Isolation, primary structure characterization and identification of the glycosylation pattern of recombinant goldfish neurolin, a neuronal cell adhesion protein
✍ Scribed by Denzinger, Thomas; Diekmann, Heike; Bruns, Kai; Laessing, Ute; Stuermer, Claudia A.; Przybylski, Michael
- Publisher
- John Wiley and Sons
- Year
- 1999
- Tongue
- English
- Weight
- 328 KB
- Volume
- 34
- Category
- Article
- ISSN
- 1076-5174
No coin nor oath required. For personal study only.
✦ Synopsis
Neurolin is a growth-associated cell surface glycoprotein from goldÐsh and zebra Ðsh which has been shown to be involved in axonal path-Ðnding in the goldÐsh 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 Ðve 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-Ðnding errors and disturbance of axonal fasciculation. In order to obtain a complete structural characterization and a molecular basis for structure-function 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 identiÐed using speciÐc lectin assays, MALDI/MS peptide mapping analysis by proteolytic and in-gel digestion, electrospray ionization MS and MALDI/MS in combination with speciÐc glycosidase degradation. HPLC isolation of glycosylated peptide fragments and MS after selective deglycosylation revealed heterogeneous glycosylations at all Ðve N-glycosylation consensus sites. All attached N-glycans are of the complex type and show a mainly biantennary structure ; they are fucosylated with a(2,3)-terminal neuraminic acid. These data serve as a Ðrst detailed model to characterize the molecular recognition structures exhibited by the extracellular domains.