title: Localization and characterization of polysialic acid-containing N-linked glycans from bovine NCAM.
abstract: 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 alpha 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.
authors.affiliation: Zentrum für Molekulare Neurobiologie, University of Hamburg, D-20246 Hamburg, Germany.
authors.forename: Maren
authors.initials: M
authors.lastname: von Der Ohe
authors.forename: Susan F
authors.initials: SF
authors.lastname: Wheeler
authors.forename: Manfred
authors.initials: M
authors.lastname: Wuhrer
authors.forename: David J
authors.initials: DJ
authors.lastname: Harvey
authors.forename: Steffen
authors.initials: S
authors.lastname: Liedtke
authors.forename: Martina
authors.initials: M
authors.lastname: Mühlenhoff
authors.forename: Rita
authors.initials: R
authors.lastname: Gerardy-Schahn
authors.forename: Hildegard
authors.initials: H
authors.lastname: Geyer
authors.forename: Raymond A
authors.initials: RA
authors.lastname: Dwek
authors.forename: Rudolf
authors.initials: R
authors.lastname: Geyer
authors.forename: David R
authors.initials: DR
authors.lastname: Wing
authors.forename: Melitta
authors.initials: M
authors.lastname: Schachner
mesh_terms: D000818:Animals; D002417:Cattle; D019006:Neural Cell Adhesion Molecules / Q000737:chemistry / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D012794:Sialic Acids / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D019006:Neural Cell Adhesion Molecules; D011134:Polysaccharides; D012794:Sialic Acids; C021319:polysialic acid