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Isolation, Primary Structure Characterization and Identification of the Glycosylation Pattern of Recombinant Goldfish Neurolin, a Neuronal Cell Adhesion Protein

Isolation, Primary Structure Characterization and Identification of the Glycosylation Pattern of Recombinant Goldfish Neurolin, a Neuronal Cell Adhesion Protein

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DENZINGER, Thomas, Heike DIEKMANN, Kai BRUNS, Ute LAESSING, Claudia STÜRMER, Michael PRZYBYLSKI, 1999. Isolation, Primary Structure Characterization and Identification of the Glycosylation Pattern of Recombinant Goldfish Neurolin, a Neuronal Cell Adhesion Protein. In: Journal of Mass Spectrometry. 34(4), pp. 435-446. ISSN 1076-5174. eISSN 1096-9888

@article{Denzinger1999Isola-9684, title={Isolation, Primary Structure Characterization and Identification of the Glycosylation Pattern of Recombinant Goldfish Neurolin, a Neuronal Cell Adhesion Protein}, year={1999}, doi={10.1002/(SICI)1096-9888(199904)34:4<435::AID-JMS803>3.0.CO;2-2}, number={4}, volume={34}, issn={1076-5174}, journal={Journal of Mass Spectrometry}, pages={435--446}, author={Denzinger, Thomas and Diekmann, Heike and Bruns, Kai and Laessing, Ute and Stürmer, Claudia and Przybylski, Michael} }

<rdf:RDF xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#" xmlns:bibo="http://purl.org/ontology/bibo/" xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:dcterms="http://purl.org/dc/terms/" xmlns:xsd="http://www.w3.org/2001/XMLSchema#" > <rdf:Description rdf:about="https://kops.uni-konstanz.de/rdf/resource/123456789/9684"> <dc:creator>Diekmann, Heike</dc:creator> <bibo:uri rdf:resource="http://kops.uni-konstanz.de/handle/123456789/9684"/> <dc:creator>Laessing, Ute</dc:creator> <dc:creator>Stürmer, Claudia</dc:creator> <dc:contributor>Laessing, Ute</dc:contributor> <dc:rights>deposit-license</dc:rights> <dc:contributor>Stürmer, Claudia</dc:contributor> <dcterms:issued>1999</dcterms:issued> <dcterms:title>Isolation, Primary Structure Characterization and Identification of the Glycosylation Pattern of Recombinant Goldfish Neurolin, a Neuronal Cell Adhesion Protein</dcterms:title> <dc:contributor>Denzinger, Thomas</dc:contributor> <dc:contributor>Bruns, Kai</dc:contributor> <dcterms:rights rdf:resource="https://creativecommons.org/licenses/by-nc-nd/2.0/legalcode"/> <dcterms:abstract xml:lang="eng">Neurolin is a growth-associated cell surface glycoprotein from goldfish and zebra fish which has been shown to be involved in axonal path-finding in the goldfish retina and suggested to function as a receptor for axon guidance molecules. Being a member of the immunoglobulin superfamily of cell adhesion proteins, neurolin consists of five N-terminal extracellular immunoglobulin (Ig)-like domains, a transmembrane and a short cytoplasmatic domain. Repeated injections of polyclonal Fab fragments against neurolin and of monoclonal antibodies against either Ig domains cause path-finding errors and disturbance of axonal fasciculation. In order to obtain a complete structural characterization and a molecular basis for structurefunction determination, recombinant neurolin with the complete extracellular part but lacking the transmembrane and cytoplasmatic domain was expressed in Chinese hamster ovary (CHO) cells (CHO-neurolin). The isolation of CHO-neurolin was carried out by Ni-affinity chromatography and subsequent high-performance liquid chromatography (HPLC). An exact molecular mass determination was obtained by matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) and revealed 60.9 kDa, which suggested that 10 kDa are due to glycosylation. The predicted molecular mass is 51.5 kDa, whereas sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) yielded an apparent molecular mass of 72 kDa. Gel shift assays using SDS-PAGE and Western blot analysis with anti-neurolin antibodies provided consistent molecular mass data. The complete primary structure and N-glycosylation patterns were identified using specific lectin assays, MALDI/MS peptide mapping analysis by proteolytic and in-gel digestion, electrospray ionization MS and MALDI/MS in combination with specific glycosidase degradation. HPLC isolation of glycosylated peptide fragments and MS after selective deglycosylation revealed heterogeneous glycosylations at all five N-glycosylation consensus sites. All attached N-glycans are of the complex type and show a mainly biantennary structure; they are fucosylated with alpha-(2,3)-terminal neuraminic acid. These data serve as a first detailed model to characterize the molecular recognition structures exhibited by the extracellular domains.</dcterms:abstract> <dcterms:bibliographicCitation>First publ. in: Journal of Mass Spectrometry 34 (1999), pp. 435-446</dcterms:bibliographicCitation> <dc:format>application/pdf</dc:format> <dc:language>eng</dc:language> <dc:creator>Denzinger, Thomas</dc:creator> <dcterms:available rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2011-03-24T18:13:41Z</dcterms:available> <dc:contributor>Diekmann, Heike</dc:contributor> <dc:contributor>Przybylski, Michael</dc:contributor> <dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2011-03-24T18:13:41Z</dc:date> <dc:creator>Bruns, Kai</dc:creator> <dc:creator>Przybylski, Michael</dc:creator> </rdf:Description> </rdf:RDF>

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