Time and space-resolved quantification of plasma membrane sialylation for measurements of cell function and neurotoxicity.

Publisher: Springer-Verlag Country of Publication: Germany NLM ID: 0417615 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1432-0738 (Electronic) Linking ISSN: 03405761 NLM ISO Abbreviation: Arch Toxicol Subsets: MEDLINE

Original Publication: Berlin, New York, Springer-Verlag.

Cell Membrane/*metabolism ; Drug Evaluation, Preclinical/*methods ; Molecular Biology/*methods ; N-Acetylneuraminic Acid/*metabolism ; Neurotoxicity Syndromes/*pathology; Bortezomib/pharmacology ; Cell Line ; Glycoconjugates/chemistry ; Glycoconjugates/metabolism ; Green Fluorescent Proteins/genetics ; Green Fluorescent Proteins/metabolism ; Hexosamines/chemistry ; Hexosamines/metabolism ; Hexosamines/pharmacology ; Humans ; Neurites/chemistry ; Neurites/metabolism ; Neurons/drug effects ; Neurons/metabolism ; Neurons/pathology ; Neurotoxicity Syndromes/metabolism ; Tunicamycin/pharmacology

While there are many methods to quantify the synthesis, localization, and pool sizes of proteins and DNA during physiological responses and toxicological stress, only few approaches allow following the fate of carbohydrates. One of them is metabolic glycoengineering (MGE), which makes use of chemically modified sugars (CMS) that enter the cellular biosynthesis pathways leading to glycoproteins and glycolipids. The CMS can subsequently be coupled (via bio-orthogonal chemical reactions) to tags that are quantifiable by microscopic imaging. We asked here, whether MGE can be used in a quantitative and time-resolved way to study neuronal glycoprotein synthesis and its impairment. We focused on the detection of sialic acid (Sia), by feeding human neurons the biosynthetic precursor N-acetyl-mannosamine, modified by an azide tag. Using this system, we identified non-toxic conditions that allowed live cell labeling with high spatial and temporal resolution, as well as the quantification of cell surface Sia. Using combinations of immunostaining, chromatography, and western blotting, we quantified the percentage of cellular label incorporation and effects on glycoproteins such as polysialylated neural cell adhesion molecule. A specific imaging algorithm was used to quantify Sia incorporation into neuronal projections, as potential measure of complex cell function in toxicological studies. When various toxicants were studied, we identified a subgroup (mitochondrial respiration inhibitors) that affected neurite glycan levels several hours before any other viability parameter was affected. The MGE-based neurotoxicity assay, thus allowed the identification of subtle impairments of neurochemical function with very high sensitivity.

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(NEURODEG) International the land-BW; (NeuriTox) International Bundesministerium für Bildung und Forschung; (MST-667-00205) International the DK-EPA; KPK InViTe Sigmaringen/Konstanz International Ministerium für Wissenschaft, Forschung und Kunst Baden-Württemberg; 681002 International EU-ToxRisk

Keywords: Confocal imaging; Cytotoxicity; Metabolic glycoengineering; Neurite integrity; Neurotoxicity; Sialic acid

0 (Glycoconjugates)
0 (Hexosamines)
11089-65-9 (Tunicamycin)
147336-22-9 (Green Fluorescent Proteins)
69G8BD63PP (Bortezomib)
GZP2782OP0 (N-Acetylneuraminic Acid)
X80PR7P73R (N-acetylmannosamine)

Date Created: 20191213 Date Completed: 20210223 Latest Revision: 20210617

20231215

10.1007/s00204-019-02642-z

31828357