Publikation: Isolation, Primary Structure Characterization and Identification of the Glycosylation Pattern of Recombinant Goldfish Neurolin, a Neuronal Cell Adhesion Protein
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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.
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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. 1999, 34(4), pp. 435-446. ISSN 1076-5174. eISSN 1096-9888. Available under: doi: 10.1002/(SICI)1096-9888(199904)34:4<435::AID-JMS803>3.0.CO;2-2BibTex
@article{Denzinger1999Isola-9684, year={1999}, doi={10.1002/(SICI)1096-9888(199904)34:4<435::AID-JMS803>3.0.CO;2-2}, title={Isolation, Primary Structure Characterization and Identification of the Glycosylation Pattern of Recombinant Goldfish Neurolin, a Neuronal Cell Adhesion Protein}, 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} }
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