|Titel:||Synapsin I released via exosomes is an oligomannose bearing glycoprotein and an oligomannose binding lectin that promotes neurite outgrowth in Mus musculus (Linnaeus, 1758).||Sonstige Titel:||Synapsin I, ein Oligomannose-tragendes Glykoprotein und Oligomannose-bindendes Lektin, wird aus Exosomen freigesetzt und stimuliert Neuritenwachstum in Mus musculus (Linnaeus, 1758).||Sprache:||Englisch||Autor*in:||Wang, Shiwei||Erscheinungsdatum:||2009||Tag der mündlichen Prüfung:||2009-05-15||Zusammenfassung:||
Oligomannoses are expressed on developing and mature brains and involved in multiple important physiological and pathological processes. In order to circumvent low availability of natural saccharides for functional analysis, a phage-displayed random peptide library was screened to search for peptide mimics that interact specifically with L3 and L4 antibodies, which recognize oligomannosidic epitopes. During the panning procedures, two specific competitive elution conditions (antibodies and saccharides) were applied to increase specificity of elution. A phage clone expressing the sequence TISWWHLWPSPA emerged from all three panning protocols. The peptide was confirmed to be a peptide mimic of oligomannose by ELISA experiments. The synthetic peptide coupled to catalase for increasing solubility promoted neurite outgrowth of hippocampal neurons and reduced adhesion between cerebellar neurons and astrocytes, while a corresponding scrambled peptide showed no such activities. To identify novel oligomannose specific lectin, cross-linking, gel electrophoresis and mass spectrometry were performed and synapsin I was identified as a potential oligomannose binding protein. This was further confirmed by oligomannose dependent binding between synapsin I and adhesion molecule of glia in ELISA. In addition, synapsin I was eluted from L3 and L4 antibodies columns. When purified bovine synapsin I was treated with Endoglycosidase H and N-glycosidase F which removed high oligomannose glycans and N-linked glycans individually from glycoproteins, L3 and L4 antibodies lost their reactivities with synapsin I. Synapsin I was also recognized by some distinct lectins and the L5 antibody, which recognized LewisX. These results indicated complex glycosylation of synapsin I. Synapsin I was expressed and released via exosomes from cultured astrocytes and the astroglial cell line C6. Exosomes are small membrane vesicles, they are formed in multivesicular bodies by inward budding and secreted into extracellular milieu upon fusion of multivesicular bodies with the plasma membrane. Electron microscopy revealed that a preparation of exosome isolated from the cell culture medium of C6 cells showed a high degree of homogeneity of vesicles with a diameter of 80-100 nm. Incubation of astrocyte-derived exosomes in the presence of 40 mM KCl or LiCl in phosphate buffered saline released no synapsin I from exosomes, when increasing the concentrations of KCl or LiCl to 80 mM, most of synapsin I in exosomes was released into the soluble phases. Under same osmotic strength conditions (230 mM sucrose) synapsin I was not released from exosomes. C6 exosomes released synapsin I already when incubated with PBS. Proteomic analysis of C6 exosomes showed that a number of molecules including secreted, membrane and cytosolic proteins were released from exosomes under KCl stimulation. Substrate coated synapsin I which mimics synapsin I released from exosomes to the extracellular milieu promoted neurite outgrowth of hippocampal neurons in an oligomannose dependent way. When oligomannoses were removed, synapsin I lost its promotive effects on neurite outgrowth. Synapsin I stimulates neurite outgrowth probably by interacting with the neural cell adhesion molecule NCAM, because it binds with extracellular domain of NCAM in an oligomannose dependent way and loses its promotive effects on NCAM-deficient hippocampal neurons. The release of synapsin I from astrocyte-derived exosomes under high ionic strength conditions indicates that extracellular synapsin I plays a role in modulating interactions and communication between astrocytes and neurons, in particular under conditions of high neuronal or synaptic activities.
|URL:||https://ediss.sub.uni-hamburg.de/handle/ediss/2623||URN:||urn:nbn:de:gbv:18-41813||Dokumenttyp:||Dissertation||Betreuer*in:||Schachner, Melitta (Prof. Dr.)|
|Enthalten in den Sammlungen:||Elektronische Dissertationen und Habilitationen|
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