Glycobiology, 2002, Vol. 12, No. 1 47-63
© 2002 Oxford University Press
Localization and characterization of polysialic acid–containing N-linked glycans from bovine NCAM
5Zentrum für Molekulare Neurobiologie, University of Hamburg, D-20246 Hamburg, Germany; 6Oxford Glycobiology Institute, Department of Biochemistry, South Parks Road, Oxford, OX1 3QU, UK; 7Institute of Biochemistry, University of Giessen, D-35392 Giessen, Germany; and 8Institut für Medizinische Mikrobiologie, Medizinische Hochschule Hannover, D-30625 Hannover, Germany
The neural cell adhesion molecule (NCAM) plays important roles during development, plasticity, and regeneration in the adult nervous system. Its function is strongly influenced by attachment of the unusual 
2-8-linked polysialic acid (PSA). Here we analyzed the N-glycosylation pattern of polysialylated NCAM from brains of newborn calves. Purified PSA-NCAM glycoprotein was digested with trypsin, and PSA-glycopeptides were separated by immunoaffinity chromatography. For determining the N-glycosylation sites, PNGase F-treated glycopeptides were analyzed by Edman degradation and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). They were found to be exclusively linked to the fifth (Asn 439) and sixth (Asn 468) N-glycosylation sites in the fifth immunoglobulin-like domain of NCAM. The chain length of PSA consisted of at least 30 sialic acid residues, as shown by anion exchange chromatography. For analysis of the core structures, endoneuraminidase N–treated PSA-NCAM was separated by SDS–PAGE and digested with PNGase F. The core structures of polysialylated glycans were characterized by MALDI-MS combined with exoglycosidase digestions and chromatographic fractionation. They include hybrid, di-, tri-, and small amounts of tetraantennary carbohydrates, which were all fucosylated at the innermost N-acetylglucosamine. For the triantennary glycans, the "2,6" arm was preferred in polysialylated structures. High levels of sulfated groups were found on polysialylated structures and to a lower extent also on nonpolysialylated glycans. In addition, high-mannose-type glycans could be detected on PSA-NCAM glycoforms ranging from (GlcNAc)2(Man)5 up to (GlcNAc)2(Man)9. In conclusion, we observed a structural variability and high regional selectivity for the PSA-glycans attached to the NCAM molecule that are most likely influencing its biological functions.
1 These authors contributed equally to this paper.
2 Present address: Mermaid Pharmaceuticals, Falkenreid 88, D-20251 Hamburg, Germany
3 Present address: Neuroscience Research Centre, Merck, Sharp and Dohme, Terlings Park, Harlow, Essex CM20 2QR, UK
4 To whom correspondence should be addressed
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