Characterization of N-glycans from mouse brain neural cell adhesion molecule.
The N-glycosylation pattern of the neural cell adhesion molecule (NCAM), isolated from brains of newborn mice, has been analyzed. Following digestion with trypsin, generated glycopeptides were fractionated by serial immunoaffinity chromatography using immobilized monoclonal antibodies specifically recognizing polysialic acid (PSA) units or the HNK1-carbohydrate epitope. Subsequent analyses of the resulting (glyco)peptides by Edman degradation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) revealed polysialylated glycans to be exclusively linked to glycosylation sites 5 (Asn(431)) and 6 (Asn(460)), whereas glycans carrying the HNK1-epitope could be assigned to sites 2 (Asn(297)), 5, 6, and, to a lesser extent, site 3 (Asn(329)). PSA-, HNK1-, and non-PSA/HNK1-glycan fractions were characterized by carbohydrate constituent and methylation analyses as well as MALDI-TOF-MS in conjunction with chromatographic fractionation techniques. The results revealed that the core structures of PSA-glycans represented predominantly fucosylated, partially sulfated 2,6-branched isomers of triantennary as well as tetraantennary complex-type glycans, whereas carbohydrate chains bearing the HNK1-epitope were dominated by diantennary species carrying in part bisecting GlcNAc residues. Non-PSA/HNK1-glycans exhibited a highly heterogeneous pattern of partially truncated, mostly diantennary structures being characterized by the presence of additional fucose, bisecting GlcNAc and/or sulfate residues. In conclusion, our results revealed that the glycosylation pattern of murine NCAM displays high structural and regional selectivity, which might play an important role in controlling the biological activities of this molecule.
Liedtke
S
S
Zentrum für Molekulare Neurobiologie, Universität Hamburg, D-20246 Hamburg, Germany.
Geyer
H
H
Wuhrer
M
M
Geyer
R
R
Frank
G
G
Gerardy-Schahn
R
R
Zähringer
U
U
Schachner
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D001923:Brain Chemistry; D018959:CD57 Antigens / Q000737:chemistry; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D006020:Glycopeptides / Q000737:chemistry; D006031:Glycosylation; D051379:Mice; D008969:Molecular Sequence Data; D019006:Neural Cell Adhesion Molecules / Q000737:chemistry*; D011134:Polysaccharides / Q000737:chemistry*; D012794:Sialic Acids / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D018959:CD57 Antigens; D006020:Glycopeptides; D019006:Neural Cell Adhesion Molecules; D011134:Polysaccharides; D012794:Sialic Acids; C021319:polysialic acid