ST3GAL3 mutations impair the development of higher cognitive functions.

The genetic variants leading to impairment of intellectual performance are highly diverse and are still poorly understood. ST3GAL3 encodes the Golgi enzyme ß-galactoside-α2,3-sialyltransferase-III that in humans predominantly forms the sialyl Lewis a epitope on proteins. ST3GAL3 resides on chromosome 1 within the MRT4 locus previously identified to associate with nonsyndromic autosomal recessive intellectual disability. We searched for the disease-causing mutations in the MRT4 family and a second independent consanguineous Iranian family by using a combination of chromosome sorting and next-generation sequencing. Two different missense changes in ST3GAL3 cosegregate with the disease but were absent in more than 1000 control chromosomes. In cellular and biochemical test systems, these mutations were shown to cause ER retention of the Golgi enzyme and drastically impair ST3Gal-III functionality. Our data provide conclusive evidence that glycotopes formed by ST3Gal-III are prerequisite for attaining and/or maintaining higher cognitive functions.

Polysialic acid controls NCAM signals at cell-cell contacts to regulate focal adhesion independent from FGF receptor activity.

The polysialic acid (polySia) modification of the neural cell adhesion molecule NCAM is a key regulator of cell migration. Yet its role in NCAM-dependent or NCAM-independent modulation of motility and cell-matrix adhesion is largely unresolved. Here, we demonstrate that loss of polySia attenuates tumour cell migration and augments the number of focal adhesions in a cell-cell contact- and NCAM-dependent manner. In the presence or absence of polySia, NCAM never colocalised with focal adhesions but was enriched at cell-cell contacts. Focal adhesion of polySia- and NCAM-negative cells was enhanced by incubation with soluble NCAM or by removing polySia from heterotypic contacts with polySia-NCAM-positive cells. Focal adhesion was compromised by the src-family kinase inhibitor PP2, whereas loss of polySia or exposure to NCAM promoted the association of p59(Fyn) with the focal adhesion scaffolding protein paxillin. Unlike other NCAM responses, NCAM-induced focal adhesion was not prevented by inhibiting FGF receptor activity and could be evoked by NCAM fragments comprising immunoglobulin domains three and four but not by the NCAM fibronectin domains alone or by an NCAM-derived peptide known to interact with and activate FGF receptors. Together, these data indicate that polySia regulates cell motility through NCAM-induced but FGF-receptor-independent signalling to focal adhesions.

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.

A universal fluorescent acceptor for high-performance liquid chromatography analysis of pro- and eukaryotic polysialyltransferases.

Polysialyltransferases (polySTs) play critical roles in diverse biological processes, including neural development, tumorigenesis, and bacterial pathogenesis. Although the bacterial enzymes are presumed to have evolved to provide molecular mimics of the host-specific polysialic acid, no analytical technique is currently available to facilitate a direct comparison of the bacterial and vertebrate enzymes. Here we describe a new fluorescent acceptor, a 1,2-diamino-4,5-methylenedioxybenzene (DMB)-labeled trimer of α2,8-linked sialic acid (DMB-DP3), which primes both pro- and eukaryotic polySTs. High-performance liquid chromatography separation and fluorescence detection (HPLC-FD) of reaction products enabled the sensitive and quantitative detection of polyST activity, even using cell lysates as enzyme source, and revealed product profiles characteristic of each enzyme. Single product resolution afforded by this assay system revealed mechanistic insights into a kinetic lag phase exhibited by the polyST from Neisseria meningitidis serogroup B during chain elongation. DMB-DP3 is the first fluorescent acceptor shown to prime the mammalian polySTs. Moreover, product profiles obtained for the two murine polySTs provided direct biochemical evidence for enzymatic properties that had, until now, only been inferred from the analysis of biological samples. With DMB-DP3, we introduce a universal acceptor that provides an easy, fast, and reliable system for the comprehensive mechanistic and comparative analysis of polySTs.