Polysialylated neural cell adhesion molecule (NCAM) was immunoaffinity-purified from the brains of newborn calves. A degree of polymerization of up to 40 was chromatographically determined for released polysialic acid (PSA) chains. For characterization of N-glycan structures and attachment sites, PSA-NCAM was digested with trypsin, and the generated glycopeptides were fractionated by serial immunoaffinity chromatography using immobilized monoclonal antibodies specific for PSA or the HNK1 epitope, i.e., HSO3-3GlcA(β1-3)Gal(β1-4)GlcNAc(β1-), yielding PSA-glycopeptides, HNK-glycopeptides and non-PSA/HNK1-(glyco) peptides. Using a combination of enzymatic deglycosylation, peptide fractionation, mass spectrometry and Edman degradation, HNK1-N-glycans could be assigned to glycosylation sites 2, 4, 5 and 6. Non-PSA/HNK1-glycans were assigned to glycosylation site 2, whereas PSA-N-glycans of bovine NCAM had been already previously shown to be restricted to glycosylation sites 5 and 6 (Glycobiology 12 (2002) 47). Respective oligosaccharides were enzymatically released, labeled with 2-aminopyridine and characterized by linkage analysis and mass spectrometry. Carbohydrate chains bearing PSA or the HNK1 epitope comprised mainly fucosylated, partially sulfated diantennary, triantennary or tetraantennary glycans without bisecting GlcNAc or fucosylated diantennary and triantennary species carrying, in part, bisecting GlcNAc residues, respectively. Some N-glycans simultaneously contained both the HNK1-epitope and PSA. Non-PSA/HNK1-glycans exhibited a heterogeneous pattern of partially truncated, mostly diantennary structures with one to three fucose residues, bisecting GlcNAc and/or sulfate residues. In addition, they were demonstrated to carry, to some extent, the Lewis X epitope. When compared with previous data on murine NCAM glycosylation, our results indicate a conservation of structural features and attachment sites for the different types of NCAM N-glycans.
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