生物化学细胞分解检测.pptVIP

Metabolic Labeling and Imaging of N-Linked Glycans in ArabidopsisThaliana Date:2016.10.28 KEY POINT: 1. Metabolic glycan labeling in Arabidopsisthaliana by using Nazidoacetylglucosamine(GlcNAz) as the chemical reporter isreported. 2. GlcNAz is metabolized through the salvage pathway of N-acetylglucosamine (GlcNAc) and incorporated into Nlinked glycans. 3. Clicklabeling with fluorescent probes enables visualization of newly synthesized N-linked glycans. WHY: Glycoproteins modified with N- or/and O-linked glycans are synthesized in plants and play important functional roles. Comparing to glycans in animals, plant glycosylation has received relatively less attention for the past several decades. The glycan labeling and Imaging techniques developed in animal systems, if can beadapted for plants, will be Invaluable for probing biosynthesis and biological function of plant glycans. N-linked glycosylation, the attachment of N- glycans to asparagine residues within the N- !P-S/T (where !P is not proline) consensus sequence of cell surface and secreted proteins, is one of the most prominent protein posttranslational modifications, which occurs in all eukaryotes.Plant N-linked glycosylation has been implicated in protein quality control, development, innate immunity, and stress response. WAY Evaluation of unnatural sugarc reporters in plants is critical for developing metabolic glycan labeling methods for plants. For example, 6-alkynyl fucose (FucAl), a fucose analogue containing an alkyne, was used to metabolically label the fucose-containing pectin in cell walls,and no incorporation of FucAl into fucosylated proteins was observed.[14] Distinctly, FucAl primarily labels Fucosylated proteins in mammalian cells. GlcNAz was chemically synthesized and globally acetylated as Ac4GlcNAz to facilitate the cellular uptake. Arabidopsis Col-0 seedlings were incubated with 100 mm Ac4GlcNAz in liquid Murashige and Skoog (MS) mineral medium for 48h (Figure 1a