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Background: In order to develop novel clinical applications and to gain insights into possible therapeutic mechanisms, detailed molecular characterization of human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) is needed. Neural cell adhesion molecule (NCAM, CD56) is a transmembrane glycoprotein modulating cell-cell and cell-matrix interactions. An additional post-translational modification of NCAM is the α2,8-linked polysialic acid (polySia). Because of its background, NCAM is often considered a marker of neural lineage commitment. Generally, hBM-MSCs are considered to be devoid of NCAM expression, but more rigorous characterization is needed. Methods: We have studied NCAM and polySia expression in five hBM-MSC lines at mRNA and protein levels. Cell surface localization was confirmed by immunofluorescence staining and expression frequency in the donor-specific lines by flow cytometry. For the detection of poorly immunogenic polySia, a fluorochrome-tagged catalytically defective enzyme was employed. Results: All five known NCAM isoforms are expressed in these cells at mRNA level and the three main isoforms are present at protein level. Both polysialyltransferases, generally responsible for NCAM polysialylation, are expressed at mRNA level, but only very few cells express polySia at the cell surface. Conclusions: Our results underline the need for a careful control of methods and conditions in the characterization of MSCs. This study shows that, against the generally held view, clinical-grade hBM-MSCs do express NCAM. In contrast, although both polysialyltransferase genes are transcribed in these cells, very few express polySia at the cell surface. NCAM and polySia represent new candidate molecules for influencing MSC interactions. [ABSTRACT FROM AUTHOR]

Polysialic acid (polySia) is a unique linear homopolymer of α2,8-linked sialic acid that has been studied extensively as a posttranslational modification of neural cell adhesion molecule in the central nervous system. Only two proteins are known to be polysialylated in cells of the immune system: CD56 on human natural killer cells and murine bone marrow (BM) leukocytes, and neuropilin-2 (NRP-2) on dendritic cells (DCs). We tested the hypothesis that polySia expression is regulated during maturation and migration of leukocytes and plays a role in functional activity. Using wild-type and NCAM−/− mice, we show that BM neutrophils express only polysialylated CD56, whereas a subset of BM monocytes expresses polysialylated CD56 and/or another polysialylated protein(s). We demonstrate that polysialylated CD56 expression is progressively down-regulated in wild-type monocytes and monocyte-derived cells during migration from BM through peripheral blood to pulmonary and peritoneal sites of inflammation. Freshly isolated monocyte-derived peritoneal macrophages are devoid of polySia yet re-express polySia on NRP-2 and an additional protein(s) after maintenance in culture. Removal of polySia from these cells enhances phagocytosis of Klebsiella pneumoniae, suggesting that down-regulation of polySia on macrophages facilitates bacterial clearance. Using wild-type and NRP-2−/− mice, we demonstrate that NRP-2 and an additional protein(s) are polysialylated by ST8 SiaIV in BM-derived DCs. We conclude that polySia expression in monocyte-derived cells is dynamically regulated by ST8 SiaIV activity and by expression of carrier proteins during recruitment to sites of inflammation and influences cellular interactions with microbes, contributing to innate and adaptive immune responses. [ABSTRACT FROM AUTHOR]

Background: The aim of the study was to assess the localization of Polysialic acid (polySia) and Neural cell adhesion molecule (NCAM) in grade I-IV astrocytomas by confocal microscopy, and also to clarify and compare their relationship to conventional clinicopathological features in these tumors. Methods: Study material was stained immunohistochemically for polySia, NCAM and IDH1-R132H point mutation. Confocal microscopy of polySia and NCAM staining was performed on tissue micro-array samples (TMA) of 242 diffusely infiltrating astrocytomas (grade II: 28; grade III: 33; grade IV: 181) and 82 pilocytic astrocytomas. The results were statistically correlated to clinicopathological factors and survival data. Results: PolySia was observed in 45 cases (19%) and NCAM positivity in 92 cases (38%). All 45 tumors with polySia positivity were also positive for NCAM whereas there were 47 tumors which contained positive staining for NCAM but not for polySia. The simultaneous expression was concomitant and colocalized suggesting polysialyated NCAM (polySia-NCAM). PolySia expression was significantly stronger in IDH1 mutated tumors than in IDH1 non-mutated (p = 0.001, chi-square test). There were no significant differences in polySia-NCAM between primary tumors or recurrences (p = n.s., chi-square test). PolySia positivity was associated with longer patient survival in relation to total tumor material (p = 0.020, log-rank test). Furthermore, when only glioblastomas were assessed, patients with positive polySia had significantly better prognosis (p = 0.006, log-rank test). In multivariate survival analysis, polySia was found to be an independent prognostic factor. PolySia was nearly absent in grade I pilocytic astrocytomas (1 immunopositive tumor of 82). Conclusions: Expression of polySia is common in adult grade II-IV astrocytomas, whereas it is nearly absent in pediatric grade I pilocytic astrocytomas. PolySia positivity is associated with longer survival rates in patients with a grade II-IV astrocytomas and also grade IV glioblastomas assessed separately. The results of this study suggest that IDH1 mutation may be associated with polySia expression pathways in malignant gliomas. [ABSTRACT FROM AUTHOR]

Polysialic acid (polySia) is mainly described as a glycan modification of the neural cell adhesion molecule NCAM1. PolySia-NCAM1 has multiple functions during the development of vertebrate nervous systems including axon extension and fasciculation. Phylogenetic analyses reveal the presence of two related gene clusters, NCAM1 and NCAM2, in tetrapods and fishes. Within the ncam1 cluster, teleost fishes express ncam1a (ncam) and ncam1b (pcam) as duplicated paralogs which arose from a second round of ray-finned fish-specific genome duplication. Tetrapods, in contrast, express a single NCAM1 gene. Using the zebrafish model, we identify Ncam1b as a novel major carrier of polySia in the nervous system. PolySia-Ncam1a is expressed predominantly in rostral regions of the developing nervous system, whereas polySia-Ncam1b prevails caudally. We show that ncam1a and ncam1b have different expression domains which only partially overlap. Furthermore, Ncam1a and Ncam1b and their polySia modifications serve different functions in axon guidance. Formation of the posterior commissure at the forebrain/midbrain junction requires polySia-Ncam1a on the axons for proper fasciculation, whereas Ncam1b, expressed by midbrain cell bodies, serves as an instructive guidance cue for the dorso-medially directed growth of axons. Spinal motor axons, on the other hand, depend on axonally expressed Ncam1b for correct growth toward their target region. Collectively, these findings suggest that the genome duplication in the teleost lineage has provided the basis for a functional diversification of polySia carriers in the nervous system. [ABSTRACT FROM PUBLISHER]

Abstract: We have used a conceptually novel way to construct antibody mimics based on the binding of a noncatalytic enzyme to its substrate. Bacteriophage-derived endosialidase cleaves polysialic acid (polySia), an important oncofetal and bacterial antigen, which is poorly immunogenic. We fused to green fluorescent protein (GFP) a catalytically inactive endosialidase known to bind but not degrade polysialic acid. The fusion protein is a convenient single-step reagent in fluorescence microscopy, binding assays and immunoblots. It efficiently and specifically detected polysialic acid in developing brain, neuroblastoma cells and bacteria causing meningitis. Enzyme–substrate interactions represent an unexploited source of molecular recognition events. Some of these could be used in designing well-defined substitute antibodies for the study of target molecules which are difficult to purify, available in low quantities, are unstable or have poor immunogenity. [Copyright &y& Elsevier]

Long-chain polysialic acid (PSA) is expressed on the vertebrate neural cell adhesion molecule (NCAM) during neuronal plasticity. Its structural similarity to the capsular PSAs of some pathogenic bacteria has hampered the development of polysaccharide vaccines against meningitis. The antibodies formed during immunization require a long epitope for binding, and cross-react with host tissue PSA. The nature of the epitope and possible external effectors involved are still unclear. We have evaluated the interaction of PSA with its antibody mAb735 by surface plasmon resonance. The influences of PSA chain length, pH, temperature, ionic environment, and polyamines were also determined.The antibody binding affinity was found to dramatically increase with PSA chain length. A sub-nanomolar dissociation constant (KD=8.5×10−10 M) was obtained for the binding of very long chain native MenB polysaccharides (∼200 Neu5Ac-residues). Colominic acid from Escherichia coli K1 (∼100 residues) and shorter polymers exhibited progressively weaker affinities. The antibody also bound tightly (KD∼5×10−9 M) to polysialylated glycopeptides from human embryonal brain. The effects of pH and ionic strength suggested that the interaction is largely electrostatic. Ca2+ and Mn2+ ions promoted the observed surface plasmon resonance response in a concentration dependent fashion. Spermine increased the response in a similar way. Our results suggest that divalent cations and polyamines may play significant role in the regulation of the PSA epitope presentation in vivo. [Copyright &y& Elsevier]