Poly-␣2,8-sialic acid (polySia) is a unique modification of the neural cell adhesion molecule, NCAM, tightly associated with neural development and plasticity. However, the vital role attributed to this carbohydrate polymer has been challenged by the mild phenotype of mice lacking polySia due to NCAM-deficiency. To dissect polySia and NCAM functions, we generated polySia-negative but NCAM-positive mice by simultaneous deletion of the two polysialyltransferase genes, St8sia-II and St8sia-IV. Beyond features shared with NCAM-null animals, a severe phenotype with specific brain wiring defects, progressive hydrocephalus, postnatal growth retardation, and precocious death was observed. These drastic defects were selectively rescued by additional deletion of NCAM, demonstrating that they originate from a gain of NCAM functions because of polySia deficiency. The data presented in this study reveal that the essential role of polySia resides in the control and coordination of NCAM interactions during mouse brain development. Moreover, this first demonstration in vivo that a highly specific glycan structure is more important than the glycoconjugate as a whole provides a novel view on the relevance of protein glycosylation for the complex process of building the vertebrate brain.The cellular glycosylation machinery is a most impressive example of how cells enhance structural and functional complexity by use of only limited parts (Ͻ10%) of the genome. Glycans conjugated to lipids and proteins form the glycocalyx, the outer rim and prominent communication structure of animal cells (1, 2). Their paramount importance is emphasized by the growing group of congenital disorders of glycosylation, which manifest as severe multisystemic diseases including neuropathological symptoms (3).Poly-␣2,8-linked sialic acid (polySia) 3 is a unique glycan added to the neural cell adhesion molecule, NCAM, and is known to exert an important influence on the development and function of the nervous system (4 -6). The intriguing role assigned to polySia in promoting neurogenesis, migration, axon outgrowth, and synaptic plasticity has been explained predominantly by a negative regulation of cell-cell interactions due to the stereochemical properties of the large polyanion (shown schematically in Fig. 1A) (4, 7). Recent x-ray and neutron reflectivity data as well as direct force measurements confirm that an increased intermembrane repulsion in the presence of polySia overcomes homophilic NCAM binding and attenuates cadherin-dependent adhesion (8, 9). On the other hand, polySia is known to exert highly specific functions. For instance, NCAM trans-interactions with heparan sulfate proteoglycans involved in the formation and remodeling of hippocampal synapses depend on the presence of polySia (10, 11). Finally, competition experiments carried out with exogenously added polySia indicate that the carbohydrate polymer mediates autonomous, NCAM-independent functions. These concern the development of commissural axons in zebrafish (12), the strengthening of ...

Phages infecting the polysialic acid (polySia)-encapsulated human pathogen Escherichia coli K1 are equipped with capsule-degrading tailspikes known as endosialidases, which are the only identified enzymes that specifically degrade polySia. As polySia also promotes cellular plasticity and tumor metastasis in vertebrates, endosialidases are widely applied in polySia-related neurosciences and cancer research. Here we report the crystal structures of endosialidase NF and its complex with oligomeric sialic acid. The structure NF, which reveals three distinct domains, indicates that the unique polySia specificity evolved from a combination of structural elements characteristic of exosialidases and bacteriophage tailspike proteins. The endosialidase assembles into a catalytic trimer stabilized by a triple beta-helix. Its active site differs markedly from that of exosialidases, indicating an endosialidase-specific substrate-binding mode and catalytic mechanism. Residues essential for endosialidase activity were identified by structure-based mutational analysis.

A mechanism of capsular polysaccharide phase variation in Neisseria meningitidis is described. Meningococcal cells of an encapsulated serogroup B strain were used in invasion assays. Only unencapsulated variants were found to enter epithelial cells. Analysis of one group of capsule-deficient variants indicated that the capsular polysaccharide was re-expressed at a frequency of 10(-3). Measurement of enzymatic activities involved in the biosynthesis of the alpha-2,8 polysialic acid capsule showed that polysialyltransferase (PST) activity was absent in these capsule-negative variants. Nucleotide sequence analysis of siaD revealed an insertion or a deletion of one cytidine residue within a run of (dC)7 residues at position 89, resulting in a frameshift and premature termination of translation. We analysed unencapsulated isolates from carriers and encapsulated case isolates collected during an outbreak of meningococcal disease. Further paired blood-culture isolates and unencapsulated nasopharyngeal isolates from patients with meningococcal meningitis were examined. In all unencapsulated strains analysed we found an insertion or deletion within the oligo-(dC) stretch within siaD, resulting in a frameshift and loss of capsule formation. All encapsulated isolates, however, had seven dC residues at this position, indicating a correlation between capsule phase variation and bacterial invasion and the outbreak of meningococcal disease.