Martina Mühlenhoff

Genetic Ablation of Polysialic Acid Causes Severe Neurodevelopmental Defects Rescued by Deletion of the Neural Cell Adhesion Molecule

Poly-α2,8-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.

Polysialic acid: three-dimensional structure, biosynthesis and function

Polysialic acid is a unique cell surface polysaccharide found in the capsule of neuroinvasive bacteria and as a highly regulated post-translational modification of the neural cell adhesion molecule. Recent progress has been achieved in research on both the physicochemical properties of polysialic acid and the biosynthetic pathways leading to polysialic acid expression in bacteria and mammals.

Crystal structure of the polysialic acid-degrading endosialidase of bacteriophage K1F.

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 β-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.

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.

Dissecting polysialic acid and NCAM functions in brain development

The unique modification of the neural cell adhesion molecule (NCAM) by polysialic acid (polySia) is tightly associated with nervous system development and plasticity. The prevailing view that this large carbohydrate polymer acts as an anti‐adhesive factor seems straightforward at first sight. However, during almost 25 years of polySia research it became increasingly clear that the impact of polySia on cell surface interactions can not be explained by one unifying mechanism. Recent progress in the generation of mouse models, which partially or completely lack polySia due to ablation of one or both of the two polySia synthesizing enzymes, provides novel insights into the function of this unique post‐translational modification. The present review is focused on a phenotype comparison between the newly established mouse strains which combine polySia‐deficiency with normal NCAM expression and the well‐characterized NCAM negative mouse model. Analysis of shared and individual phenotypes allows a clear distinction between NCAM and polySia functions and revealed that polySia plays a vital role as a specific control element of NCAM‐mediated interactions.

Autocatalytic polysialylation of polysialyltransferase-1.

Polysialic acid (PSA) is a specific and highly regulated post‐translational modification of the neural cell adhesion molecule NCAM. Synthesis of PSA depends on the activity of a single enzyme, the polysialyltransferase‐1 (PST‐1), recently cloned from three mammalian species. The present study was carried out to investigate the catalytic mechanism of PST‐1. Using a newly developed in vitro assay system, we demonstrate autopolysialylation for PST‐1. The synthesis of PSA chains, which involved N‐glycosylation sites, occurred immediately after contact with the activated sugar donor CMP‐Neu5Ac. In contrast to the polysialylation of NCAM, where terminal sialylation in either the alpha2,3 or alpha2,6 position is required, the autopolysialylation could be started in the asialo‐PST‐1 isolated from CHO cells of the Lec2 complementation group. Pre‐formed PSA chains were not transferred to NCAM. Nevertheless, the autocatalytic step is likely to be a prerequisite for enzymatic activity, since agalacto‐PST‐1 isolated from Lec8 cells was functionally inactive. Our data describe a novel route of autocatalytic maturation of a glycosyltransferase and thereby provide a new basis for studies aimed at elucidating and influencing the catalytic functions of PST‐1.

Impact of the Polysialyltransferases ST8SiaII and ST8SiaIV on Polysialic Acid Synthesis during Postnatal Mouse Brain Development

Polysialic acid (polySia), a post-translational modification of the neural cell adhesion molecule (NCAM), is the key regulator of NCAM-mediated functions and crucial for normal brain development, postnatal growth, and survival. Two polysialyltransferases, ST8SiaII and ST8SiaIV, mediate polySia biosynthesis. To dissect the impact of each enzyme during postnatal brain development, we monitored the developmental changes in NCAM polysialylation in wild-type, ST8SiaII-, and ST8SiaIV-deficient mice using whole brain lysates obtained at 10 time points from postnatal days 1 to 21 and from adult mice. In wild-type and ST8SiaIV-null brain, polySia biosynthesis kept pace with the rapid increase in brain weight until day 9, and nearly all NCAM was polysialylated. Thereafter, polySia dropped by ∼70% within 1 week, accompanied by the first occurrence of polySia-free NCAM-140 and NCAM-180. In ST8SiaII-null brain, polySia declined immediately after birth, leading to 60% less polySia at day 9 combined with the untimely appearance of polySia-free NCAM. Polysialyltransferase deficiency did not alter NCAM expression level or isoform pattern. In all three genotypes, NCAM-140 and NCAM-180 were expressed at constant levels from days 1 to 21 and provided the major polySia acceptors. By contrast, NCAM-120 first appeared at day 5, followed by a strong up-regulation inverse to the decrease in polySia. Together, we provide a comprehensive quantitative analysis of the developmental changes in polySia level, NCAM polysialylation status, and polysialyltransferase transcript levels and show that the predominant role of ST8SiaII during postnatal brain development is restricted to the first 15 days.

Evolution of bacteriophages infecting encapsulated bacteria: lessons from Escherichia coli K1‐specific phages

Bacterial capsules are not only important virulence factors, but also provide attachment sites for bacteriophages that possess capsule degrading enzymes as tailspike proteins. To gain insight into the evolution of these specialized viruses, we studied a panel of tailed phages specific for Escherichia coli K1, a neuroinvasive pathogen with a polysialic acid capsule. Genome sequencing of two lytic K1‐phages and comparative analyses including a K1‐prophage revealed that K1‐phages did not evolve from a common ancestor. By contrast, each phage is related to a different progenitor type, namely T7‐, SP6‐, and P22‐like phages, and gained new host specificity by horizontal uptake of an endosialidase gene. The new tailspikes emerged by combining endosialidase domains with the capsid binding module of the respective ancestor. For SP6‐like phages, we identified a degenerated tailspike protein which now acts as versatile adaptor protein interconnecting tail and newly acquired tailspikes and demonstrate that this adapter utilizes an N‐terminal undecapeptide interface to bind otherwise unrelated tailspikes. Combining biochemical and sequence analyses with available structural data, we provide new molecular insight into basic mechanisms that allow changes in host specificity while a conserved head and tail architecture is maintained. Thereby, the present study contributes not only to an improved understanding of phage evolution and host‐range extension but may also facilitate the on purpose design of therapeutic phages based on well‐characterized template phages.

Molecular cloning and functional expression of bacteriophage PK1E‐encoded endoneuraminidase Endo NE

Homopolymeric α‐2,8‐linked sialic acid (PSA) has been found as a capsular component of sepsis‐ and meningitis‐causing bacterial pathogens, and on eukaryotic cells as a post‐translational modification of the neural cell adhesion molecule (NCAM). The polysaccharide is specifically recognized and degraded by a phage‐encoded enzyme, the endo‐N‐acetylneuraminidase E (Endo NE). Endo NE therefore has become a valuable tool in the study of bacterial pathogenesis and eukaryotic morphogenesis. In this report we describe the molecular cloning of Endo NE and the expression of a functionally active recombinant enzyme. The cloned DNA sequence (2436 bp) encodes a polypeptide of 811 amino acids, which at the 5′ end contains a totally conserved neuraminidase motif. Expressed in Escherichia coli, the enzyme migrates as a single band of approximately 74 kDa in SDS‐PAGE. A central domain of 669 amino acid residues is about 90% homologous to the recently cloned Endo NF. Both phage‐induced lysis of bacteria and the catalysis of PSA degradation by the recombinant enzyme are efficiently inhibited by a polyclonal antiserum raised against the intact phage particle. The C‐terminal region seems to be essential to enzymatic functions, as truncation of 32 amino acids outside the homology domain completely abolishes Endo NE activity. Our data also indicate that the 38 kDa protein, previously assumed to be a subunit of the Endo NE holoenzyme, is the product of a separate gene locus and is not necessary for in vitro depolymerase activity.

Biochemical characterization of a Neisseria meningitidis polysialyltransferase reveals novel functional motifs in bacterial sialyltransferases

The extracellular polysaccharide capsule is an essential virulence factor of Neisseria meningitidis, a leading cause of severe bacterial meningitis and sepsis. Serogroup B strains, the primary disease causing isolates in Europe and America, are encapsulated in α‐2,8 polysialic acid (polySia). The capsular polymer is synthesized from activated sialic acid by action of a membrane‐associated polysialyltransferase (NmB‐polyST). Here we present a comprehensive characterization of NmB‐polyST. Different from earlier studies, we show that membrane association is not essential for enzyme functionality. Recombinant NmB‐polyST was expressed, purified and shown to synthesize long polySia chains in a non‐processive manner in vitro. Subsequent structure–function analyses of NmB‐polyST based on refined sequence alignments allowed the identification of two functional motifs in bacterial sialyltransferases. Both (D/E‐D/E‐G and HP motif) are highly conserved among different sialyltransferase families with otherwise little or no sequence identity. Their functional importance for enzyme catalysis and CMP‐Neu5Ac binding was demonstrated by mutational analysis of NmB‐polyST and is emphasized by structural data available for the Pasteurella multocida sialyltransferase PmST1. Together our data are the first description of conserved functional elements in the highly diverse families of bacterial (poly)sialyltransferases and thus provide an advanced basis for understanding structure–function relations and for phylogenetic sorting of these important enzymes.