Isolation, primary structure characterization and identification of the glycosylation pattern of recombinant goldfish neurolin, a neuronal cell adhesion protein

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 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 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 α(2,3)‐terminal neuraminic acid. These data serve as a first detailed model to characterize the molecular recognition structures exhibited by the extracellular domains. Copyright © 1999 John Wiley & Sons, Ltd.