Localization and characterization of polysialic acid-containing N-linked glycans from bovine NCAM

Maren Von Der Ohe, Susan F. Wheeler, Manfred Wuhrer, David J. Harvey, Steffen Liedtke, Martina Mühlenhoff, Rita Gerardy-Schahn, Hildegard Geyer, Raymond A. Dwek, Rudolf Geyer, David R. Wing, Melitta Camartin

Research output: Contribution to journalArticle

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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 α2-8-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.

Original languageEnglish (US)
Pages (from-to)47-63
Number of pages17
JournalGlycobiology
Volume12
Issue number1
StatePublished - Mar 23 2002
Externally publishedYes

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Neural Cell Adhesion Molecules
Polysaccharides
Glycosylation
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
Glycopeptides
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Chromatography
Ionization
Mass spectrometry
Desorption
Acetylglucosamine
Lasers
Glycoside Hydrolases
Neurology
N-Acetylneuraminic Acid
Mannose
Fractionation
polysialic acid
Chain length
Trypsin

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Von Der Ohe, M., Wheeler, S. F., Wuhrer, M., Harvey, D. J., Liedtke, S., Mühlenhoff, M., ... Camartin, M. (2002). Localization and characterization of polysialic acid-containing N-linked glycans from bovine NCAM. Glycobiology, 12(1), 47-63.
Von Der Ohe, Maren ; Wheeler, Susan F. ; Wuhrer, Manfred ; Harvey, David J. ; Liedtke, Steffen ; Mühlenhoff, Martina ; Gerardy-Schahn, Rita ; Geyer, Hildegard ; Dwek, Raymond A. ; Geyer, Rudolf ; Wing, David R. ; Camartin, Melitta. / Localization and characterization of polysialic acid-containing N-linked glycans from bovine NCAM. In: Glycobiology. 2002 ; Vol. 12, No. 1. pp. 47-63.
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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 α2-8-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.",
author = "{Von Der Ohe}, Maren and Wheeler, {Susan F.} and Manfred Wuhrer and Harvey, {David J.} and Steffen Liedtke and Martina M{\"u}hlenhoff and Rita Gerardy-Schahn and Hildegard Geyer and Dwek, {Raymond A.} and Rudolf Geyer and Wing, {David R.} and Melitta Camartin",
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Von Der Ohe, M, Wheeler, SF, Wuhrer, M, Harvey, DJ, Liedtke, S, Mühlenhoff, M, Gerardy-Schahn, R, Geyer, H, Dwek, RA, Geyer, R, Wing, DR & Camartin, M 2002, 'Localization and characterization of polysialic acid-containing N-linked glycans from bovine NCAM', Glycobiology, vol. 12, no. 1, pp. 47-63.

Localization and characterization of polysialic acid-containing N-linked glycans from bovine NCAM. / Von Der Ohe, Maren; Wheeler, Susan F.; Wuhrer, Manfred; Harvey, David J.; Liedtke, Steffen; Mühlenhoff, Martina; Gerardy-Schahn, Rita; Geyer, Hildegard; Dwek, Raymond A.; Geyer, Rudolf; Wing, David R.; Camartin, Melitta.

In: Glycobiology, Vol. 12, No. 1, 23.03.2002, p. 47-63.

Research output: Contribution to journalArticle

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T1 - Localization and characterization of polysialic acid-containing N-linked glycans from bovine NCAM

AU - Von Der Ohe, Maren

AU - Wheeler, Susan F.

AU - Wuhrer, Manfred

AU - Harvey, David J.

AU - Liedtke, Steffen

AU - Mühlenhoff, Martina

AU - Gerardy-Schahn, Rita

AU - Geyer, Hildegard

AU - Dwek, Raymond A.

AU - Geyer, Rudolf

AU - Wing, David R.

AU - Camartin, Melitta

PY - 2002/3/23

Y1 - 2002/3/23

N2 - 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 α2-8-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.

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Von Der Ohe M, Wheeler SF, Wuhrer M, Harvey DJ, Liedtke S, Mühlenhoff M et al. Localization and characterization of polysialic acid-containing N-linked glycans from bovine NCAM. Glycobiology. 2002 Mar 23;12(1):47-63.