Branched Sialylated N-glycans Are Accumulated in Brain Synaptosomes and Interact with Siglec-H.

Proper N-glycosylation of proteins is important for normal brain development and nervous system function. Identification of the localization, carrier proteins and interacting partners of N-glycans is essential for understanding the roles of glycoproteins. The present study examined the N-glycan A2G'2F (Galß1-3GlcNAcß1-2Manα1-6[Galß1-3GlcNAcß1-2Manα1-3]Manß1-4GlcNAcß1-4[Fucα1-6]GlcNAc-). A2G'2F has a branched sialic acid structural feature, and branched sialylated A2G'2F is a major N-glycan in the mouse brain. Its expression in the mouse brain increases during development, suggesting that branched sialylated N-glycans play essential roles during brain development. However, the carrier proteins, interacting partners and localization of branched sialylated N-glycans remain unknown. We previously improved our method for analyzing N-glycans from trace samples, and here we succeeded in detecting A2G'2F in small fragments excised from the two-dimensional electrophoresis gels of subcellular fractionated mouse brain proteins. A2G'2F was accumulated in mouse brain synaptosomes. We identified calreticulin as one of the candidate A2G'2F carriers and found calreticulin expression in both the endoplasmic reticulum and synaptosomal fractions. Calreticulin was observed in dendritic spines of cultured cortical neurons. Synthesized branched sialylated glycan clusters interacted with sialic acid-binding immunoglobulin-like lectin H (Siglec-H), which is known to be a microglia-specific molecule. Taken together, these results suggest that branched sialylated A2G'2F in synaptosomes plays a role in the interaction of dendritic spines with microglia.Key words: N-glycan, subcellular fractionation, calreticulin, dendritic spine, Siglec-H.

GlcNAc6ST-1 regulates sulfation of N-glycans and myelination in the peripheral nervous system.

Highly specialized glial cells wrap axons with a multilayered myelin membrane in vertebrates. Myelin serves essential roles in the functioning of the nervous system. Axonal degeneration is the major cause of permanent neurological disability in primary myelin diseases. Many glycoproteins have been identified in myelin, and a lack of one myelin glycoprotein results in abnormal myelin structures in many cases. However, the roles of glycans on myelin glycoproteins remain poorly understood. Here, we report that sulfated N-glycans are involved in peripheral nervous system (PNS) myelination. PNS myelin glycoproteins contain highly abundant sulfated N-glycans. Major sulfated N-glycans were identified in both porcine and mouse PNS myelin, demonstrating that the 6-O-sulfation of N-acetylglucosamine (GlcNAc-6-O-sulfation) is highly conserved in PNS myelin between these species. P0 protein, the most abundant glycoprotein in PNS myelin and mutations in which at the glycosylation site cause Charcot-Marie-Tooth neuropathy, has abundant GlcNAc-6-O-sulfated N-glycans. Mice deficient in N-acetylglucosamine-6-O-sulfotransferase-1 (GlcNAc6ST-1) failed to synthesize sulfated N-glycans and exhibited abnormal myelination and axonal degeneration in the PNS. Taken together, this study demonstrates that GlcNAc6ST-1 modulates PNS myelination and myelinated axonal survival through the GlcNAc-6-O-sulfation of N-glycans on glycoproteins. These findings may provide novel insights into the pathogenesis of peripheral neuropathy.

Determination of major sialylated N-glycans and identification of branched sialylated N-glycans that dynamically change their content during development in the mouse cerebral cortex.

Oligosaccharides of glycoproteins expressed on the cell surface play important roles in cell-cell interactions, particularly sialylated N-glycans having a negative charge, which interact with sialic acid-binding immunoglobulin-like lectins (siglecs). The entire structure of sialylated N-glycans expressed in the mouse brain, particularly the linkage type of sialic acid residues attached to the backbone N-glycans, has not yet been elucidated. An improved method to analyze pyridylaminated sugar chains using high performance liquid chromatography (HPLC) was developed to determine the entire structure of sialylated N-linked sugar chains expressed in the adult and developing mouse cerebral cortices. Three classes of sialylated sugar chains were prevalent: 1) N-glycans containing α(2-3)-sialyl linkages on a type 2 antennary (Galß(1-4)GlcNAc), 2) sialylated N-glycans with α(2-6)-sialyl linkages on a type 2 antennary, and 3) a branched sialylated N-glycan with a [Galß(1-3){NeuAcα(2-6)}GlcNAc-] structure, which was absent at embryonic day 12 but then increased during development. This branched type sialylated N-glycan structure comprised approximately 2 % of the total N-glycans in the adult brain. Some N-glycans (containing type 2 antennary) were found to change their type of sialic acid linkage from α(2-6)-Gal to α(2-3)-Gal. Thus, the linkages and expression levels of sialylated N-glycans change dramatically during brain development.

Detection of N-glycans on small amounts of glycoproteins in tissue samples and sodium dodecyl sulfate-polyacrylamide gels.

N-linked glycans harbored on glycoproteins profoundly affect the character of proteins by altering their structure or capacity to bind to other molecules. Specific knowledge of the role of N-glycans in these changes is limited due to difficulties in identifying precise carbohydrate structures on a given glycoprotein, which arises from the large amounts of glycoprotein required for N-glycan structural determination. Here, we refined a simple method to purify and detect trace amounts of N-glycans. During the N-glycan purification step, most contaminants were removed by two kinds of columns: a graphite carbon column and a cellulose column. N-Glycans were identified with a three-dimensional high-performance liquid chromatography (HPLC) system. Using our method, a global analysis of N-glycans from human muscle biopsy samples and mouse brain sections was possible. By combining sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with our method, we refined analytical procedures for N-glycans from SDS-PAGE gels using hydrazinolysis to achieve a high N-glycan recovery rate. N-Glycans on as little as 1 µg of the target protein transferrin or immunoglobulin G (IgG) were easily detected. These methods allowed us to efficiently determine glycoprotein N-glycans at picomole (pmol) levels.