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.

Polysialylation of the Synaptic Cell Adhesion Molecule 1 (SynCAM 1) Depends Exclusively on the Polysialyltransferase ST8SiaII in Vivo

“…The extracellular part of NCAM is composed of five Ig-like domains followed by two fibronectin type III repeats (FnIII-1 and -2) (52). Among the six N-glycosylation sites of NCAM, only two particular sites located in Ig5 are used for polysialylation (53)(54)(55). Nevertheless, Ig5 alone did not serve as an acceptor substrate for the polySTs, whereas a truncated NCAM variant encompassing the tandem domains Ig5 and FnIII-1 was efficiently polysialylated (56).…”

Pericytes limit tumor cell metastasis

“…First, NCAM itself could directly bind and enhance PDGF-B function in a retention motif-dependent manner. Although untested, this is a possibility, as NCAM has been found to be heavily sulfated at an unusual poly-sialic acid group linked to one of the extracellular Ig domains (37). This sialyl-type sulfated N-linked glycan should, in theory, provide excellent retention of growth factors that contain a basic amino acid retention motif.…”

High affinity binding of long-chain polysialic acid to antibody, and modulation by divalent cations and polyamines

“…After lyophilization and desalting, the presence and sialylation degree of the brain polysialylated glycans were confirmed by FID-GLC, GLC-MS and 1 H-NMR. Calculations of the concentration of the glycans were based on a previously suggested trimannosyl core glycan structure (Finne, 1982;Kudo et al, 1996;Nelson et al, 1995;Zamze et al, 1998;von der Ohe et al, 2002).…”