Brain structure, cognition and negative symptoms in schizophrenia are associated with serum levels of polysialic acid-modified NCAM.

The neural cell adhesion molecule (NCAM) is a glycoprotein implicated in cell-cell adhesion, neurite outgrowth and synaptic plasticity. Polysialic acid (polySia) is mainly attached to NCAM (polySia-NCAM) and has an essential role in regulating NCAM-dependent developmental processes that require plasticity, that is, cell migration, axon guidance and synapse formation. Post-mortem and genetic evidence suggests that dysregulation of polySia-NCAM is involved in schizophrenia (SZ). We enrolled 45 patients diagnosed with SZ and 45 healthy individuals who were submitted to polySia-NCAM peripheral quantification, cognitive and psychopathological assessment and structural neuroimaging (brain volumes and diffusion tensor imaging). PolySia-NCAM serum levels were increased in SZ patients, independently of antipsychotic treatment, and were associated with negative symptoms, blunted affect and declarative memory impairment. The increased polySia-NCAM levels were associated with decreased volume in the left prefrontal cortex, namely Brodmann area 46, in patients and increased volume in the same brain area of healthy individuals. As this brain region is involved in the pathophysiology of SZ and its associated phenomenology, the data indicate that polySia-NCAM deserves further scrutiny because of its possible role in early neurodevelopmental mechanisms of the disorder.

Deficiency of neural cell adhesion molecule or its polysialylation modulates pharmacological effects of the AMPA receptor antagonist NBQX.

The neural cell adhesion molecule NCAM and its dynamically regulated posttranslational modification polysialic acid (PSA) are major determinants of cellular interactions during ontogeny. While NCAM in the absence of PSA stabilizes cell-cell interactions, the attachment of the large and polyanionic PSA negatively influences cell adhesion and promotes plasticity. Disease-associated changes in the polysialylation state of NCAM raise the question whether the PSA-NCAM system can affect CNS pharmacology. Here we investigated the pharmacological effects of the competitive AMPA antagonist NBQX in genetic mouse models either lacking NCAM and PSA (female NCAM knockout mice) or being drastically reduced in the level of PSA expression (female ST8SiaIV knockout mice). Studies were carried out with the respective wildtype littermate controls. In mice lacking NCAM and PSA, NBQX-induced ataxia proved to be more intense as compared with wild-type mice. On both mutant backgrounds, NBQX significantly elevated seizure thresholds during i.v. infusion of the chemoconvulsant pentylenetetrazole. In summary, the data demonstrate that the PSA-NCAM system impacts AMPA receptor pharmacology under in vivo conditions. The fact that comparable effects were observed in NCAM- and ST8SiaIV-knockout mice indicates that this impact is not due to a stabilizing effect of NCAM in the absence of PSA. Thus, disease-related changes in the polysialylation of NCAM are likely to be associated with effects on the efficacy and tolerability of AMPA receptor antagonists.

Selective learning and memory impairments in mice deficient for polysialylated NCAM in adulthood.

The neural cell adhesion molecule (NCAM) has been implicated in regulating synaptic plasticity mechanisms as well as memory consolidation processes. Attachment of polysialic acid to NCAM (PSA-NCAM) has been reported to down-regulate its adhesive forces, a process hypothesized to be implicated in synapse selection after learning experiences. PSA-NCAM has been critically implicated in hippocampus-related synaptic plasticity and memory storage, but information about its functional role in other brain areas remains scarce. Here, we studied mice deficient for polysialyltransferase-1 (ST8SialV/PST-1), an enzyme which attaches PSA to NCAM during postnatal development and adulthood, and whose deficiency results in a drastic reduction of PSA-NCAM expression throughout the brain in adulthood. Mice were tested for their performance in the water maze and auditory fear conditioning (AFC). We report that ST8SiaIV knockout mice were impaired in spatial as well as reversal learning in the water maze. On the other hand, AFC was intact and ST8SiaIV mice exhibited no impairments in the acquisition or retention of cued fear memories. Spatial orientation learning and reversal learning require complex integration of spatial information and response selection involving the hippocampus and prefrontal cortex, whereas cued fear conditioning is an associative type of emotional memory that highly depends on amygdala function. Therefore, our results indicate that PSA-NCAM contributes differentially to learning processes that differ in the nature of the neural computations involved, which probably reflects a differential role of this molecule in different brain regions.

Culturing of glial and neuronal cells on polysialic acid.

Although peripheral nerves exhibit regeneration capacities after transection injuries, the success of nerve repair depends crucially on the length of the gap. In addition to autologous nerve grafting as the conventional neurosurgical treatment to overcome long gaps, alternative strategies are needed. Numerous experimental studies have been undertaken to find the optimal material for production of artificial prostheses, which can be introduced as conduits between the nerve stumps. The current study follows the aim to establish polysialic acid (polySia), a homopolymer of alpha2,8-linked sialic acid residues, as a novel, biocompatible, and bioresorbable material for nerve tissue engineering. As a first step towards this goal, protocols for efficient coating of cell culture dishes with soluble polySia were established. In addition, primary nerve cells which are candidates for reconstructive therapies, including neonatal and adult Schwann cells, neural progenitor cells, spinal ganglionic neurons and motoneurons were cultured on polySia substrates. Cultures were evaluated with regard to cell survival and cell proliferation capacities. polySia turned out to be stable under cell culture conditions, and induced degradable and degradation products had no negative effects on cell cultures. Furthermore, polySia revealed its compatibility for several cell types derived from rat embryonic, postnatal and adult nervous tissue when used as a substrate.

Immunoelectron microscopic localization of the neural recognition molecules L1, NCAM, and its isoform NCAM180, the NCAM-associated polysialic acid, beta1 integrin and the extracellular matrix molecule tenascin-R in synapses of the adult rat hippocampus.

We have investigated the possibility that morphologically different excitatory glutamatergic synapses of the "trisynaptic circuit" in the adult rodent hippocampus, which display different types of long-term potentiation (LTP), may express the immunoglobulin superfamily recognition molecules L1 and NCAM, the extracellular matrix molecule tenascin-R, and the extracellular matrix receptor constituent beta1 integrin in a differential manner. The neural cell adhesion molecules L1, NCAM (all three major isoforms), NCAM180 (the largest major isoform with the longest cytoplasmic domain), beta1 integrin, polysialic acid (PSA) associated with NCAM, and tenascin-R were localized by pre-embedding immunostaining procedures in the CA3/CA4 region (mossy fiber synapses) and in the dentate gyrus (spine synapses) of the adult rat hippocampus. Synaptic membranes of mossy fiber synapses where LTP is expressed presynaptically did not show detectable levels of immunoreactivity for any of the molecules/epitopes studied. L1, NCAM, and PSA, but not NCAM180 or beta1 integrin, were detectable on axonal membranes of fasciculating mossy fibers. In contrast to mossy fiber synapses, spine synapses in the outer third of the molecular layer of the dentate gyrus, which display postsynaptic expression mechanisms of LTP, were both immunopositive and immunonegative for NCAM, NCAM180, beta1 integrin, and PSA. Those spine synapses postsynaptically immunoreactive for NCAM or PSA also showed immunoreactivity on their presynaptic membranes. NCAM180 was not detectable presynaptically in spine synapses. L1 could not be found in spine synapses either pre- or postsynaptically. Also, the extracellular matrix molecule tenascin-R was not detectable in synaptic clefts of all synapses tested, but was amply present between fasciculating axons, axon-astrocyte contact areas, and astrocytic gap junctions. Differences in expression of the membrane-bound adhesion molecules at both types of synapses may reflect the different mechanisms for induction and/or maintenance of synaptic plasticity.

Nucleotide sugar transporters: biological and functional aspects.

The Golgi apparatus serves as the major site of glycosylation reactions. Nucleotide sugars which are substrates of the Golgi localized glycosyltransferases are synthesized in the cytoplasm (cell nucleus in case of CMP-sialic acid) and must be transported into the compartment lumen. This transport function is carried out by nucleotide sugar transporters. The first genes were cloned in the year 1996 and revealed a family of structurally conserved multi-transmembrane-spanning proteins. Due to the high structural and functional conservation, the identification of many putative nucleotide sugar transporter sequences has become possible in the existing gene data bases and accelerates the increase in knowledge on structure-function-relationships. Recent developments in the nucleotide sugar transporter field are discussed in this article.

Molecular cloning of a unique CMP-sialic acid synthetase that effectively utilizes both deaminoneuraminic acid (KDN) and N-acetylneuraminic acid (Neu5Ac) as substrates.

2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) is a sialic acid (Sia) that is ubiquitously expressed in vertebrates during normal development and tumorigenesis. Its expression is thought to be regulated by multiple biosynthetic steps catalyzed by several enzymes, including CMP-Sia synthetase. Using crude enzyme preparations, it was shown that mammalian CMP-Sia synthetases had very low activity to synthesize CMP-KDN from KDN and CTP, and the corresponding enzyme from rainbow trout testis had high activity to synthesize both CMP-KDN and CMP-N-acetylneuraminic acid (Neu5Ac) (Terada et al. [1993] J. Biol. Chem., 268, 2640-2648). To demonstrate if the unique substrate specificity found in the crude trout enzyme is conveyed by a single enzyme, cDNA cloning of trout CMP-Sia synthetase was carried out by PCR-based strategy. The trout enzyme was shown to consist of 432 amino acids with two potential nuclear localization signals, and the cDNA sequence displayed 53.8% identity to that of the murine enzyme. Based on the Vmax/Km values, the recombinant trout enzyme had high activity toward both KDN and Neu5Ac (1.1 versus 0.68 min(-1)). In contrast, the recombinant murine enzyme had 15 times lower activity toward KDN than Neu5Ac (0.23 versus 3.5 min(-1)). Northern blot analysis suggested that several sizes of the mRNA are expressed in testis, ovary, and liver in a tissue-specific manner. These results indicate that at least one cloned enzyme has the ability to utilize both KDN and Neu5Ac as substrates efficiently and is useful for the production of CMP-KDN.

Characterization of N-glycans from mouse brain neural cell adhesion molecule.

The N-glycosylation pattern of the neural cell adhesion molecule (NCAM), isolated from brains of newborn mice, has been analyzed. Following digestion with trypsin, generated glycopeptides were fractionated by serial immunoaffinity chromatography using immobilized monoclonal antibodies specifically recognizing polysialic acid (PSA) units or the HNK1-carbohydrate epitope. Subsequent analyses of the resulting (glyco)peptides by Edman degradation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) revealed polysialylated glycans to be exclusively linked to glycosylation sites 5 (Asn(431)) and 6 (Asn(460)), whereas glycans carrying the HNK1-epitope could be assigned to sites 2 (Asn(297)), 5, 6, and, to a lesser extent, site 3 (Asn(329)). PSA-, HNK1-, and non-PSA/HNK1-glycan fractions were characterized by carbohydrate constituent and methylation analyses as well as MALDI-TOF-MS in conjunction with chromatographic fractionation techniques. The results revealed that the core structures of PSA-glycans represented predominantly fucosylated, partially sulfated 2,6-branched isomers of triantennary as well as tetraantennary complex-type glycans, whereas carbohydrate chains bearing the HNK1-epitope were dominated by diantennary species carrying in part bisecting GlcNAc residues. Non-PSA/HNK1-glycans exhibited a highly heterogeneous pattern of partially truncated, mostly diantennary structures being characterized by the presence of additional fucose, bisecting GlcNAc and/or sulfate residues. In conclusion, our results revealed that the glycosylation pattern of murine NCAM displays high structural and regional selectivity, which might play an important role in controlling the biological activities of this molecule.

The impact of N-glycosylation on the functions of polysialyltransferases.

Poly-alpha-2,8-sialic acid (polysialic acid) is a post-translational modification of the neural cell adhesion molecule (NCAM) and an important regulator of neuronal cell-cell interactions. The synthesis of polysialic acid depends on the two polysialyltransferases ST8SiaII and ST8SiaIV. Understanding the catalytic mechanisms of the polysialyltransferases is critical toward the aim of influencing physiological and pathophysiological functions mediated by polysialic acid. We recently demonstrated that polysialyltransferases are bifunctional enzymes exhibiting auto- and NCAM polysialylation activity. Autopolysialylation occurs on N-glycans of the enzymes, and glycosylation variants lacking sialic acid and galactose were found to be inactive for both auto- and NCAM polysialylation. In the present study, we have analyzed the number and functional importance of N-linked oligosaccharides present on polysialyltransferases. We demonstrate that autopolysialylation depends on specific N-glycans attached to Asn(74) in ST8SiaIV and Asn(89) and Asn(219) in ST8SiaII. Deletion of polysialic acid acceptor sites by site-directed mutagenesis rendered the polysialyltransferases inactive in vitro and in vivo. The inactivity of autopolysialylation-negative polysialyltransferases in vivo was not caused by the absence or default targeting of the enzymes. The data presented in this study clearly show that active polysialyltransferases are competent to perform autopolysialylation and provide strong evidence for a tight functional link between the two catalytic functions.

The gene defective in leukocyte adhesion deficiency II encodes a putative GDP-fucose transporter.

Leukocyte adhesion deficiency II (LAD II) is characterized by the lack of fucosylated glycoconjugates, including selectin ligands, causing immunodeficiency and severe mental and growth retardation. No deficiency in fucosyltransferase activities or in the activities of enzymes involved in GDP-fucose biosynthesis has been found. Instead, the transport of GDP-fucose into isolated Golgi vesicles of LAD II cells appeared to be reduced. To identify the gene mutated in LAD II, we cloned 12 cDNAs from Caenorhabditis elegans, encoding multi-spanning transmembrane proteins with homology to known nucleotide sugar transporters, and transfected them into fibroblasts from an LAD II patient. One of these clones re-established expression of fucosylated glycoconjugates with high efficiency and allowed us to identify a human homolog with 55% identity, which also directed re-expression of fucosylated glycoconjugates. Both proteins were localized to the Golgi. The corresponding endogenous protein in LAD II cells had an R147C amino acid change in the conserved fourth transmembrane region. Overexpression of this mutant protein in cells from a patient with LAD II did not rescue fucosylation, demonstrating that the point mutation affected the activity of the protein. Thus, we have identified the first putative GDP-fucose transporter, which has been highly conserved throughout evolution. A point mutation in its gene is responsible for the disease in this patient with LAD II.

Point mutations identified in Lec8 Chinese hamster ovary glycosylation mutants that inactivate both the UDP-galactose and CMP-sialic acid transporters.

Nucleotide-sugar transporters (NSTs) are critical components of glycosylation pathways in eukaryotes. The identification of structural elements that are involved in NST functions provides an important task. Chinese hamster ovary glycosylation mutants defective in nucleotide-sugar transport provide access to inactive transporters that can define such structure/function relationships. In this study, we have cloned the hamster UDP-galactose transporter (UGT) and identified defects in UGT gene transcripts from nine independent Chinese hamster ovary mutants that belong to the Lec8 complementation group. Reverse transcription polymerase chain reaction with primers that span the UGT open reading frame showed that three Lec8 mutants express a full-length open reading frame, while six Lec8 mutants predominantly express truncated UGT gene transcripts. Sequencing identified different single or triplet nucleotide changes in full-length UGT transcripts from three of the mutants. These mutations translate into three different amino acid changes at positions that are highly conserved in all the known mammalian NSTs. Transfection of a cDNA encoding either of the mutations Delta serine 213 or G281D failed to correct the UDP-galactose transport defect in Lec8 transfectants. Most importantly, introducing these same mutations into the homologous region of the murine CMP-sialic acid transporter caused inactivation of this transporter. Thus, identifying point mutations that inactivate UGT in Lec8 mutants resulted in the discovery of amino acids that are critical to the activity of both UGT and CST, the two most divergent mammalian NSTs.

Control of NCAM polysialylation by the differential expression of polysialyltransferases ST8SiaII and ST8SiaIV.

Polysialic acid (PSA) is a developmentally regulated carbohydrate consisting of alpha-2,8-linked sialic acid residues attached to the neural cell adhesion molecule NCAM. PSA promotes plasticity of cell-cell interactions in the nervous system and appears linked to the malignant potential of several tumors. Two enzymes, the polysialyltransferases ST8SiaII (STX) and ST8SiaIV (PST) have been identified and shown to be independently able to synthesize PSA. However, in vivo studies have demonstrated that in the majority of PSA-positive tissues the two polysialyltransferases are expressed simultaneously. Therefore, this study was undertaken to elucidate in which way the individual enzymes contribute to PSA expression under in vivo conditions. Using a semiquantitative RT-PCR strategy PSA-positive human tumor cell lines were screened for expression of ST8SiaII and ST8SiaIV at the mRNA level. Divergent patterns observed in some cell lines suggest that polysialyltransferases are independently regulated at the transcriptional level. In subsequent analyses the different mRNA levels of ST8SiaII and ST8SiaIV in these tumor cells were correlated with the degree of PSA expression and the cellular capacity to rapidly synthesize PSA. Our data indicate that ST8SiaIV is the major regulator of NCAM polysialylation in vivo.

A high-throughput assay for rat liver golgi and Saccharomyces cerevisiae-expressed murine CMP-N-acetylneuraminic acid transport proteins.

Rat liver Golgi and Saccharomyces cerevisiae-expressed CMP-Neu5Ac transport protein were reconstituted in phosphatidylcholine liposomes and transport of CMP-Neu5Ac into these proteoliposomes was determined. The separation of transported substrate from free substrate was performed using Multiscreen minicolumns loaded with Sephadex G-50 resin (fine). The CMP-Neu5Ac transport characteristics of the rat liver Golgi and S. cerevisiae-expressed transporters, determined using this separation system, were very similar to those previously reported. Inhibition studies, utilizing the above procedure, revealed that the main structural features required for recognition of glycosyl nucleosides by the rat liver Golgi CMP-Neu5Ac transport protein were the nature of the nucleoside base and the anomeric configuration of the associated carbohydrate. In general, pyrimidine-based glycosyl nucleosides were found to inhibit transport to a far greater extent than purine-based glycosyl nucleosides, an observation that is in good agreement with previous reports. These results indicate that the reconstitution procedure, in conjunction with Multiscreen minicolumns, is an effective high-throughput method for the determination of CMP-Neu5Ac transport.

Molecular defects that cause loss of polysialic acid in the complementation group 2A10.

Polysialic acid (PSA) is a dynamically regulated posttranslational modification of the neural cell adhesion molecule (NCAM), which modulates NCAM binding functions. PSA biosynthesis is catalyzed by two polysialyltransferases, ST8SiaII and ST8SiaIV. The catalytic mechanisms of these enzymes are unknown. In Chinese hamster ovary cells, ST8SiaIV is responsible for PSA expression. In the complementation group 2A10, the ST8SiaIV gene is disrupted. Investigating the molecular defects in this complementation group, seven clones with missense mutations in ST8SiaIV were found. Mutations cause replacement of amino acids that are highly conserved in alpha2,8-sialyltransferases. To verify the physiological relevance of identified mutations, identical amino acid substitutions were introduced into epitope-tagged variants of hamster ST8SiaIV and murine ST8SiaII and recombinant proteins were tested in vivo and in vitro. None of these constructs reconstituted PSA synthesis in 2A10 cells, although the proteins were expressed and with the exception of the cysteine variants ST8SiaIV-C356F and ST8SiaII-C371F correctly targeted to the Golgi apparatus. Interestingly, two mutations (ST8SiaIV-R277G and -M333V and the corresponding mutants ST8SiaII-R292G and -M348V) could be partially rescued if tested in vitro. Although these mutants were negative for autopolysialylation, partial reconstitution of both auto- and NCAM polysialylation was achieved in the presence of NCAM. The data presented in this study suggest a functional link between auto- and NCAM polysialylation.

Mice deficient in the polysialyltransferase ST8SiaIV/PST-1 allow discrimination of the roles of neural cell adhesion molecule protein and polysialic acid in neural development and synaptic plasticity.

Functional properties of the neural cell adhesion molecule (NCAM) are strongly influenced by polysialylation. We used gene-targeting to generate mice lacking ST8SiaIV/PST-1, one of the polysialyltransferases responsible for addition of polysialic acid (PSA) to NCAM. Mice homozygous for the null mutation reveal normal development of gross anatomical features. In contrast to NCAM-deficient mice, olfactory precursor cells in the rostral migratory stream express PSA and follow their normal pathway. Furthermore, delamination of mossy fibers in the hippocampal CA3 region, as found in NCAM-deficient mice, does not occur in ST8SiaIV mutants. However, during postnatal development these animals show a decrease of PSA in most brain regions compared to wild-type animals. Loss of PSA in the presence of NCAM protein but in the absence of obvious histological changes allowed us to directly address the role of PSA in synaptic plasticity. Schaffer collateral-CA1 synapses, which express PSA in wild types, showed impaired long-term potentiation (LTP) and long-term depression (LTD) in adult mutants. This impairment was age-dependent, following the time course of developmental disappearance of PSA. Contrary to NCAM mutant mice, LTP in ST8SiaIV mutants was undisturbed at mossy fiber-CA3 synapses, which do not express PSA in wild-type mice. The results demonstrate an essential role for ST8SiaIV in synaptic plasticity in hippocampal CA1 synapses, whereas PSA produced by different polysialyltransferase or polysialyltransferases at early stages of differentiation regulates migration of neural precursor cells and correct lamination of mossy fibers. We suggest that NCAM but not PSA is likely to be important for LTP in the hippocampal CA3 region.

Membrane topology of the mammalian CMP-sialic acid transporter.

Nucleotide sugar transporters form a family of distantly related membrane proteins of the Golgi apparatus and the endoplasmic reticulum. The first transporter sequences have been identified within the last 2 years. However, information about the secondary and tertiary structure for these molecules has been limited to theoretical considerations. In the present study, an epitope-insertion approach was used to investigate the membrane topology of the CMP-sialic acid transporter. Immunofluorescence studies were carried out to analyze the orientation of the introduced epitopes in semipermeabilized cells. Both an amino-terminally introduced FLAG sequence and a carboxyl-terminal hemagglutinin tag were found to be oriented toward the cytosol. Results obtained with CMP-sialic acid transporter variants that contained the hemagglutinin epitope in potential intermembrane loop structures were in good correlation with the presence of 10 transmembrane regions. This building concept seems to be preserved also in other mammalian and nonmammalian nucleotide sugar transporters. Moreover, the functional analysis of the generated mutants demonstrated that insertions in or very close to membrane-spanning regions inactivate the transport process, whereas those in hydrophilic loop structures have no detectable effect on the activity. This study points the way toward understanding structure-function relationships of nucleotide sugar transporters.

Expression of the polysialyltransferase ST8SiaIV: polysialylation interferes with adhesion of PC12 cells in vitro.

Addition of polysialic acid (PSA) to the neural cell adhesion molecule, NCAM, represents a unique posttranslational modification. Polysialylation of NCAM is developmentally regulated and associated with neural regeneration and plastic processes, as well as learning and memory. Two enzymes, the polysialyltransferases ST8SiaII and ST8SiaIV, are known to be involved in the polysialylation of NCAM. Both enzymes are individually capable of catalyzing polysialylation of NCAM, but their time of occurrence and their tissue expression are different. In this study the influence of polysialylation on the nerve growth factor-induced differentiation of PC12 cells was investigated. For this purpose, PC12 cells, which endogenously express NCAM, were transfected with ST8SiaIV to produce, for the first time, a stable polysialylated PC12 cell. We demonstrate that integrin-dependent adhesion to collagen I is reduced in PSA-expressing PC12 cells. Furthermore, polysialylated cell membranes as matrix are a poor substrate for the adhesion and differentiation of PC12 cells, compared with normal cell membranes.

The presence of N-acetylneuraminic acid in Malpighian tubules of larvae of the cicada Philaenus spumarius.

Sialic acid-containing glycoconjugates are generally considered to be unique to the deuterostomes, a lineage of the animal kingdom which includes animals from the echinoderms up to the vertebrates. There are, however, two isolated reports of sialic acid occurring in the insect species Drosophila melanogaster and Galleria mellonella. Since insects are classified as protostomes, these findings call previous assumption on the phylogenetic distribution and thus on the evolution of sialic acids into question. Here, we report the occurrence of N-acetylneuraminic acid (Neu5Ac) in larvae of the cicada Philaenus spumarius. Cytochemical analysis of larval sections with lectins from Sambucus nigra and Limax flavus suggested the presence of sialic acids in the concrement vacuoles of the Malpighian tubules. The monoclonal antibody MAb 735, which is specific for polysialic acid, labelled the same structures. A chemical analysis performed by HPLC of fluorescent derivatives of sialic acids and by GLC-MS provided sound evidence for the presence of Neu5Ac in the Philaenus spumarius larvae. These data suggest that in this cicada Neu5Ac occurs in alpha2,8-linked polysialic acid structures and in alpha2,6-linkages. The results provide further evidence for the existence of sialic acids in insects and in linkages known to occur in glycoconjugates of deuterostomate origin.

Heterogeneous expression of the polysialyltransferases ST8Sia II and ST8Sia IV during postnatal rat brain development.

Polysialic acid on the neural cell adhesion molecule is developmentally regulated and has been implicated in the plasticity of cell-cell interactions. The sialyltransferases ST8Sia II and ST8Sia IV are able to catalyze the synthesis of polysialic acid. This study compares the expression of ST8Sia II and ST8Sia IV mRNA during postnatal rat brain development. Northern blot analysis indicated a substantial down-regulation of ST8Sia II from high expression at postnatal day 2 to almost undetectable levels at the age of 6 months. In contrast, the decline of ST8Sia IV content was moderate. In the mature brain, ST8Sia IV is the predominant polysialyltransferase. In situ hybridization of selected brain regions at postnatal days 2, 11, and 21 confirmed the decline of ST8Sia II level in isocortex, hippocampus, and cerebellum. ST8Sia II was not detectable at any time point in the subependymal layer and the layers of the olfactory bulb. Persistent ST8Sia IV expression was localized in the subependymal layer, the glomerular layer of the olfactory bulb, and the granule cell layer of the dentate gyrus and in some widely dispersed cells of the isocortex. The distinct expression patterns of ST8Sia II and ST8Sia IV suggest their differential regulation. As discussed with regard to the persistent polysialic acid expression, ST8Sia IV should receive particular attention in the mature brain.

Polysialylated neural cell adhesion molecule serum levels in normal children.

Serum concentrations of polysialylated neural cell adhesion molecule (PSA-NCAM), a developmentally regulated form of the NCAM, have been recently described to be elevated in children with rhabdomyosarcoma and neuroblastoma, proving PSA-NCAM to be a tumor marker of diagnostic relevance to these malignancies. The present investigation was undertaken to define age-dependent reference intervals in normal children. Serum concentrations of polysialylated NCAM were determined in 366 children aged newborn to 17 y and in 18 adult patients by an immunoluminescence assay using the polysialic acid-specific MAb 735. Serum levels in newborn children were 51.7 kU/L (mean +/- 12.0 kU/L SD), whereas in adult patients they were 9.9 kU/L (mean +/- 3.5 kU/l SD). Assigning the patients to 14 different age groups, a gradual decay of PSA-NCAM serum concentrations was observed, and therefore, mean levels and empirical interpolated percentiles were determined for every age group. Applying specially fitted logistic functions, two different sigmoid graphs were obtained describing the age-dependent decrease of serum PSA-NCAM during the neonatal period and during childhood. The age at which the levels reach half the initial value was located at 3.1 d (mean +/- 2 d SE) and 14 y (mean +/- 1 y SE), respectively. There was no difference between male and female individuals. Repeated measurements revealed variations below 10%. For the first time, our study describes serum levels of PSA-NCAM in children of different age and their gradual decay until adulthood.