Chemical glycomics enrichment: imaging the recycling of sialic acid in living cells

Abstract

The development of metabolic oligosaccharide engineering (MOE) over the past two decades enabled the bioimaging studies of glycosylation processes in physio-pathological contexts. Herein, we successfully applied the chemical reporter strategy to image the fate of sialylated glycoconjugates in healthy and sialin-deficient patient fibroblasts. This chemical glycomics enrichment is a powerful tool for tracking sialylated glycoconjugates and probing lysosomal recycling capacities. Thus, such strategies appear fundamental for the characterization of lysosomal storage diseases.

Fig. 1

Schematic representation of the biosynthetic (green arrow) and the recycling (blue dashed arrow) pathways of sialic acids into mammalian cells. The alkynyl reporter ManNAl enters the cell and is enzymatically modified into SiaNAl in the cytosol. Then, its activation is performed in the nucleus before transport into the Golgi and incorporation into the glycans through the action of sialyltransferases. The incorporated alkyne can then be detected via chemical ligation with a specific fluorescent probe. The reinternalization of the glycoconjugates is achieved by endocytosis. After maturation of the endosomes into lysosomes, specific lysosomal sialidases are able to cleave the terminal sialic acids residues which are subsequently transported from the lysosome to the cytosol by the specific transporter sialin

Fig. 2

Fibroblasts from healthy individuals and sialin deficient patient were metabolically labeled with 500 μM of ManNAl for 8 h (T₀) and then chased for 6, 12, 24, 48 and 72 h. The sialylated glycoconjugates (in red) were visualized by confocal microscopy after staining with azido 545 fluorescent probe (Scale bar, 25 μm)

Fig. 3

Fibroblasts from healthy individual were metabolically labeled with 500 μM of SiaNAl overnight and then fixed and submitted to CuAAC reaction with Azido 545 fluorescent probe (red signal) and DAPI (blue signal). Immediately after fixation, cells were submitted to either no treatment (a) or Arthrobacter ureafaciens sialidase treatment (b) (Scale bar, 20 μm)

Fig. 4

Fibroblasts from healthy individuals and sialin deficient patient were metabolically labeled with 500 μM of ManNAl for 3, 6, and 8 h respectively and stained with azido 545 fluorescent probe (sialic acid in glycoconjugates in red) or antibodies against a lysosomal marker (LAMP-2 in green). Staining was then visualized using confocal microscopy. It could be noticed that this co-localization of the MOE signal with the lysosomal LAMP-2 marker was seen in all investigated cells (Scale bar, 25 μm)

Fig. 5

Fibroblasts from healthy individuals and sialin deficient patients were metabolically labeled with 500 μM of ManNAl for 8 h in absence or presence of 40 μM chloroquine and stained with azido 545 fluorescent probes (sialic acid in glycoconjugates in red). Staining was visualized using confocal microscopy (Scale bar, 10 μm except for image on the up-right, 25 μm)