Tissue-based metabolic labeling of polysialic acids in living primary hippocampal neurons

Abstract

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 PSANCAMs 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.

In vivo metabolic labeling of sialoglycans in the mouse brain by using a liposome-assisted bioorthogonal reporter strategy

“…The LABOR strategy developed herein overcomes this obstacle by exploiting liposomes to shuttle 9AzSia into the brain. Previous in vitro studies using neuron cell culture (33) and brain tissue culture (34) have shown that neurons can uptake azidosugars and metabolically incorporate them into sialoglycans. Our results demonstrate that LABOR-mediated delivery of 9AzSia into the brain results in robust metabolic incorporation of azides into brain sialoglycans in living mice.…”

Direct Visualization of Live Zebrafish Glycan via Single-step Metabolic Labeling with Fluorophore-tagged Nucleotide Sugars

“…Metabolic oligosaccharide engineering (MOE) coupled with the bioorthogonal chemical reporter strategy has opened an avenue for labeling and visualizing glycans in living organisms. [1][2][3][4][5] In this approach, the cell's glycan biosynthetic machinery is exploited to install a biorthogonal chemical group onto cell surface glycans, which is then covalently labeled in a secondary step with a complementary probe. 6 However, this strategy has a few intrinsic limitations, such as the biocompatibility of a chosen bioorthogonal reaction and poor deep-tissue penetration of the probes.…”

Multiplexed Metabolic Labeling of Glycoconjugates in Polarized Primary Cerebral Cortical Neurons

“…The application of metabolic glycan labeling to neurons has been challenging because of the apparent neurotoxicity of unnatural monosaccharide precursors (e.g., N-glycolylmannosamine pentaacetate and peracetylated N-azidoacetyl-d-mannosamine (Ac 4 ManNAz)). [18][19][20] In this work, we utilized ar ecently developed strategy forn eurocompatible metabolic glycanl abeling of primary cortical neurons(metabolism-by-tissues, MbT, Figure 1) [19] to acquire multiplexed informationo nt he spatiotemporal distribution of two different glycoconjugates-one with sialic acid( Sia5Ac) and the other with N-acetylgalactosamine/glucosamine (GalNAc/GlcNAc). Specifically,w es imultaneously fed ac ortical tissue (befored issociation into cortical neurons) with two different unnatural monosaccharides:p eracetylated N-(4-pentynoyl)-d-mannosamine (Ac 4 ManNAl) as aS ia5Ac precursor and peracetylated N-azidoacetyl-d-galactosamine (Ac 4 GalNAz) as am etabolic precursor of GalNAc/GlcNAc.…”

“…Ac 4 ManNAz was additionally examined for neurotoxicity studies, because it had been used most intensively for metabolic glycanl abeling in the previous reports and considered as an eurotoxic probe. [19] As shown in Figure 2, the viability of the cortical neuronsd ecreasedg radually over time, when they weref ed with Ac 4 ManNAz, Ac 4 ManNAl, or Ac 4 GalNAz( 50 mm)u nder the conventional protocol (see the Supporting Information for the experimental details). For example, the relative viability of Ac 4 ManNAz-or Ac 4 GalNAz-fed cortical neurons to non-treated cells as ar eference was 5.5 %o r1 2.0 %a t4DIV (DIV:d ays in vitro) (Figure 2a and b).…”

“…Therefore, the information on the relative spatial-distribution of glycoconjugates in the polarized neurons would be highly beneficial in neuroglycomics and neural physiology.The application of metabolic glycan labeling to neurons has been challenging because of the apparent neurotoxicity of unnatural monosaccharide precursors (e.g., N-glycolylmannosamine pentaacetate and peracetylated N-azidoacetyl-d-mannosamine (Ac 4 ManNAz)). [18][19][20] In this work, we utilized ar ecently [a] J.…”