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

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1999

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Denzinger, Thomas
Diekmann, Heike
Bruns, Kai
Laessing, Ute

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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;2-2

Zusammenfassung

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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570 Biowissenschaften, Biologie

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neurolin, neuronal cell adhesion protein, glycosylation, structure

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ISO 690DENZINGER, 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;2-2
BibTex
@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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