia II mRNA. In situ hybridization of postnatal day 3 mouse brain showed high levels of ST8Sia II mRNA expression in the cerebral neocortex, striatum, hippocampus, subiculum, medial habenular nucleus, thalamus, pontine nuclei, and inferior colliculus; intermediate-level expression in the olfactory bulb, hypothalamus, superior colliculus, and cerebellum; and low-level expression in other regions. The distribution of ST8Sia II mRNA in the neocortex and cerebellum coincided with the immunohistochemical localization of PSA. During brain development, ST8Sia II mRNA started decreasing and had almost disappeared by postnatal day 14. Comparison between ST8Sia II and IV mRNA expression was also undertaken by northern blot analysis and competitive PCR analysis. During the late embryonic to early postnatal stages of the mouse CNS, the ST8Sia II mRNA showed abundant mRNA expression compared with the ST8Sia IV mRNA. Competitive PCR analysis of the adult mouse CNS showed weak expression of the two genes in the olfactory bulb, thalamus, hippocampus, and eyes. The regional and transient expression of ST8Sia II mRNA coincides with that of PSA, suggesting that ST8Sia II is closely involved in the biosynthesis and expression of PSA in the developing mouse CNS.     

ST8Sia II and ST8Sia IV polysialyltransferases exhibit marked differences in utilizing various acceptors containing oligosialic acid and short polysialic acid. The basis for cooperative polysialylation by two enzymes

Polysialylation of the neural cell adhesion molecule (NCAM) is thought to play a critical role in neural development. Two polysialyltransferases, ST8Sia II and ST8Sia IV, play dominant roles in polysialic acid synthesis on NCAM. However, the individual roles and mechanisms by which these two enzymes form large amounts of polysialic acid on NCAM were heretofore unknown. Previous studies indicate that ST8Sia IV forms more highly polysialylated N-glycans on NCAM than ST8Sia II in vitro. In the present study, we first demonstrated that a combination of ST8Sia II and ST8Sia IV cooperatively polysialylated NCAM, resulting in NCAM N-glycans containing more, and thus longer, polysialic acid than when the enzymes were used individually. There was also an increase in polysialylated NCAM when we used ST8Sia II and ST8Sia IV sequentially, whereas there appeared to be a subtle increase when the enzymes were used in the reverse order. Furthermore, ST8Sia IV was able to add polysialic acid to oligosialylated oligosaccharides and unpolysialylated antennas in N-glycans attached to NCAM, even when polysialic acid was attached to at least one of the other antennas. By contrast, ST8Sia II added little polysialic acid to the same acceptors. On the other hand, neither ST8Sia II nor ST8Sia IV could add polysialic acid to a polysialylated antenna of NCAM N-glycans. These combined results indicate that the synergistic effect of ST8Sia II and ST8Sia IV is caused by: 1) the ability of ST8Sia IV to add polysialic acid to oligosialic acid formed by ST8Sia II, 2) the potential of ST8Sia IV to act on more antennas of N-glycans than ST8Sia II, and 3) the ability of ST8Sia II and ST8Sia IV in combination to act on the fifth and sixth N-glycosylation sites of NCAM.     

Differential biosynthesis of polysialic acid on neural cell adhesion molecule (NCAM) and oligosaccharide acceptors by three distinct alpha 2,8-sialyltransferases, ST8Sia IV (PST), ST8Sia II (STX), and ST8Sia III

Polysialylated neural cell adhesion molecule (NCAM) is thought to play a critical role in neural development. Polysialylation of NCAM was shown to be achieved by two alpha2,8-polysialyltransferases, ST8Sia IV (PST) and ST8Sia II (STX), which are moderately related to another alpha2,8-sialyltransferase, ST8Sia III. Here we describe that all three alpha2,8-sialyltransferases can utilize oligosaccharides as acceptors but differ in the efficiency of adding polysialic acid on NCAM. First, we found that ST8Sia III can form polysialic acid on the enzyme itself (autopolysialylation) but not on NCAM. These discoveries prompted us to determine if ST8Sia IV and ST8Sia II share the property of ST8Sia III in utilizing low molecular weight oligosaccharides as acceptors. By using a newly established method, we found that ST8Sia IV, ST8Sia II, and ST8Sia III all add oligosialic and polysialic acid on various sialylated N-acetyllactosaminyl oligosaccharides, including NCAM N-glycans, fetuin N-glycans, synthetic sialylated N-acetyllactosamines, and on alpha(2)-HS-glycoprotein. Our results also showed that monosialyl and disialyl N-acetyllactosamines can serve equally as an acceptor, suggesting that no initial addition of alpha2,8-sialic acid is necessary for the action of polysialyltransferases. Polysialylation of NCAM by ST8Sia IV and ST8Sia II is much more efficient than polysialylation of N-glycans isolated from NCAM. Moreover, ST8Sia IV and ST8Sia II catalyze polysialylation of NCAM much more efficiently than ST8Sia III. These results suggest that no specific acceptor recognition is involved in polysialylation of low molecular weight sialylated oligosaccharides, whereas the enzymes exhibit pronounced acceptor specificities if glycoproteins are used as acceptors.     

Differential biosynthesis of polysialic or disialic acid Structure by ST8Sia II and ST8Sia IV

ST8Sia II (STX) and ST8Sia IV (PST) are polysialic acid (polySia) synthases that catalyze polySia formation of neural cell adhesion molecule (NCAM) in vivo and in vitro. It still remains unclear how these structurally similar enzymes act differently in vivo. In the present study, we performed the enzymatic characterization of ST8Sia II and IV; both ST8Sia II and IV have pH optima of 5.8-6.1 and have no requirement of metal ions. Because the pH dependence of ST8Sia II and IV enzyme activities and the pK profile of His residues are similar, we hypothesized that a histidine residue would be involved in their catalytic activity. There is a conserved His residue (cf. His(348) in ST8Sia II and His(331) in ST8Sia IV, respectively) within the sialyl motif VS in all sialyltransferase genes cloned to date. Mutant ST8Sia II and IV enzymes in which this His residue was changed to Lys showed no detectable enzyme activity, even though they were folded correctly and could bind to CDP-hexanolamine, suggesting the importance of the His residue for their catalytic activity. Next, the degrees of polymerization of polySia in NCAM catalyzed by ST8Sia II and IV were compared. ST8Sia IV catalyzed larger polySia formation of NCAM than ST8Sia II. We also analyzed the (auto)polysialylated enzymes themselves. Interestingly, when ST8Sia II or IV itself was sialylated under conditions for polysialylation, the disialylated compound was the major product, even though polysialylated compounds were also observed. These results suggested that both ST8Sia II and IV catalyze polySia synthesis toward preferred acceptor substrates such as NCAM, whereas they mainly catalyze disialylation, similarly to ST8Sia III, toward unfavorable substrates such as enzyme themselves.     

The polysialyltransferase ST8Sia II/STX: posttranslational processing and role of autopolysialylation in the polysialylation of neural cell adhesion molecule.

The presence of alpha2,8-linked polysialic acid on the neural cell adhesion molecule (NCAM) is known to modulate cell interactions during development and oncogenesis. Two enzymes, the alpha2,8-polysialyltransferases ST8Sia IV()/PST and ST8Sia II()/STX are responsible for the polysialylation of NCAM. We previously reported that both ST8Sia IV/PST and ST8Sia II/STX enzymes are themselves modified by alpha2,8-linked polysialic acid chains, a process called autopolysialylation. In the case of ST8Sia IV/PST, autopolysialylation is not required for enzymatic activity. However, whether the autopolysialylation of ST8Sia II/STX is required for its ability to polysialylate NCAM is unknown. To understand how autopolysialylation impacts ST8Sia II/STX enzymatic activity, we employed a mutagenesis approach. We found that ST8Sia II/STX is modified by six Asn-linked oligosaccharides and that polysialic acid is distributed among the oligosaccharides modifying Asn 89, 219, and 234. Coexpression of a nonautopolysialylated ST8Sia II/STX mutant with NCAM demonstrated that autopolysialylation is not required for ST8Sia II/STX polysialyltransferase activity. In addition, catalytically active, nonautopolysialylated ST8Sia II/STX does not polysialylate any endogenous COS-1 cell proteins, highlighting the protein specificity of polysialylation. Furthermore, immunoblot analysis of NCAM polysialylation by autopolysialylated and nonautopolysialylated ST8Sia II/STX suggests that the NCAM is polysialylated to a higher degree by autopolysialylated ST8Sia II/STX. Therefore, we conclude that autopolysialylation of ST8Sia II/STX, like that of ST8Sia IV/PST, is not required for, but does enhance, NCAM polysialylation.     

Molecular dissection of the ST8Sia IV polysialyltransferase. Distinct domains are required for neural cell adhesion molecule recognition and polysialylation

Polysialic acid, a homopolymer of alpha2,8-linked sialic acid expressed on the neural cell adhesion molecule (NCAM), is thought to play critical roles in neural development. Two highly homologous polysialyltransferases, ST8Sia II and ST8Sia IV, which belong to the sialyltransferase gene family, synthesize polysialic acid on NCAM. By contrast, ST8Sia III, which is moderately homologous to ST8Sia II and ST8Sia IV, adds oligosialic acid to itself but very inefficiently to NCAM. Here, we report domains of polysialyltransferases required for NCAM recognition and polysialylation by generating chimeric enzymes between ST8Sia IV and ST8Sia III or ST8Sia II. We first determined the catalytic domain of ST8Sia IV by deletion mutants. To identify domains responsible for NCAM polysialylation, different segments of the ST8Sia IV catalytic domain, identified by the deletion experiments, were replaced with corresponding segments of ST8Sia II and ST8Sia III. We found that larger polysialic acid was formed on the enzymes themselves (autopolysialylation) when chimeric enzymes contained the carboxyl-terminal region of ST8Sia IV. However, chimeric enzymes that contain only the carboxyl-terminal segment of ST8Sia IV and the amino-terminal segment of ST8Sia III showed very weak activity toward NCAM, even though they had strong activity in polysialylating themselves. In fact, chimeric enzymes containing the amino-terminal portion of ST8Sia IV fused to downstream sequences of ST8Sia III inhibited NCAM polysialylation in vitro, although they did not polysialylate NCAM. These results suggest that in polysialyltransferases the NCAM recognition domain is distinct from the polysialylation domain and that some chimeric enzymes may act as a dominant negative enzyme for NCAM polysialylation.     

The minimal structural domains required for neural cell adhesion molecule polysialylation by PST/ST8Sia IV and STX/ST8Sia II

A limited number of mammalian proteins are modified by polysialic acid, with the neural cell adhesion molecule (NCAM) being the most abundant of these. We hypothesize that polysialylation is a protein-specific glycosylation event and that an initial protein-protein interaction between polysialyltransferases and glycoprotein substrates mediates this specificity. To evaluate the regions of NCAM required for recognition and polysialylation by PST/ST8Sia IV and STX/ST8Sia II, a series of domain deletion proteins were generated, co-expressed with each enzyme, and their polysialylation analyzed. A protein consisting of the fifth immunoglobulin-like domain (Ig5), which contains the reported sites of polysialylation, and the first fibronectin type III repeat (FN1) was polysialylated by both enzymes, whereas a protein consisting of Ig5 alone was not polysialylated by either enzyme. This demonstrates that the Ig5 domain of NCAM and FN1 are sufficient for polysialylation, and suggests that the FN1 may constitute an enzyme recognition and docking site. Two other NCAM mutants, NCAM-6 (Ig1-5) and NCAM-7 (FN1-FN2), were weakly polysialylated by PST/ST8Sia IV, suggesting that a weaker enzyme recognition site may exist within the Ig domains, and that glycans in the FN region are polysialylated. Further analysis indicated that O-linked oligosaccharides in NCAM-7, and O-linked and N-linked glycans in full-length NCAM, are polysialylated when these proteins are co-expressed with the polysialyltransferases in COS-1 cells. Our data support a model in which the polysialyltransferases bind to the FN1 of NCAM to polymerize polysialic acid chains on appropriately presented glycans in adjacent regions.     

Polysialyltransferase ST8Sia II (STX) polysialylates all of the major isoforms of NCAM and facilitates neurite outgrowth

The neural cell adhesion molecule (NCAM) has different isoforms due to different sizes in its polypeptide and plays a significant role in neural development. In neural development, the function of NCAM is modified by polysialylation catalyzed by two polysialyltransferases, ST8Sia II and ST8Sia IV. Previously, it was reported by others that ST8Sia II polysialylates only transmembrane isoforms of the NCAM, such as NCAM-140 and NCAM-180, but not NCAM-120 and NCAM-125 anchored by a glycosylphosphotidylinositol. In the present study, we first discovered that ST8Sia II polysialylates all isoforms of the NCAM examined, and we demonstrated that polysialylation of NCAM expressed on 3T3 cells facilitates neurite outgrowth regardless of isoforms of NCAM, where polysialic acid is attached. We then show that neurite outgrowth is significantly facilitated only when polysialylated NCAM is present in cell membranes. Moreover, the soluble NCAM coated on plates did not have an effect on neurite outgrowth exerted by soluble L1 adhesion molecule coated on plates. These results, taken together, indicate that ST8Sia II plays critical roles in modulating the function of all major isoforms of NCAM. The results also support previous studies showing that a signal cascade initiated by NCAM differs from that initiated by L1 molecule.     

Unique disulfide bond structures found in ST8Sia IV polysialyltransferase are required for its activity

NCAM polysialylation plays a critical role in neuronal development and regeneration. Polysialylation of the neural cell adhesion molecule (NCAM) is catalyzed by two polysialyltransferases, ST8Sia II (STX) and ST8Sia IV (PST), which contain sialylmotifs L and S conserved in all members of the sialyltransferases. The members of the ST8Sia gene family, including ST8Sia II and ST8Sia IV are unique in having three cysteines in sialylmotif L, one cysteine in sialylmotif S, and one cysteine at the COOH terminus. However, structural information, including how disulfide bonds are formed, has not been determined for any of the sialyltransferases. To obtain insight into the structure/function of ST8Sia IV, we expressed human ST8Sia IV in insect cells, Trichoplusia ni, and found that the enzyme produced in the insect cells catalyzes NCAM polysialylation, although it cannot polysialylate itself ("autopolysialylation"). We also found that ST8Sia IV does not form a dimer through disulfide bonds. By using the same enzyme preparation and performing mass spectrometric analysis, we found that the first cysteine in sialylmotif L and the cysteine in sialylmotif S form a disulfide bridge, whereas the second cysteine in sialylmotif L and the cysteine at the COOH terminus form a second disulfide bridge. Site-directed mutagenesis demonstrated that mutation at cysteine residues involved in the disulfide bridges completely inactivated the enzyme. Moreover, changes in the position of the COOH-terminal cysteine abolished its activity. By contrast, the addition of green fluorescence protein at the COOH terminus of ST8Sia IV did not render the enzyme inactive. These results combined indicate that the sterical structure formed by intramolecular disulfide bonds, which bring the sialylmotifs and the COOH terminus within close proximity, is critical for the catalytic activity of ST8Sia IV.     

Polysialyltransferases: major players in polysialic acid synthesis on the neural cell adhesion molecule

Polysialic acid is a unique carbohydrate composed of a linear homopolymer of alpha2,8-linked sialic acid, and is mainly attached to the fifth immunoglobulin-like domain of the neural cell adhesion molecule (NCAM) via a typical N-linked glycan in vertebrate neural system. Polysialic acid plays critical roles in neural development by modulating adhesive property of NCAM such as neural cell migration, neurite outgrowth, neural pathfinding, and synaptogenesis. The expression of polysialic acid is temporally and spatially regulated during neural development. Polysialylation of NCAM is catalyzed by two polysialyltransferases, ST8Sia II (STX) and ST8Sia IV (PST), which belong to the family of six genes encoding alpha 2,8-sialyltransferases. ST8Sia II and IV are expressed differentially in tissue-specific and cell-specific manners, and they apparently have distinct roles in development and organogenesis. The presence of polysialic acid is always associated with expression of ST8Sia II and/or IV, suggesting that ST8Sia II and IV are the key enzymes that control the expression of polysialic acid. Both ST8Sia II and IV can transfer multiple alpha 2,8-linked sialic acid residues to an acceptor N-glycan containing a NeuNAc alpha 2-->3 (or 6) Gal beta 1-->4GlcNAc beta 1-->R structure without participation of other enzymes. The two enzymes differently but cooperatively act on NCAM and the amount of polysialic acid synthesized by both enzymes together is greater than that synthesized by either enzyme alone. The polysialyltransferases are thus important regulators in polysialic acid synthesis and contribute to neural development in the vertebrate.     

Differential expression of disialic acids in the cerebellum of senile mice

It is known that disialic acids (diSia) are present in the mammalian brain. However, the precise anatomical distribution and the chronology of its expression along life are not well studied yet. It is accepted that the transfer of diSia in the brain is mediated mainly by the enzyme ST8Sia III (α2,8-sialyltransferase III). We studied the expression of diSia glycoepitopes and of the ST8Sia III gene in different structures of the mouse brain at different postnatal stages by immunohistochemistry and real-time polymerase chain reaction, respectively. C57BL/6 mice of different stages were used. Samples of hippocampus, olfactory bulb, cortex and cerebellum were processed for studies of molecular biology and immunohistochemistry. Histological analysis revealed an important decrease in diSia labeling in the senile cerebellum compared with other structures and stages (P???0.001). In concordance with these results, a significant decrease in ST8Sia III gene expression was found in the cerebellum of senile animals (P?相似文献   

Fjx1, the murine homologue of the Drosophila four-jointed gene, codes for a putative secreted protein expressed in restricted domains of the developing and adult brain

The Drosophila gene four jointed (fj) codes for a secreted or cell surface protein important for growth and differentiation of legs and wings and for proper development of the eyes. Here we report the cloning of the mouse four-jointed gene (fjx1) and its pattern of expression in the brain during embryogenesis and in the adult. In the neural plate, fjx1 is expressed in the presumptive forebrain and midbrain, and in rhombomere 4, however a small rostral/medial area of the forebrain primordium is devoid of expression. Expression of fjx1 in the neural tube can be divided into three phases. (1) In the embryonic brain fjx1 is expressed in two patches of neuroepithelium: in the midbrain tectum and the telencephalic vesicles. (2) In fetal and early postnatal brain fjx1 is expressed mainly by the primordia of layered telencephalic structures: cortex (ventricular layer and cortical plate), olfactory bulb (subependymal layer and in the mitral cell layer). In addition expression is observed in the superior colliculus. (3) In the adult, fjx1 is expressed by neurons evenly distributed in the telencephalon (isocortex, striatum, hippocampus, olfactory bulb, piriform cortex), in the Purkinje cell layer of the cerebellum, and numerous medullary nuclei. In the embryo, strong expression can further be seen in the apical ectodermal ridge of fore- and hindlimbs and in the ectoderm of the branchial arches.     

Identification of Sequences in the Polysialyltransferases ST8Sia II and ST8Sia IV That Are Required for the Protein-specific Polysialylation of the Neural Cell Adhesion Molecule, NCAM

The polysialyltransferases ST8Sia II and ST8Sia IV polysialylate the glycans of a small subset of mammalian proteins. Their most abundant substrate is the neural cell adhesion molecule (NCAM). An acidic surface patch and a novel α-helix in the first fibronectin type III repeat of NCAM are required for the polysialylation of N-glycans on the adjacent immunoglobulin domain. Inspection of ST8Sia IV sequences revealed two conserved polybasic regions that might interact with the NCAM acidic patch or the growing polysialic acid chain. One is the previously identified polysialyltransferase domain (Nakata, D., Zhang, L., and Troy, F. A. (2006) Glycoconj. J. 23, 423–436). The second is a 35-amino acid polybasic region that contains seven basic residues and is equidistant from the large sialyl motif in both polysialyltransferases. We replaced these basic residues to evaluate their role in enzyme autopolysialylation and NCAM-specific polysialylation. We found that replacement of Arg²⁷⁶/Arg²⁷⁷ or Arg²⁶⁵ in the polysialyltransferase domain of ST8Sia IV decreased both NCAM polysialylation and autopolysialylation in parallel, suggesting that these residues are important for catalytic activity. In contrast, replacing Arg⁸²/Arg⁹³ in ST8Sia IV with alanine substantially decreased NCAM-specific polysialylation while only partially impacting autopolysialylation, suggesting that these residues may be particularly important for NCAM polysialylation. Two conserved negatively charged residues, Glu⁹² and Asp⁹⁴, surround Arg⁹³. Replacement of these residues with alanine largely inactivated ST8Sia IV, whereas reversing these residues enhanced enzyme autopolysialylation but significantly reduced NCAM polysialylation. In sum, we have identified selected amino acids in this conserved polysialyltransferase polybasic region that are critical for the protein-specific polysialylation of NCAM.Polysialic acid is a linear homopolymer of α2,8-linked sialic acid that is added to a small subset of mammalian glycoproteins by the polysialyltransferases (polySTs)3 ST8Sia II (STX) and ST8Sia IV (PST) (1–4). Substrates for the polySTs include the neural cell adhesion molecule (NCAM) (5, 6), the α-subunit of the voltage-dependent sodium channel (7, 8), CD36, a scavenger receptor found in milk (9), neuropilin-2 expressed by dendritic cells (10), and the polySTs themselves, which can polysialylate their own N-glycans in a process called autopolysialylation (11, 12). This small number of polysialylated proteins and other evidence from our laboratory (13–15) suggest that polysialylation is a protein-specific modification that requires an initial protein-protein interaction between the polySTs and their glycoprotein substrates.The most abundant polysialylated protein is NCAM. The three major NCAM isoforms consist of five Ig domains, two fibronectin type III repeats, and a transmembrane domain and cytoplasmic tail (NCAM140 and NCAM180) or a glycosylphosphatidylinositol anchor (NCAM120) (16). Polysialylation takes place primarily on two N-linked glycans in the Ig5 domain (17). We have previously shown that a truncated NCAM140 protein consisting of Ig5, the first fibronectin type III repeat (FN1), the transmembrane region, and cytoplasmic tail is fully polysialylated (13). However, a protein consisting of Ig5, the transmembrane region, and cytoplasmic tail is not polysialylated (13). This suggests that the polySTs recognize and bind the FN1 domain to polysialylate N-glycans on the adjacent Ig5 domain. We subsequently identified an acidic patch unique to NCAM FN1, consisting of Asp⁴⁹⁷, Asp⁵¹¹, Glu⁵¹², and Glu⁵¹⁴ (15).⁴ When three of these residues (Asp⁵¹¹, Glu⁵¹², and Glu⁵¹⁴) are mutated to alanine or arginine, NCAM polysialylation is reduced or abolished, suggesting that the acidic patch is part of a larger recognition region. We anticipate that within this putative recognition region there will be amino acids required for mediating polyST-NCAM binding, and those that do not mediate binding per se but instead are required for correct positioning of the enzyme-substrate complex for polysialylation. For example, we have identified a novel α-helix in the FN1 domain that when replaced leads to polysialylation of O-glycans found on the FN1 domain rather than N-glycans on the Ig5 domain (14). This helix may mediate an interdomain interaction that positions the Ig5 N-glycans for polysialylation by an enzyme bound to the FN1 domain (14). Alternatively, the helix could act as a secondary interaction site that positions the polyST properly on the substrate.The expression of the polySTs is developmentally regulated with high levels of STX and moderate levels of PST expressed throughout the developing embryo (2, 18, 19). STX levels decline after birth, although PST expression persists in specific regions of the adult brain where polysialylated NCAM is involved in neuronal regeneration and synaptic plasticity (18–23). The large size and negative charge of polysialic acid disrupt NCAM-dependent and NCAM-independent interactions, thereby negatively modulating cell adhesion (24–26). Simultaneous disruption of both PST and STX in mice results in severe neuronal defects and death usually within 4 weeks after birth (27). Interestingly, when NCAM expression is also eliminated in these mice, they have a nearly normal phenotype, suggesting the main function of polysialic acid is to modulate NCAM-mediated cell adhesion during development (27). In addition, re-expression of highly polysialylated NCAM has been associated with several cancers, including neuroblastomas, gliomas, small cell lung carcinomas, and Wilms tumor. The presence of polysialic acid is thought to promote cancer cell growth and invasiveness (28–35).Sialyltransferases, including the polySTs, have three motifs required for catalytic activity (36–38) (see Fig. 1A). Sialyl motif Large (SML) is thought to bind the donor substrate CMP-sialic acid (39), whereas sialyl motif Small (SMS) is believed to bind both donor and carbohydrate acceptor substrates (40). The sialyl motif Very Small (SMVS) has a conserved His residue that is required for catalytic activity (38, 41). However, the precise function of this motif is unknown. An additional 4-amino acid motif, motif III, is conserved in the sialyltransferases (42–44). It was suggested that this motif, and particularly His and Tyr residues within its sequence, may be required for optimal activity and acceptor recognition (42).Open in a separate windowFIGURE 1.PST and STX polybasic regions and mutants generated for this study. A, representation of the polySTs and their polybasic regions and sialyl motifs. The PBR is a 35-amino acid region present in both PST and STX, equidistant from the SML of each enzyme and rich in conserved positively charged amino acids. The PSTD is a region identified by Nakata et al. (47) that is 32 amino acids in length, rich in basic residues, and contiguous with the SMS of the enzymes. The sialyl motifs (SML, SMS, SMVS, and motif III) are regions of homology found in all sialyltransferases that are believed to be involved in substrate and donor interactions. B, PSTD of PST and the mutants made in this region that are used in this study. C, PBR of PST and STX and the mutants made in this region that are used in this study.Angata et al. (45) used chimeric enzymes to identify regions within the polySTs required for catalytic activity and NCAM polysialylation. Sequences from PST, STX, and ST8Sia III were used to construct the chimeric proteins. ST8Sia III is an α2,8-sialyltransferase that typically adds one or two sialic acid residues to glycoprotein or glycolipid substrates, can autopolysialylate its own glycans, but cannot polysialylate NCAM (46). Deletion analysis showed that amino acids 62–356 are required for PST catalytic activity. Replacement of segments of this region with corresponding STX or ST8Sia III sequences led to the suggestion that amino acids 62–127 and possibly 194–267 of PST may be required for NCAM recognition (45).Recently, Troy and co-workers (47, 48) identified a stretch of basic residues, termed the polysialyltransferase domain (PSTD), which is only observed in the two polySTs and not in other sialyltransferases. The PSTD is contiguous with SMS and extends from amino acids 246–277 in PST and 261–292 in STX. Mutation analysis demonstrated that the overall positive charge of this motif is important for activity and identified specific residues required for NCAM polysialylation (Arg²⁵², Ile²⁷⁵, Lys²⁷⁶, and Arg²⁷⁷) (47).In this study, we have scanned the critical polyST regions identified by the work of Angata et al. (45) for sequences that may be involved in protein-protein recognition and NCAM polysialylation. We identified a second polybasic motif that we named the polybasic region (PBR). The PBR is conserved in PST and STX and is located equidistant from the SML of each enzyme. It consists of 35 amino acids of which 7 are the basic amino acids Arg and Lys. We found that the replacement of two specific residues within the PBR (Arg⁸² and Arg⁹³ of PST and Arg⁹⁷ and Lys¹⁰⁸ of STX) have a greater negative effect on NCAM polysialylation than on autopolysialylation. Replacement of acidic residues surrounding PST Arg⁹³ led to a similar disparate effect on these processes. Comparison of the critical residues in both the PSTD and PBR demonstrated that the replacement of PSTD residues had an equally negative impact on both NCAM polysialylation and enzyme autopolysialylation, whereas replacement of selected PBR residues more severely impacted NCAM polysialylation, suggesting that the PBR residues may play important roles in NCAM-specific polysialylation.     

Experimental approaches to interfere with the polysialylation of the neural cell adhesion molecule in vitro and in vivo

Sialic acid (Sia) is expressed as terminal sugar in many glycoconjugates and plays an important role during development and regeneration. Addition of homopolymers of Sia (polysialic acid; polySia/PSA) is a unique and highly regulated post-translational modification of the neural cell adhesion molecule (NCAM). The presence of polySia affects NCAM-dependent cell adhesion and plays an important role during brain development, neural regeneration, and plastic processes including learning and memory. PolySia-NCAM is expressed on several neuroendocrine tumors of high malignancy and correlates with poor prognosis. Two closely related enzymes, the polysialyltransferases ST8SiaII and ST8SiaIV, catalyze the biosynthesis of polySia. This review summarizes recent knowledge on Sia biosynthesis and the correlation between Sia biosynthesis and polysialylation of NCAM and report on approaches to modify the degree of polySia on NCAM in vitro and in vivo. First, we describe the inhibition of polysialylation of NCAM in ST8SiaII-expressing cells using synthetic Sia precursors. Second, we demonstrate that the key enzyme of the Sia biosynthesis (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase) regulates and limits the synthesis of polySia by controlling the cellular Sia concentration.     

The postnatal development of the main olfactory bulb of the rat

The postnatal development from birth to 1 year of the main olfactory bulb was examined quantitatively. The volume of the main olfactory bulb increased over seven-fold by day 30 and remained unchanged thereafter. During the same period the volume of the granular layer increased 18-fold and the mean areas of the olfactory glomeruli increased seven-fold. The mean areas of mitral cell perikarya doubled between the neonatal and juvenile periods. The total number of the mitral cells, however, declined during the first three postnatal weeks. In the internal granular layer of the main olfactory bulb, 89% of the granule cells were acquired postnatally. Much of the cellular gain occurred during the first 3 weeks, with the period of maximum acquisition between days 8 and 14. The number of subependymal cells, the precursors of granule cells, reached a peak at 12 days and gradually declined. But some primitive cells could still be found at one year of age and there was an increase in the total number of granule cells beyond day 30. The mean nuber of internal granular layer cells in a single main olfactory bulb of adult rats was about 5 X 10(6); the number of mitral cells about 4 X 10(4). In the animals injected with 3H-thymidine on day 20 and killed 2 h after injection a small but significant proportion of cells was labelled in the subependymal layer but few in the internal granular layer. In the animals killed 20 and 40 days after injection there was a 10--11-fold rise in the proportion of labelled internal granular layer cells. The proportion of labelled internal granular layer cells decreased in longer survival groups but the total number of labelled cells remained the same, even in year-old animals. However, the total number of internal granular layer cells in the sections examined increased with age.     

Differential impact of polysialyltransferase ST8SiaII and ST8SiaIV knockout on social interaction and aggression

Previous studies using neuronal cell adhesion molecule (NCAM) ?/? knockout (KO) mice provided evidence for a role of NCAMs in social behaviors. However, polysialic acid (PSA), the most important post‐translational modification of NCAM, was also absent in these mice, which makes it difficult to distinguish between the specific involvement of either PSA or NCAM in social interactions. To address this issue, we assessed two lines of mice deficient for one of the two sialyltransferase enzymes required for the polysialylation of NCAM, sialyltransferase‐X (St8SiaII or STX) and polysialyltransferase (ST8SiaIV or PST), in a series of tests for social behaviors. Results showed that PST KO mice display a decreased motivation in social interaction. This deficit can be partly explained by olfactory deficits and was associated with a clear decrease in PSA‐NCAM expression in all brain regions analyzed (amygdala, septum, bed nucleus of the stria terminalis and frontal cortices). STX KO mice displayed both a decreased social motivation and an increased aggressive behavior that cannot be explained by olfactory deficits. This finding might be related to the reduced anxiety‐like behavior, increased locomotion and stress‐induced corticosterone secretion observed in these mice. Moreover, STX KO mice showed mild increase of PSA‐NCAM expression in the lateral septum and the orbitofrontal cortex. Altogether, these findings support a role for PSA‐NCAM in the regulation of social behaviors ranging from a lack of social motivation to aggression. They also underscore STX KO mice as an interesting animal model that combines a behavioral profile of violence and hyperactivity with reduced anxiety‐like behavior.     

Differential expression of mRNAs for sialyltransferase isoenzymes induced in the hippocampus of mouse following kindled seizures

Sialic acids play important roles in various biological functions. In the brain, evidence suggests that sialylation of glycoproteins and glycolipids affects neural plasticity. While the 18 sialyltransferase isoenzymes (STs) identified to date synthesize individual sialyl-oligosaccharide structures, they each exhibit activity toward more than one substrate and can overlap in their specificity. Therefore, the distribution of STs is a secondary factor in the study of specific sialylation. Here, seven STs; ST3Gal I-IV, ST8Sia IV, ST6Gal I and ST6GalNAc II, the expressions of which were identified in the adult hippocampus by RT-PCR, showed diverse localization patterns in the hippocampus on in situ hybridization, suggesting that the individual cells expressed relevant STS: Furthermore, to assay activity-related changes in ST expression, we used amygdaloid-kindling among models of neural plasticity. Differential expression of the STs participating in the kindling, notably, up-regulation of ST3Gal IV and ST6GalNAc II mRNAs, and down-regulation of ST3Gal I and ST8Sia IV mRNAs, were observed in the hippocampus following kindled seizures. These results indicate that ST expressions are regulated by physiological activity and may play a role in neural plasticity.