Laser microdissection of paraffin embedded tissue as a tool to estimate the sialylation status of selected cell populations.

In vertebrates, sialic acids occur at the terminal end of glycans mediating numerous biological processes like cell differentiation or tumor metastasis. Consequently, the cellular sialylation status under healthy and pathological conditions is of high interest. Existing analytical strategies to determine sialylation patterns are mostly applied to tissue samples consisting of a mixture of different cell types. Alterations in the sialylation status in a distinct area of tissues or in a specific cell population may, therefore, be easily overlooked. Likewise, estimated variations in sialylation in tissue homogenates might be simply the result of a changed cell composition. To overcome these limitations, we employed laser microdissection to isolate defined cell types or functional subunits and cell populations of paraffin embedded specimens which represent the most abundant supply of human tissue associated with clinical records. For qualitative and quantitative estimation of the sialylation status, sialic acids were released, fluorescently labeled, and analyzed by an online high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) system. As a proof of principle, this strategy was successfully applied to characterize the sialylation of the apical region of epididymal epithelial cells. Furthermore, it was possible to detect an impaired sialylation during kidney maturation in a transgenic mouse model, which was restricted to glomeruli, whereas no differences in sialylation were observed when whole kidney homogenates were used. Thus, starting from paraffin embedded tissue samples, the outlined approach offers a sensitive method to detect and quantify sialic acids on defined cell populations, which may be useful to explore novel sialic acid dependent roles during physiological and pathological processes.

Identification and biochemical characterization of two functional CMP-sialic acid synthetases in Danio rerio.

Sialic acids (Sia) form the nonreducing end of the bulk of cell surface-expressed glycoconjugates. They are, therefore, major elements in intercellular communication processes. The addition of Sia to glycoconjugates requires metabolic activation to CMP-Sia, catalyzed by CMP-Sia synthetase (CMAS). This highly conserved enzyme is located in the cell nucleus in all vertebrates investigated to date, but its nuclear function remains elusive. Here, we describe the identification and characterization of two Cmas enzymes in Danio rerio (dreCmas), one of which is exclusively localized in the cytosol. We show that the two cmas genes most likely originated from the third whole genome duplication, which occurred at the base of teleost radiation. cmas paralogues were maintained in fishes of the Otocephala clade, whereas one copy got subsequently lost in Euteleostei (e.g. rainbow trout). In zebrafish, the two genes exhibited a distinct spatial expression pattern. The products of these genes (dreCmas1 and dreCmas2) diverged not only with respect to subcellular localization but also in substrate specificity. Nuclear dreCmas1 favored N-acetylneuraminic acid, whereas the cytosolic dreCmas2 showed highest affinity for 5-deamino-neuraminic acid. The subcellular localization was confirmed for the endogenous enzymes in fractionated zebrafish lysates. Nuclear entry of dreCmas1 was mediated by a bipartite nuclear localization signal, which seemed irrelevant for other enzymatic functions. With the current demonstration that in zebrafish two subfunctionalized cmas paralogues co-exist, we introduce a novel and unique model to detail the roles that CMAS has in the nucleus and in the sialylation pathways of animal cells.

Synaptic cell adhesion molecule SynCAM 1 is a target for polysialylation in postnatal mouse brain.

Among the large set of cell surface glycan structures, the carbohydrate polymer polysialic acid (polySia) plays an important role in vertebrate brain development and synaptic plasticity. The main carrier of polySia in the nervous system is the neural cell adhesion molecule NCAM. As polySia with chain lengths of more than 40 sialic acid residues was still observed in brain of newborn Ncam(-/-) mice, we performed a glycoproteomics approach to identify the underlying protein scaffolds. Affinity purification of polysialylated molecules from Ncam(-/-) brain followed by peptide mass fingerprinting led to the identification of the synaptic cell adhesion molecule SynCAM 1 as a so far unknown polySia carrier. SynCAM 1 belongs to the Ig superfamily and is a powerful inducer of synapse formation. Importantly, the appearance of polysialylated SynCAM 1 was not restricted to the Ncam(-/-) background but was found to the same extent in perinatal brain of WT mice. PolySia was located on N-glycans of the first Ig domain, which is known to be involved in homo- and heterophilic SynCAM 1 interactions. Both polysialyltransferases, ST8SiaII and ST8SiaIV, were able to polysialylate SynCAM 1 in vitro, and polysialylation of SynCAM 1 completely abolished homophilic binding. Analysis of serial sections of perinatal Ncam(-/-) brain revealed that polySia-SynCAM 1 is expressed exclusively by NG2 cells, a multifunctional glia population that can receive glutamatergic input via unique neuron-NG2 cell synapses. Our findings sug-gest that polySia may act as a dynamic modulator of SynCAM 1 functions during integration of NG2 cells into neural networks.

Quantification of nucleotide-activated sialic acids by a combination of reduction and fluorescent labeling.

Sialic acids usually represent the terminal monosaccharide of glycoconjugates and are directly involved in many biological processes. The cellular concentration of their nucleotide-activated form is one pacemaker for the highly variable sialylation of glycoconjugates. Hence, the determination of CMP-sialic acid levels is an important factor to understand the complex glycosylation machinery of cells and to standardize the production of glycotherapeutics. We have established a highly sensitive strategy to quantify the concentration of nucleotide-activated sialic acid by a combination of reduction and fluorescent labeling using the fluorophore 1,2-diamino-4,5-methylenedioxybenzene (DMB). The labeling with DMB requires free keto as well as carboxyl groups of the sialic acid molecule. Reduction of the keto group prior to the labeling process precludes the labeling of nonactivated sialic acids. Since the keto group is protected against reduction by the CMP-substitution, labeling of nucleotide-activated sialic acids is still feasible after reduction. Subsequent combination of the DMB-high-performance liquid chromatography (HPLC) application with mass spectrometric approaches, such as matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOF-MS) and electrospray-ionization (ESI)-MS, allows the unambiguous identification of both natural and modified CMP-sialic acids and localization of potential substituents. Thus, the described strategy offers a sensitive detection, identification, and quantification of nucleotide-activated sialic acid derivatives in the femtomole range without the need for nucleotide-activated standards.

Polysialic acid governs T-cell development by regulating progenitor access to the thymus.

Although the polysialyltransferase ST8Sia IV is expressed in both primary and secondary human lymphoid organs, its product, polysialic acid (polySia), has been largely overlooked by immunologists. In contrast, polySia expression and function in the nervous system has been well characterized. In this context, polySia modulates cellular adhesion, migration, cytokine response, and contact-dependent differentiation. Provocatively, these same processes are vital components of immune development and function. We previously established that mouse multipotent hematopoietic progenitors use ST8Sia IV to express polySia on their cell surfaces. Here, we demonstrate that, relative to wild-type controls, ST8Sia IV(-/-) mice have a 30% reduction in total thymocytes and a concomitant deficiency in the earliest thymocyte precursors. T-cell progenitors originate in the bone marrow and are mobilized to the blood at regular intervals by unknown signals. We performed in vivo reconstitution experiments in which ST8Sia IV(-/-) progenitors competed with wild-type cells to repopulate depleted or deficient immune subsets. Progenitors lacking polySi exhibited a specific defect in T-cell development because of an inability to access the thymus. This phenotype probably reflects a decreased capacity of the ST8Sia IV(-/-) progenitors to escape from the bone marrow niche. Collectively, these results provide evidence that polySia is involved in hematopoietic development.

Polysialic acid, a glycan with highly restricted expression, is found on human and murine leukocytes and modulates immune responses.

Polysialic acid (polySia) is a large glycan with restricted expression, typically found attached to the protein scaffold neural cell adhesion molecule (NCAM). PolySia is best known for its proposed role in modulating neuronal development. Its presence and potential functions outside the nervous systems are essentially unexplored. Herein we show the expression of polySia on hematopoietic progenitor cells, and demonstrate a role for this glycan in immune response using both acute inflammatory and tumor models. Specifically, we found that human NK cells modulate expression of NCAM and the degree of polymerization of its polySia glycans according to activation state. This contrasts with the mouse, where polySia and NCAM expression are restricted to multipotent hematopoietic progenitors and cells developing along a myeloid lineage. Sialyltransferase 8Sia IV(-/-) mice, which lacked polySia expression in the immune compartment, demonstrated an increased contact hypersensitivity response and decreased control of tumor growth as compared with wild-type animals. This is the first demonstration of polySia expression and regulation on myeloid cells, and the results in animal models suggest a role for polySia in immune regulation.

Polysialylated neural cell adhesion molecule is involved in induction of long-term potentiation and memory acquisition and consolidation in a fear-conditioning paradigm.

Polysialic acid (PSA) regulates functions of the neural cell adhesion molecule (NCAM) during development and in neuroplasticity in the adult; the underlying mechanisms at different phases of learning and memory consolidation are, however, unknown. To investigate the contributions of PSA versus the extracellular domain of the NCAM glycoprotein backbone to synaptic plasticity, we applied NCAM, PSA-NCAM, and PSA to acute slices of the hippocampal CA1 region of NCAM-deficient mice and measured their effects on long-term potentiation (LTP). Remarkably, only PSA and PSA-NCAM, but not NCAM restored normal LTP. Application of these molecules to the dorsal hippocampus of wild-type mice showed that PSA-NCAM and PSA, but not NCAM, injected before fear conditioning, impaired formation of hippocampus-dependent contextual memory. Consolidation of contextual memory was affected by PSA-NCAM only when injected during its late, but not early phases. None of the tested compounds disturbed extrahippocampal-cued memory. Mice lacking the polysialyltransferase (ST8SialV/PST) responsible for attachment of PSA to NCAM in adulthood showed a mild deficit only in hippocampal contextual learning, when compared with NCAM-deficient mice that were disturbed in both contextual and cued memories. Contextual and tone memory in NCAM-deficient mice could be partially restored by injection of PSA-NCAM, but not of NCAM, into the hippocampus, suggesting that the impact of PSA-NCAM in synaptic plasticity and learning is not mediated by modulation of NCAM-NCAM homophilic interactions. In conclusion, our data support the view that polysialylated NCAM is involved in both formation and late consolidation of contextual memory.

In vivo synaptic plasticity in the dentate gyrus of mice deficient in the neural cell adhesion molecule NCAM or its polysialic acid.

The neural cell adhesion molecule NCAM and its associated polysialic acid (PSA) play important roles in synaptic plasticity in the CA1 and/or CA3 regions of the hippocampus in vitro. Here, we address the question of whether NCAM and PSA are involved in regulation of synaptic transmission and plasticity also in vivo at synapses formed by entorhinal cortex axons in the dentate gyrus of mice anaesthetized with urethane. We show that basal synaptic transmission, measured as the slope of field excitatory postsynaptic potentials, was reduced strongly in mice lacking ST8SiaII/STX, the enzyme involved in polysialylation of NCAM in stem cell-derived immature granule cells, but not in mice deficient either in the NCAM glycoprotein or the enzyme ST8SiaIV/PST involved in polysialylation of NCAM in mature neurons. Strikingly, only mice deficient in NCAM, but not in PST or STX, were impaired in long-term potentiation (LTP) induced by theta-burst stimulation, suggesting that LTP in the dentate gyrus depends on the NCAM glycoprotein alone rather than on its associated PSA. As also patterns of synaptic activity during and immediately after induction of LTP were impaired in NCAM-deficient mice, it is likely that induction of LTP requires NCAM. These data are the first to describe that NCAM is necessary for induction of synaptic plasticity in identified synapses in vivo and suggest that polysialylation of NCAM expressed by immature granule cells in the dentate gyrus supports development of basal excitatory synaptic transmission in this region.