title: Isolation, primary structure characterization and identification of the glycosylation pattern of recombinant goldfish neurolin, a neuronal cell adhesion protein.
abstract: 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 approximately 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.
authors.affiliation: Fakultät für Chemie, Universität Konstanz, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Denzinger
authors.forename: H
authors.initials: H
authors.lastname: Diekmann
authors.forename: K
authors.initials: K
authors.lastname: Bruns
authors.forename: U
authors.initials: U
authors.lastname: Laessing
authors.forename: C A
authors.initials: CA
authors.lastname: Stuermer
authors.forename: M
authors.initials: M
authors.lastname: Przybylski
mesh_terms: D020410:Activated-Leukocyte Cell Adhesion Molecule / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D006031:Glycosylation; D006054:Goldfish; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D020410:Activated-Leukocyte Cell Adhesion Molecule; D011994:Recombinant Proteins