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Cellular interactions with the extracellular environment are modulated by cell surface polysialic acid (PSA) carried by the neural cell adhesion molecule (NCAM). PSA-NCAM is involved in cellular processes such as differentiation, plasticity, and migration, and is elevated in Alzheimer's disease as well as in metastatic tumour cells. Our previous work demonstrated that insulin enhances the abundance of cell surface PSA by inhibiting PSA-NCAM endocytosis. In the present study we have identified a mechanism for insulin-dependent inhibition of PSA-NCAM turnover affecting cell migration. Insulin enhanced the phosphorylation of the focal adhesion kinase leading to dissociation of αv-integrin/PSA-NCAM clusters, and promoted cell migration. Our results show that αv-integrin plays a key role in the PSA-NCAM turnover process. αv-integrin knockdown stopped PSA-NCAM from being endocytosed, and αv-integrin/PSA-NCAM clusters co-labelled intracellularly with Rab5, altogether indicating a role for αv-integrin as a carrier for PSA-NCAM during internalisation. Furthermore, inhibition of p-FAK caused dissociation of αv-integrin/PSA-NCAM clusters and counteracted the insulin-induced accumulation of PSA at the cell surface and cell migration was impaired. Our data reveal a functional association between the insulin/p-FAK-dependent regulation of PSA-NCAM turnover and cell migration through the extracellular matrix. Most importantly, they identify a novel mechanism for insulin-stimulated cell migration., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Sialic acid sugars are vital regulators of the immune system through binding to immunosuppressive sialic acid-binding immunoglobulin-like lectin (Siglec) receptors on immune cells. Aberrant sialic acid-Siglec interactions are associated with an increasing number of pathologies including infection, autoimmunity, and cancer. Therefore, the sialic acid-Siglec axis is an emerging target to prevent or affect the course of several diseases. Chemical modifications of the natural sialic acid ligands have led to sialic acid mimetics (SAMs) with improved binding affinity and selectivity towards Siglecs. Recent progress in glycobiotechnology allows the presentation of these SAMs on nanoparticles, polymers, and living cells via bioorthogonal synthesis. These developments now enable the detailed study of the sialic acid-Siglec axis including its therapeutic potential as an immune modulator., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Sapovirus, a member of the Caliciviridae family, is an important cause of acute gastroenteritis in humans and pigs. Currently, the porcine sapovirus (PSaV) Cowden strain remains the only cultivable member of the Sapovirus genus. While some caliciviruses are known to utilize carbohydrate receptors for entry and infection, a functional receptor for sapovirus is unknown. To characterize the functional receptor of the Cowden strain of PSaV, we undertook a comprehensive series of protein-ligand biochemical assays in mock and PSaV-infected cell culture and/or piglet intestinal tissue sections. PSaV revealed neither hemagglutination activity with red blood cells from any species nor binding activity to synthetic histo-blood group antigens, indicating that PSaV does not use histo-blood group antigens as receptors. Attachment and infection of PSaV were markedly blocked by sialic acid and Vibrio cholerae neuraminidase (NA), suggesting a role for α2,3-linked, α2,6-linked or α2,8-linked sialic acid in virus attachment. However, viral attachment and infection were only partially inhibited by treatment of cells with sialidase S (SS) or Maackia amurensis lectin (MAL), both specific for α2,3-linked sialic acid, or Sambucus nigra lectin (SNL), specific for α2,6-linked sialic acid. These results indicated that PSaV recognizes both α2,3- and α2,6-linked sialic acids for viral attachment and infection. Treatment of cells with proteases or with benzyl 4-O-β-D-galactopyranosyl-β-D-glucopyranoside (benzylGalNAc), which inhibits O-linked glycosylation, also reduced virus binding and infection, whereas inhibition of glycolipd synthesis or N-linked glycosylation had no such effect on virus binding or infection. These data suggest PSaV binds to cellular receptors that consist of α2,3- and α2,6-linked sialic acids on glycoproteins attached via O-linked glycosylation.

Retinal ganglion cells transmit the visual signal from the retina to the brain. We have previously shown that the activator protein 2 (AP-2)δ (TFAP2D) transcription factor is expressed in one third of ganglion cells in developing retina suggesting a specialized role for these AP-2δ-expressing cells. Here, we address the role of AP-2δ in retina by in ovo electroporation of RCAS/AP-2δ retroviral constructs into the eyes of chick embryos at day 2 of gestation. Ectopic expression of AP-2δ does not affect lineage differentiation in the developing retina. However, immunostaining of retinal tissue with markers associated with axonal growth such as growth-associated protein 43 and polysialic acid-neural cell adhesion molecule (PSA-NCAM) demonstrates axonal misrouting and abnormal axonal bundling. Treatment of AP-2δ-misexpressing retinal cell cultures with endoneuraminidase, an enzyme that removes PSA from NCAM, decreases AP-2δ-induced axonal bundling. Our data suggest a role for AP-2δ in polysialylation of NCAM, with ectopic expression of AP-2δ resulting in premature bundling of emerging axons and misrouting of axons. We propose that expression of AP-2δ in a subset of ganglion cells contributes to the fine-tuning of axonal growth in the developing retina., (© 2013 International Society for Neurochemistry.)

Background: Transmissible gastroenteritis virus (TGEV) has a sialic acid binding activity that is believed to be important for enteropathogenicity, but that has so far appeared to be dispensable for infection of cultured cells. The aims of this study were to determine the effect of sialic acid binding for the infection of cultured cells under unfavorable conditions, and comparison of TGEV strains and mutants, as well as the avian coronavirus IBV concerning their dependence on the sialic acid binding activity., Methods: The infectivity of different viruses was analyzed by a plaque assay after adsorption times of 5, 20, and 60 min. Prior to infection, cultured cells were either treated with neuraminidase to deplete sialic acids from the cell surface, or mock-treated. In a second approach, pre-treatment of the virus with porcine intestinal mucin was performed, followed by the plaque assay after a 5 min adsorption time. A student's t-test was used to verify the significance of the results., Results: Desialylation of cells only had a minor effect on the infection by TGEV strain Purdue 46 when an adsorption period of 60 min was allowed for initiation of infection. However, when the adsorption time was reduced to 5 min the infectivity on desialylated cells decreased by more than 60%. A TGEV PUR46 mutant (HAD3) deficient in sialic acid binding showed a 77% lower titer than the parental virus after a 5 min adsorption time. After an adsorption time of 60 min the titer of HAD3 was 58% lower than that of TGEV PUR46. Another TGEV strain, TGEV Miller, and IBV Beaudette showed a reduction in infectivity after neuraminidase treatment of the cultured cells irrespective of the virion adsorption time., Conclusions: Our results suggest that the sialic acid binding activity facilitates the infection by TGEV under unfavorable environmental conditions. The dependence on the sialic acid binding activity for an efficient infection differs in the analyzed TGEV strains.

Functional maturation of GABAergic innervation in the developing visual cortex is regulated by neural activity and sensory inputs and in turn influences the critical period of ocular dominance plasticity. Here we show that polysialic acid (PSA), presented by the neural cell adhesion molecule, has a role in the maturation of GABAergic innervation and ocular dominance plasticity. Concentrations of PSA significantly decline shortly after eye opening in the adolescent mouse visual cortex; this decline is hindered by visual deprivation. The developmental and activity-dependent regulation of PSA expression is inversely correlated with the maturation of GABAergic innervation. Premature removal of PSA in visual cortex results in precocious maturation of perisomatic innervation by basket interneurons, enhanced inhibitory synaptic transmission, and earlier onset of ocular dominance plasticity. The developmental and activity-dependent decline of PSA expression therefore regulates the timing of the maturation of GABAergic inhibition and the onset of ocular dominance plasticity.

The large quantities of polysialic acid (PSA) characterizing highly sialylated isoform of the neural cell adhesion molecule (PSA-NCAM), greatly reduce cell adhesion and render this particular cell surface adhesion molecule a likely candidate to intervene in dynamic neuronal phenomena, such as synaptic plasticity. The hypothalamic arcuate nucleus expresses high levels of PSA-NCAM and maintains a high capacity for neuroplastic changes in the adult. Thus, in the arcuate nucleus of female rats, varying circulating levels of estrogen give rise to a reversible reduction in the number of axo-somatic GABA synapses, together with a changing ensheathing of neuronal somata by astrocytes. To examine the role of PSA in such changes, we perturbed its expression, either by blockade with antibodies raised against this carbohydrate moiety (delivered intracerebroventricularly), or by its enzymatic cleavage after microinjection of endoneuraminidase N over the arcuate nucleus. Either procedure was performed in ovariectomized adult rats that received concurrent treatment with 17 beta-estradiol. Morphological synaptic plasticity was analysed using the unbiased disector method to assess synaptic densities in ultrathin sections of the arcuate nucleus immunogold-labelled for GABA. As expected, 17 beta-estradiol induced a significant reduction in the number of GABAergic axo-somatic synapses, a reduction which did not occur after infusion of anti-PSA antibodies or in vivo enzymatic removal of PSA from NCAM. Taken together, our results provide strong evidence that the presence of large quantities of the PSA moiety on NCAM is a necessary prerequisite for estrogen-induced phasic remodelling of synapses in the adult female arcuate nucleus.

The polysialic acid (PSA) modification of the neural cell adhesion molecule (NCAM) has been shown to alter the responses of developing axons to their environment. We have studied the potential role of PSA in regulating the innervation of the spinal cord by corticospinal axons, which occurs by a delayed formation of collateral branches from the parent axons. Developmental changes in the distribution of PSA were examined immuno-histochemically using light and electron microscopy. Whereas NCAM is distributed along the entire pathway of rat corticospinal axons as they grow from the cortex to the spinal cord, PSA-modified NCAM does not become evident until later. When PSA becomes evident, it is restricted to the distal segment of these axons from the caudal hindbrain through the spinal cord. The increase in PSA on corticospinal axons coincides with the time that they begin to form collateral branches in the spinal cord. This unique spatiotemporal distribution of PSA suggests its involvement in corticospinal axon branching. To test this hypothesis, PSA was selectively removed by an in vivo injection of endoneuraminidase N. This treatment did not seem to interfere with the pathfinding of corticospinal axons; however, PSA removal delayed the onset of collateral branching by corticospinal axons within the spinal cord and later diminished the magnitude of branching. These findings indicate a role for PSA in the regulation of interstitial axon branching, a crucial step in the process of target recognition and innervation by corticospinal axons.

The influence of polysialic acid (PSA) on the neural cell adhesion molecule on motoneuron outgrowth and pathway formation was investigated by determining its temporal and spatial pattern of expression and by the effect that its removal had on motoneuron projection patterns. Motoneurons first expressed PSA as their growth cones began to segregate into motoneuron pool-specific groups in the plexus region; furthermore, PSA levels differed between motoneurons projecting to different targets. When PSA was removed during the period of axonal segregation in the plexus region projection errors were common. However, later removal during the process of muscle nerve formation did not result in projection errors. These results suggest that PSA modulates interactions between motoneuron axons and guidance molecules in the plexus region during axonal pathfinding.

During the search for inhibitors of N-acetylneuraminic acid biosynthesis, it was shown that 3-O-methyl-N-acetylglucosamine competitively inhibits the N-acetylglucosamine kinase of rat liver in vitro with a Ki value of 17 microM. N-Acetylmannosamine kinase is inhibited non-competitively with a Ki value of 80 microM. In a human hepatoma cell line (HepG2), 3-O-methyl-N-acetyl-D-glucosamine (1 mM) inhibits the incorporation of 14C-N-acetylglucosamine and 14C-N-acetylmannosamine into cellular glycoproteins by 88% and 70%, respectively.

Polysialylation is a unique posttranslational modification of NCAM. In this report, we investigated the kinetics and localization of NCAM polysialylation in AtT-20 cells. We show that this cell line expresses both the 180 kDa and 140 kDa isoforms of NCAM, in agreement with the proposal that it belongs to a neuroendocrine lineage. The two NCAM chains bear polysialic acid (PSA) and migrate in sodium dodecyl sulfate (SDS) gels as a diffuse, high Mr component, as has been observed in fetal brain. Polysialylation of neosynthesized NCAM was found to be a rapid event, occurring within 8 to 13 min after the beginning of the pulse and appeared to be essentially complete as soon as it was detected. Treatment with endosialidase specific for PSA led to the appearance of two components of 200 and 160 kDa which still bear short sialosyl oligomers. Neither this treatment nor the slowing down of synthesis by lowering the temperature revealed any intermediate bearing oligomers of polysialic acid in the process of elongation suggesting the possibility that polysialylation may involve the transfer to NCAM of preassembled completed PSA chains. Endo H resistance preceded polysialylation, which was totally blocked by monensin and swainsonine which inhibit transport of plasma membrane or secreted proteins within the Golgi complex and the maturation of complex-type oligosaccharide chains, respectively. Depletion of cell-surface NCAM with proteinase K did not prevent the appearance of polysialylated molecules in similar amounts as in untreated cells suggesting that NCAM polysialylation occurs either in a late Golgi or in a post-Golgi compartment but before the molecules reach the plasma membrane.

Polysialic acid is a developmentally regulated carbohydrate structure found on neural cell adhesion molecules (NCAM). Expression of beta-galactoside alpha 2,6-sialyltransferase in Xenopus embryos, by injection of mRNA, prevents the polysialylation of NCAM, presumably by introducing a different type of sugar linkage that terminates chain elongation. Abnormalities in neural development result from this treatment, but in general the body plan of the injected embryos is not severely affected. The results provide evidence that the mis-expression of glycosyltransferases can be used to interfere with the normal pattern of glycosylation in whole organisms.

It has been hypothesized that the metastatic capacity of tumors may be correlated with hypersialylation of the cell surface. We used a novel inhibitor of sialic acid incorporation, KI-8110, to determine the effect of depletion of cell surface sialic acid on the metastatic behavior of three human colorectal cancer cell lines, in which hepatic seeding was related to tumor cell differentiation. Treatment of tumor cells with KI-8110 prior to intrasplenic injection prevented liver colonization. Total cellular sialic acid was reduced, as was that of the cell surface. Secreted forms of carcinoembryonic antigen also were depleted of sialic acid by this treatment. These data show that depletion of sialic acid from cell surface glycoconjugates reduces the incidence of hepatic metastases from human colorectal primary tumors and adds to the mounting evidence of the importance of sialic acid in determining the biological behavior of tumor cells.

Three-days-old White Leghorn chickens were used to study the effect of i.p.-injected chlorpromazine (CP, 2.5 or 5 mg/animal) on the incorporation of N-(3H)-acetylmannosamine into glycoprotein- and ganglioside-bound sialic acid and 14C-histidine into the proteins of the cerebrum, the optic lobes, and the cerebellum after incorporation periods of 1-24 h. All experiments were performed under constant temperature conditions. 1. The incorporation of 14C-histidine into the proteins of the optic lobes was not influenced by CP. 2. Within 24 h in the optic lobes neither the concentration of particle-bound sialic acid (mug sialic acid/mg protein) nor the content of radioactive-labeled sialic acid precursor was affected by the drug. 3. The rate of incorporation of labeled sialic acid into glycoproteins and gangliosides, however, was up to 6 h after the injection of the precursor much lower in the CP-treated animals than in controls. This may indicate, that the turnover of sialo-compounds was diminished by CP. 4. The inhibition was the most intensive in the cerebrum (up to 80%), lower in the optic lobes (up to 70%), and only small in the cerebellum (up to 20%), but was always the same for glycoproteins and gangliosides. The different inhibition rates in the different structures may indicate a correlation between the pharmacologic, sedative effects of the drug and the diminished turnover of sialo compounds.

Sialic acid is secreted by the caput, corpus and cauda epididymides of an adult mature dog. 2- alpha-Chlorohydrin administration results in a considerable reduction in the sialic acid content of the epididymides. 3- Single subcutaneous injection of CdCl2 depletes the content of caput epididymides. Whereas in the corpus and cauda region, the sialic acid contents did not change. 4- The epididymides of caput and cauda ligated side showed a marked decrease in contents of sialic acid as compared with that of non-ligated control epididymides of the same animal. Bilateral vasectomy and 72 hour vascular occlusion resulted in the reduction in the sialic acid content of the epididymides. 5- It is interesting to note that the decrease in sialic acid in the epididymides coincided with the absence of sperms and reduced secretory activity of the luminal epithelium. 6- The significance of these changes is discussed.