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 t he goldfish retina and suggested to function as a receptor for axon guidanc e molecules. Being a member of the immunoglobulin superfamily of cell adhes ion proteins, neurolin consists of five N-terminal extracellular immunoglob ulin (Ig)-like domains, a transmembrane and a short cytoplasmatic domain. R epeated injections of polyclonal Fab fragments against neurolin and of mono clonal antibodies against either Ig domains cause path-finding errors and d isturbance of axonal fasciculation. In order to obtain a complete structura l characterization and a molecular basis for structure-function determinati on, recombinant neurolin with the complete extracellular part but lacking t he transmembrane and cytoplasmatic domain was expressed in Chinese hamster ovary (CHO) cells (CHO-neurolin). The isolation of CHO-neuroIin was carried out by Ni-affinity chromatography and subsequent high-performance liquid c hromatography (HPLC). An exact molecular mass determination was obtained by matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) a nd revealed 60.9 kDa, which suggested that similar to 10 kDa are due to gly cosylation, The predicted molecular mass is 51.5 kDa, whereas sodium dodecy l sulphate polyacrylamide gel electrophoresis (SDS-PAGE) yielded an apparen t molecular mass of 72 kDa, Gel shift assays using SDS-PAGE and Western blo t analysis with anti-neurolin antibodies provided consistent molecular mass data. The complete primary structure and N-glycosylation patterns sere ide ntified using specific lectin assays, MALDI/MS peptide mapping analysis by proteolytic and in-gel digestion, electrospray ionization MS and MALDI/MS i n combination with specific glycosidase degradation HPLC isolation of glyco sylated peptide fragments and MS after selective deglycosylation revealed h eterogeneous glycosylations at all five N-glycosylation consensus sites. Al l attached N-glycans are of the complex type and show a mainly biantennary structure; they are fucosylated with alpha(2,3)-terminal neuraminic acid. T hese data serve as a first detailed model to characterize the molecular rec ognition structures exhibited by the extracellular domains. Copyright (C) 1 999 John Wiley & Sons, Ltd.