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.     

Polysialic acid and N-CAM localisation in embryonic rat kidney: mesenchymal and epithelial elements show different patterns of expression

The expression of polysialic acid (PSA) and the neural cell adhesion molecule (N-CAM) during the embryonic development of rat kidney was investigated using immunocytochemistry and immunoblotting. A monoclonal antibody (mAb 735), which recognised only long chain PSA, and polyclonal antibodies specific for N-CAM were employed. At the earliest stages of metanephros formation, PSA and N-CAM immunostaining was found in both embryonic anlagen, namely the uretic bud and the metanephrogenic mesenchyme. Reactivity in uretic bud derivatives declined during embryonic development and was generally absent in the collecting duct system by embryonic day 18 (E18). Uninduced metanephrogenic mesenchyme was immunostained throughout development while induced mesenchymal cells showed greatly increased PSA and N-CAM immunoreactivity during their transformation into epithelium. This staining declined rapidly as nephrons differentiated. These processes were preceded by sorting of PSA and N-CAM to the basolateral plasma membrane. Similar N-CAM and PSA patterns were observed in mesonephros development. In adult kidney parenchyma both PSA and N-CAM were undetectable. Immunoblotting of samples of embryonic kidney with mAb 735 revealed a broad band ranging from 140 to greater than 200 x 10(3) Mr. N-CAM antibodies revealed reactivity in a band of 140 x 10(3) Mr after removal of PSA by endoneuraminidase treatment. Expression of N-CAM and PSA in both embryonic anlagen indicates that neither molecular species acts primarily as an inductive signal. These molecules were localised in areas where changes in cell adhesion during organogenesis might be important and thus may be involved in the grouping of developing cells.     

Cloning and expression of an alpha-2,8-polysialyltransferase (STX) from Xenopus laevis

Polysialic acid (polySia) is a carbohydrate structure found on neural cell adhesion molecules (N-CAM). Two polysialyltransferases (polySiaTs) that catalyze synthesis of polySia have been described, and designated PST-1/PST/ST8SiaIV and STX/ST8SiaII. We cloned a polySiaT (xSTX) from a nonmammalian vertebrate, Xenopus laevis . xSTX had 80% amino acid similarity to the rat STX. This clone induced polySia expression when transfected into polySia-negative COS-1 cells. Northern blot analysis of whole embryos at different stages of development revealed that xSTX mRNA was most abundantly expressed in premetamorphic stages. The relative level of xSTX and N-CAM mRNAs was also examined and found to change in parallel to the extent of polysialylation on N-CAM. In adult tissues, the expression of xSTX mRNA was restricted to brain, eye and heart, which also expressed polySia. These results suggest that xSTX is the major enzyme responsible for the synthesis of polysialylated N-CAM in embryos at certain stages of development and also in adult tissues.      

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.     

Alteration of neural tissue structure by expression of polysialic acid induced by viral delivery of PST polysialyltransferase

The expression of polysialic acid (PSA) on neural cell adhesion molecule (NCAM) is known to attenuate cell-cell interactions. During neural development the widespread expression of PSA-NCAM creates permissive conditions for the migration of neuronal and glial precursors and the guidance and targeting of axons. NCAM polysialylation can occur via either of two specific sialyltransferases, ST8SiaII (STX) and ST8SiaIV (PST), and the purpose of this study was to determine if retroviral delivery of either PST or STX could induce PSA expression in vivo and thereby alter tissue plasticity. Retroviruses expressing GFP-PST or GFP-STX were injected into embryonic retina, and development was evaluated by examining neuroepithelial structure, the expression of markers for specific cell types, cellular proliferation, and apoptosis. Chick retina was chosen because it down-regulates PSA early in its development and has a highly stereotyped program of morphogenesis. Retroviral expression of PST induced PSA expression in retina and resulted in severe but localized alterations in retinal morphogenesis, including an early disruption of radial glial cell morphology, highly disorganized retinal layers, and invasion of pigmented cells into the neural retina. In contrast, retroviral delivery of STX did not induce PSA expression or affect morphogenesis. These findings demonstrate that expression of PSA is sufficient to promote morphological alterations in a relatively nonplastic neural tissue.     

Neurite outgrowth in response to transfected N-CAM changes during development and is modulated by polysialic acid

We have used monolayers of control 3T3 cells and 3T3 cells transfected with a cDNA encoding human N-CAM as a culture substrate for embryonic chick retinal ganglion cells (RGCs). At embryonic day 6 (E6), but not at E11, RGCs extended longer neurites on monolayers of N-CAM-transfected cells. This loss of RGC responsiveness was not associated with substantial changes in the level of N-CAM expression on RGC growth cones. The neurite outgrowth response from E6 RGCs could be inhibited by removal of N-CAM from the monolayer, by removal of alpha 2-8-linked polysialic acid from neuronal N-CAM, or by antibodies that bind exclusively to chick (neuronal) N-CAM. In contrast, the response was not dependent on neuronal beta 1 integrin function. These data provide substantive evidence for a homophilic binding mechanism directly mediating N-CAM-dependent neurite outgrowth, and suggest that changes in polysialic acid expression on neuronal N-CAM may modulate N-CAM-dependent axonal growth during development.     

The relationship of polysialic acid and the neural cell adhesion molecule N-CAM in Wilms tumor and their subcellular distributions

In previous studies we have reported that polysialic acid is an oncodevelopmental antigen in human kidney but its relationship to the neural cell adhesion molecule (N-CAM) remained undefined. In the present study, we showed by the combination of immunoprecipitation and immunoblotting that renal polysialic acid is a structural component of N-CAM polypeptide and that two highly sialylated N-CAM isoforms of approximately 120 kDa and 140 kDa existed in Wilms tumor. The presence of a cell surface coat composed of polysialic acid and N-CAM was revealed by immunoelectron microscopy, and morphological evidence for its involvement in modulating cell-cell adhesion has been provided. Furthermore, highly sialylated N-CAM was detectable extracellularly. N-CAM immunolabeling was present in compartments from the nuclear envelope to the plasma membrane. However, polysialic acid was only detectable at the cell surface suggesting that in Wilms tumor cells sialyl polymer synthesis may occur partially or exclusively at this site.     

Expression of polysialylated N-CAM during rat heart development

Developmental patterns of immunoreactivity for the neural cell adhesion molecule (N-CAM) and alpha 2.8-linked polysialic acid (PSA) were identified in embryonic and postnatal rat heart by immunocytochemistry and immunoblotting. Polyclonal antibodies against N-CAM and a monoclonal antibody which recognises only polymers of PSA with a chain length greater than eight units were used. Gold- and alkaline-phosphatase-labelled antibodies were used for detection. The N-CAM polypeptide isoform pattern seen by immunoblotting after endoneuraminidase treatment changed as development progressed. During embryonic development a 160-kDa polypeptide isoform was predominant. Around birth, 130-, 160- and 170-kDa polypeptide isoforms were found. The expression of the 130- and 170-kDa isoforms diminished until finally, in the adult, weak immunoreactivity for bands of 120-, 130- and 160-kDa was seen. In general the extent and intensity of PSA and N-CAM immunostaining in rat heart increased until birth and declined thereafter. Early in development prominent immunostaining for PSA and N-CAM was seen in the epicardium while later in development this area was only weakly stained. Initially myocardial cells, endocardial cells and some cells in the atrioventricular cushions were immunoreactive for both PSA and N-CAM. Later in development N-CAM immunostaining was more prominent than PSA immunoreactivity, reflecting a decrease in N-CAM polysialylation, which was also seen by immunoblotting. During innervation of the heart, nerve fibres were strongly immunostained for PSA and N-CAM, and this was the only immunostaining seen in adult heart.     

Chemical analysis of the developmental pattern of polysialylation in chicken brain. Expression of only an extended form of polysialyl chains during embryogenesis and the presence of disialyl residues in both embryonic and adult chicken brains

Recent studies have demonstrated the involvement of two polysialyltransferases in neural cell adhesion molecule (N-CAM) polysialylation. The availability of cDNAs encoding these enzymes facilitated studies on polysialylation of N-CAM. However, there is a dearth of detailed structural information on the degree of polymerization (DP), DP ranges, and the influence of embryogenesis on the DP. It is also unclear how many polysialic acid (polySia) chains are attached to a single core N-glycan. In this paper we applied new, efficient, and sensitive high pressure liquid chromatography methods to qualitatively and quantitatively analyze the polySia structures expressed on embryonic and adult chicken brain N-CAM. Our studies resulted in the following new findings. 1) The DP of the polySia chains was invariably 40-50 throughout developmental stages from embryonic day 5 to 21 after fertilization. In contrast, glycopeptides containing polySia with shorter DPs, ranging from 15 to 35, were isolated from adult brain. 2) Chemical evidence showed glycan chains abundant in Neu5Acalpha2,8Neu5Ac were expressed during all developmental stages including adult. 3) Levels of both di- and polySia were found to show distinctive changes during embryonic development.     

Promotion of Cell Migration by Neural Cell Adhesion Molecule (NCAM) Is Enhanced by PSA in a Polysialyltransferase-Specific Manner

Neural cell adhesion molecule 140 (NCAM-140) is a glycoprotein and always highly polysialylated in cancer. Functions of polysialic acid (PSA) that binds to N-glycan termini on NCAM remain unclear. ldlD-14 cells, a CHO cell mutant deficient in UDP-Gal 4-epimerase, are useful for structural and functional studies of Gal-containing glycoproteins because their abnormal glycosylation can be converted to normal status by exogenous addition of galactose (Gal). We cloned the genes for NCAM-140 and for polysialyltransferases STX and PST (responsible for PSA synthesis) from normal murine mammary gland epithelial (NMuMG) cells and transfected them into ldlD-14 and human breast cancer cells MCF-7. The effect of PSA on NCAM-mediated cell proliferation, motility, migration and adhesion was studied. We found that NCAM-140 significantly promoted cell proliferation, motility and migration, while polysialylation of NCAM-140 catalyzed by STX, but not by PST, enhanced NCAM-mediated cell migration, but not cell proliferation or motility. In addition, PSA catalyzed by different polysialyltransferases affected the adhesion of NCAM to different extracellular matrix (ECM) components.     

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 effects of unnatural sialic acids on the polysialylation of the neural cell adhesion molecule and neuronal behavior.

In this study we have examined how unnatural sialic acids can alter polysialic acid expression and influence the adhesive properties of the neural cell adhesion molecule (NCAM). Unnatural sialic acids are generated by metabolic conversion of synthetic N-acyl mannosamines and are typically incorporated into cell-surface glycoconjugates. However, N-butanoylmannosamine and N-pentanoylmannosamine are effective inhibitors of polysialic acid (PSA) synthesis in stably transfected HeLa cells expressing NCAM and the polysialyltransferase STX. These cells were used as substrates to examine the effect of inhibiting PSA synthesis on the development of neurons derived from the chick dorsal root ganglion. N-butanoylmannosamine blocked polysialylation of NCAM and significantly reduced neurite outgrowth comparable with enzymatic removal of PSA by endoneuraminidases. As a result, neurite outgrowth was similar to that observed for non-polysialylated NCAM. In contrast, previous studies have shown that N-propanoyl sialic acid (SiaProp), generated from N-propanoylmannosamine, is readily accepted by polysialyltransferases and permits the extension of poly(SiaProp) on NCAM. Despite being immunologically distinct, poly(SiaProp) can promote neurite outgrowth similarly to natural polysialic acid. Thus, subtle structural differences in PSA resulting from the incorporation of SiaProp residues do not alter the antiadhesive properties of polysialylated NCAM.     

Enzymatic Engineering of Polysialic Acid on Cells in Vitro and in Vivo Using a Purified Bacterial Polysialyltransferase

In vertebrates, polysialic acid (PSA) is typically added to the neural cell adhesion molecule (NCAM) in the Golgi by PST or STX polysialyltransferase. PSA promotes plasticity, and its enhanced expression by viral delivery of the PST or STX gene has been shown to promote cellular processes that are useful for repair of the injured adult nervous system. Here we demonstrate a new strategy for PSA induction on cells involving addition of a purified polysialyltransferase from Neisseria meningitidis (PST_Nm) to the extracellular environment. In the presence of its donor substrate (CMP-Neu5Ac), PST_Nm synthesized PSA directly on surfaces of various cell types in culture, including Chinese hamster ovary cells, chicken DF1 fibroblasts, primary rat Schwann cells, and mouse embryonic stem cells. Similarly, injection of PST_Nm and donor in vivo was able to produce PSA in different adult brain regions, including the cerebral cortex, striatum, and spinal cord. PSA synthesis by PST_Nm requires the presence of the donor CMP-Neu5Ac, and the product could be degraded by the PSA-specific endoneuraminidase-N. Although PST_Nm was able to add PSA to NCAM, most of its product was attached to other cell surface proteins. Nevertheless, the PST_Nm-induced PSA displayed the ability to attenuate cell adhesion, promote neurite outgrowth, and enhance cell migration as has been reported for endogenous PSA-NCAM. Polysialylation by PST_Nm occurred in vivo in less than 2.5 h, persisted in tissues, and then decreased within a few weeks. Together these characteristics suggest that a PST_Nm-based approach may provide a valuable alternative to PST gene therapy.     

Olfactory neurons express a unique glycosylated form of the neural cell adhesion molecule (N-CAM)

mAb-based approaches were used to identify cell surface components involved in the development and function of the frog olfactory system. We describe here a 205-kD cell surface glycoprotein on olfactory receptor neurons that was detected with three mAbs: 9-OE, 5-OE, and 13-OE. mAb 9-OE immunoreactivity, unlike mAbs 5-OE and 13-OE, was restricted to only the axons and terminations of the primary sensory olfactory neurons in the frog nervous system. The 9-OE polypeptide(s) were immunoprecipitated and tested for cross-reactivity with known neural cell surface components including HNK-1, the cell adhesion molecule L1, and the neural cell adhesion molecule (N-CAM). These experiments revealed that 9-OE-reactive molecules were not L1 related but were a subset of the 200-kD isoforms of N-CAM. mAb 9-OE recognized epitopes associated with N-linked carbohydrate residues that were distinct from the polysialic acid chains present on the embryonic form of N-CAM. Moreover, 9-OE N-CAM was a heterogeneous population consisting of subsets both with and without the HNK-1 epitope. Thus, combined immunohistochemical and immunoprecipitation experiments have revealed a new glycosylated form of N-CAM unique to the olfactory system. The restricted spatial expression pattern of this N-CAM glycoform suggests a possible role in the unusual regenerative properties of this sensory system.     

Polysialic acid is associated with sodium channels and the neural cell adhesion molecule N-CAM in adult rat brain.

We have studied alpha 2,8-linked polysialic acid (polySia) and the neural cell adhesion molecule (N-CAM) in the adult rat brain by immunohistochemistry and Western blot analysis. Both molecules were widely distributed but not ubiquitous. Various brain regions showed colocalization of polySia and N-CAM. Strong immunoreactivity for polySia was seen in regions which were negative for N-CAM, such as the main and accessory olfactory bulbs. Immunohistochemical evidence for the heterogeneity of polySia expression in different brain regions was confirmed by immunoblotting. We present evidence that N-CAM is not the only polySia bearing protein in adult rat brain. Specifically, immunoprecipitation using the polySia-specific monoclonal antibody mAb 735 precipitated not only N-CAM isoforms carrying polySia, but also the sodium channel alpha subunit. Immunoblotting using sodium channel alpha subunit antibody (SP20) revealed a smear from 250 kDa upwards. PolySia removal using an endoneuraminidase specific for alpha 2,8-linked polysialic acid of 8 or more residues long, reduced this smear to a single band at 250 kDa. Thus both N-CAM and sodium channels carry homopolymers of alpha 2,8-linked polysialic acid in adult rat brain.     

Core structures of polysialylated glycans present in neural cell adhesion molecule from newborn mouse brain.

Polysialylation of the neural cell adhesion molecule (N-CAM) is known to destabilize cell-cell adhesion and to promote plasticity in cell-cell interactions. To gain more insights into the molecular mechanisms regulating the selective expression of polysialic acid on distinct glycan chains, the underlying core structures of polysialylated N-CAM glycans from newborn mouse brain were examined. Starting from low picomolar amounts of oligosaccharides, a multistep approach was used that was based on various mass spectrometric techniques with minimized sample consumption. Evidence could be provided that polysialylated murine N-CAM glycans comprise diantennary, triantennary and tetraantennary core structures carrying, in part, type-1 N-acetyllactosamine antennae, sulfate groups linked to terminal galactose or subterminal N-acetylglucosamine residues and, as a characteristic feature, a sulfated glucuronic acid unit which was bound exclusively to C3 of terminal galactose in Manalpha3-linked type-2 antennae. Hence, our results reveal that part of the murine N-CAM carbohydrates are modified within a single oligosaccharide by polysialic acid plus a HSO3-GlcA-moiety, which is likely to represent a HNK1-epitope. As HNK1-carbohydrates are also known to modulate cell-cell interactions, the simultaneous presence of both carbohydrate epitopes may reflect a new mechanism involved in the fine-tuning of N-CAM functions.