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 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.

Internalization and recycling of ALCAM/CD166 detected by a fully human single-chain recombinant antibody

“…The immunoprecipitates from the other cell lines, although displaying slightly different molecular weights, were again identified as ALCAM/CD166. The differences in molecular weight are probably due to tissue specific variation in N-glycosylation pattern (Pourquié et al, 1992;Denziger et al, 1999).…”

Activated leukocyte cell adhesion molecule: A novel biomarker for breast cancer

“…12 The molecular weight of ALCAM is 65 kDa but with N-glycosylation at 8 putative sites, the mature ALCAM molecule has a molecular weight of 110 kDa. 13 Five extracellular Ig domains, a transmembrane region and a short cytoplasmic tail make up the ALCAM protein that resembles E-cadherin in motif-arrangement. 12 ALCAM mediates both heterophilic (ALCAM-CD6) and homophilic (ALCAM-ALCAM) cellcell interactions.…”

Identification of N -glycosylation sites of the murine neural cell adhesion molecule NCAM by MALDI-TOF and MALDI-FTICR mass spectrometry

“…We could show that all six potential N-glycosylation sites of NCAM from adult mouse brain are modified, at least one of these (N-1) only partially. The differential approach employed here should be particularly valuable in the case of highly heterogeneous glycosylations as found for glycoproteins from different cellular systems and species [15,16,17,26,27,31,32,33,34,35,36,37,38,39,40]. Direct MALDI-TOF-MS studies of glycopeptides have shown pronounced heterogeneous structures on single N-glycosylation sites of NCAM [26].…”