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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2007.03.032 Byline: Monika Marx (a), Eric Rivera-Milla (b), Katharina Stummeyer (c), Rita Gerardy-Schahn (c), Martin Bastmeyer (a) Keywords: ST8SiaII; ST8SiaIV; Polysialyltransferase; NCAM; PolySia; Morpholino; Zebrafish; Oligonucleotides; Axon guidance; Development; Evolution Abstract: Polysialic acid (PSA) is a developmentally regulated carbohydrate attached to the neural cell adhesion molecule (NCAM). PSA is involved in dynamic processes like cell migration, neurite outgrowth and neuronal plasticity. In mammals, polysialylation of NCAM is catalyzed independently by two polysialyltransferases, STX (ST8Sia II) and PST (ST8Sia IV), with STX mainly acting during early development and PST at later stages and into adulthood. Here, we functionally characterize zebrafish Stx and Pst homolog genes during fish development and evaluate their catalytic affinity for NCAM in vitro. Both genes have the typical gene architecture and share conserved synteny with their mammalian homologues. Expression analysis, gene-targeted knockdown experiments and in vitro catalytic assays indicate that zebrafish Stx is the principal-if not unique-polysialyltransferase performing NCAM-PSA modifications in both developing and adult fish. The knockdown of Stx exclusively affects PSA synthesis, producing defects in axonal growth and guidance. Zebrafish Pst is in principle capable of synthesizing PSA, however, our data argue against a fundamental function of the enzyme during development. Our findings reveal an important divergence of Stx and Pst enzymes in vertebrates, which is also characterized by a differential gene loss and rapid evolution of Pst genes within the bony-fish class. Author Affiliation: (a) Zoologisches Institut I, Lehrstuhl fur Zell-und Neurobiologie, Universitat Karlsruhe (TH), Haid-und-Neu-Strasse 9, D-76131 Karlsruhe, Germany (b) Entwicklungsneurobiologie, Fakultat fur Biologie, Universitat Konstanz, Konstanz, D-78457 Konstanz, Germany (c) Zellulare Chemie, Zentrum Biochemie, Medizinische Hochschule Hannover, Carl-Neuberg-Stra[beta]e 1, D-30625 Hannover, Germany Article History: Received 4 October 2006; Revised 20 March 2007; Accepted 23 March 2007

Sialic acids of cell surface glycoproteins and glycolipids play a pivotal role in the structure and function of animal tissues. The pattern of cell surface sialylation is species- and tissue-specific, is highly regulated during embryonic development, and changes with stages of differentiation. A prerequisite for the synthesis of sialylated glycoconjugates is the activated sugar-nucleotide cytidine 5[prime]-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac), which provides a substrate for Golgi sialyltransferases. Although a mammalian enzymatic activity responsible for the synthesis of CMP-Neu5Ac has been described and the enzyme has been purified to near homogeneity, sequence information is restricted to bacterial CMP-Neu5Ac synthetases. In this paper, we describe the molecular characterization, functional expression, and subcellular localization of murine CMP-Neu5Ac synthetase. Cloning was achieved by complementation of the Chinese hamster ovary lec32 mutation that causes a deficiency in CMP-Neu5Ac synthetase activity. A murine cDNA encoding a protein of 432 amino acids rescued the lec32 mutation and also caused polysialic acid to be expressed in the capsule of the CMP-Neu5Ac synthetase negative Escherichia coli mutant EV5. Three potential nuclear localization signals were found in the murine synthetase, and immunofluorescence studies confirmed predominantly nuclear localization of an N-terminally Flag-tagged molecule. Four stretches of amino acids that occur in the N-terminal region are highly conserved in bacterial CMP-Neu5Ac synthetases, providing evidence for an ancestral relationship between the sialylation pathways of bacterial and animal cells.

Experimental studies on polysialyl transferase-1, a vital enzyme of eukaryotic polysialic acid (PSA) synthesis, reveal that PSA synthesis in cell lines expressing neural cell adhesion molecule is induced by this enzyme. A single enzymatic activity is responsible for the polycondensation of alpha 2,8-linked sialic acids in mammals. Experimental data help understand the importance of PSA in neuro- and tumor biology.

Abstract Background Platelets are associated with HIV in the blood of infected individuals and might modulate viral dissemination, particularly if the virus is directly transmitted into the bloodstream. The C-type lectin DC-SIGN and the novel HIV attachment factor CLEC-2 are expressed by platelets and facilitate HIV transmission from platelets to T-cells. Here, we studied the molecular mechanisms behind CLEC-2-mediated HIV-1 transmission. Results Binding studies with soluble proteins indicated that CLEC-2, in contrast to DC-SIGN, does not recognize the viral envelope protein, but a cellular factor expressed on kidney-derived 293T cells. Subsequent analyses revealed that the cellular mucin-like membranous glycoprotein podoplanin, a CLEC-2 ligand, was expressed on 293T cells and incorporated into virions released from these cells. Knock-down of podoplanin in 293T cells by shRNA showed that virion incorporation of podoplanin was required for efficient CLEC-2-dependent HIV-1 interactions with cell lines and platelets. Flow cytometry revealed no evidence for podoplanin expression on viable T-cells and peripheral blood mononuclear cells (PBMC). Podoplanin was also not detected on HIV-1 infected T-cells. However, apoptotic bystander cells in HIV-1 infected cultures reacted with anti-podoplanin antibodies, and similar results were obtained upon induction of apoptosis in a cell line and in PBMCs suggesting an unexpected link between apoptosis and podoplanin expression. Despite the absence of detectable podoplanin expression, HIV-1 produced in PBMC was transmitted to T-cells in a CLEC-2-dependent manner, indicating that T-cells might express an as yet unidentified CLEC-2 ligand. Conclusions Virion incorporation of podoplanin mediates CLEC-2 interactions of HIV-1 derived from 293T cells, while incorporation of a different cellular factor seems to be responsible for CLEC-2-dependent capture of PBMC-derived viruses. Furthermore, evidence was obtained that podoplanin expression is connected to apoptosis, a finding that deserves further investigation.