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 (Galβ1-3GlcNAcβ1-2Manα1-6[Galβ1-3GlcNAcβ1-2Manα1-3]Manβ1-4GlcNAcβ1-4[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.

中文翻译：

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分支的唾液酸化的N-聚糖在脑突触体中积累，并与Siglec-H相互作用。

蛋白质的正确N-糖基化对于正常的大脑发育和神经系统功能很重要。N-聚糖的定位，载体蛋白和相互作用伴侣的鉴定对于理解糖蛋白的作用至关重要。本研究检查了N-聚糖A2G'2F（Galβ1-3GlcNAcβ1-2Manα1-6[Galβ1-3GlcNAcβ1-2Manα1-3]Manβ1-4GlcNAcβ1-4[Fucα1-6] GlcNAc-）。A2G'2F具有分支的唾液酸结构特征，而分支的唾液酸化的A2G'2F是小鼠脑中的主要N-聚糖。它在小鼠大脑中的表达在发育过程中会增加，这表明分支的唾液酸化N聚糖在大脑发育过程中起着至关重要的作用。然而，载体蛋白，相互作用的伴侣和支链唾液酸化的N-聚糖的定位仍然未知。我们以前改进了分析痕量样品中N-聚糖的方法，在这里我们成功地检测了亚细胞分离的小鼠脑蛋白的二维电泳凝胶切除的小片段中的A2G'2F。A2G'2F积累在小鼠脑突触体中。我们将钙网蛋白鉴定为候选A2G'2F携带者之一，并发现钙网蛋白在内质网和突触小体中均表达。在培养的皮质神经元的树突棘中观察到钙网蛋白。合成的分支唾液酸化聚糖簇与唾液酸结合免疫球蛋白样凝集素H（Siglec-H）相互作用，后者已知是小胶质细胞特异性分子。综上所述，这些结果表明分支的唾液酸化的A2G'