Localization of defined carbohydrates epitopes in bovine polysialylated NCAM

Manfred Wuhrer, Hildegard Geyer, Maren Von Der Ohe, Rita Gerardy-Schahn, Melitta Camartin, Rudolf Geyer

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)207-218
Number of pages12
JournalBiochimie
Volume85
Issue number1-2
DOIs
StatePublished - Jan 1 2003

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Neural Cell Adhesion Molecules
Polysaccharides
Epitopes
Carbohydrates
Glycosylation
Glycopeptides
Acids
Mass spectrometry
Mass Spectrometry
Bearings (structural)
Glycomics
Immobilized Antibodies
Peptides
Fucose
Fractionation
Chromatography
polysialic acid
Oligosaccharides
Polymerization
Trypsin

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Wuhrer, M., Geyer, H., Von Der Ohe, M., Gerardy-Schahn, R., Camartin, M., & Geyer, R. (2003). Localization of defined carbohydrates epitopes in bovine polysialylated NCAM. Biochimie, 85(1-2), 207-218. https://doi.org/10.1016/S0300-9084(03)00043-9
Wuhrer, Manfred ; Geyer, Hildegard ; Von Der Ohe, Maren ; Gerardy-Schahn, Rita ; Camartin, Melitta ; Geyer, Rudolf. / Localization of defined carbohydrates epitopes in bovine polysialylated NCAM. In: Biochimie. 2003 ; Vol. 85, No. 1-2. pp. 207-218.
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abstract = "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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Wuhrer, M, Geyer, H, Von Der Ohe, M, Gerardy-Schahn, R, Camartin, M & Geyer, R 2003, 'Localization of defined carbohydrates epitopes in bovine polysialylated NCAM', Biochimie, vol. 85, no. 1-2, pp. 207-218. https://doi.org/10.1016/S0300-9084(03)00043-9

Localization of defined carbohydrates epitopes in bovine polysialylated NCAM. / Wuhrer, Manfred; Geyer, Hildegard; Von Der Ohe, Maren; Gerardy-Schahn, Rita; Camartin, Melitta; Geyer, Rudolf.

In: Biochimie, Vol. 85, No. 1-2, 01.01.2003, p. 207-218.

Research output: Contribution to journalArticle

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T1 - Localization of defined carbohydrates epitopes in bovine polysialylated NCAM

AU - Wuhrer, Manfred

AU - Geyer, Hildegard

AU - Von Der Ohe, Maren

AU - Gerardy-Schahn, Rita

AU - Camartin, Melitta

AU - Geyer, Rudolf

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N2 - 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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