期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：3
页码：E241-E248
DOI：10.1073/pnas.1419683112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceThe roles of cell-surface glycans remain elusive compared with those of proteins or lipids because of their diverse and dynamic nature. Metabolic incorporation of unnatural monosaccharides in the biochemical synthesis of glycans as a chemical reporter has been a successful method to investigate the functions of cell-surface glycans but has also left an issue of cytotoxicity for certain cells. In this work, we developed a tissue-based strategy for metabolic incorporation of a chemical reporter to primary neurons. We let an unnatural monosaccharide be metabolized by hippocampal tissues before dissociation into individual cells, and thereby, we could eliminate cytotoxicity. We used this method to describe, for the first time to our knowledge, the real-time distribution of polysialic acids on the membranes of neurons. The posttranslational modification of neural cell-adhesion molecule (NCAM) with polysialic acid (PSA) and the spatiotemporal distribution of PSA-NCAM play an important role in the neuronal development. In this work, we developed a tissue-based strategy for metabolically incorporating an unnatural monosaccharide, peracetylated N-azidoacetyl-D-mannosamine, in the sialic acid biochemical pathway to present N-azidoacetyl sialic acid to PSA-NCAM. Although significant neurotoxicity was observed in the conventional metabolic labeling that used the dissociated neuron cells, neurotoxicity disappeared in this modified strategy, allowing for investigation of the temporal and spatial distributions of PSA in the primary hippocampal neurons. PSA-NCAM was synthesized and recycled continuously during neuronal development, and the two-color labeling showed that newly synthesized PSA-NCAMs were transported and inserted mainly to the growing neurites and not significantly to the cell body. This report suggests a reliable and cytocompatible method for in vitro analysis of glycans complementary to the conventional cell-based metabolic labeling for chemical glycobiology.
