Investigation of the glycogen-associated proteome via proximity-biotinylation

Background:

Glycogen metabolism is upregulated in many tumor types, such as hepatocellular carcinoma (HCC) of the clear cell phenotype. These cells are massively loaded with glycogen, resulting in the name-giving clear cell phenotype in HE-stained tissue. The massive storage of glycogen suggests that this may be an important aspect of the pathophysiology of cancer cells, since glucose can be provided for the aerobic glycolysis and the anabolic metabolism can be sustained via the pentose phosphate pathway. However, the impact of glycogen masses on protein distribution within the cell is not clear. Consequences could include the enrichment or depletion of certain proteins or organelles.

Aim:

For this purpose, the glycogen-associated proteome should be determined in glycogen-overloaded HepG2 cells (an immortal cell line consisting of human liver carcinoma cells).

Methods:

Therefore, we established and optimized a TurboID-system, based on the principle of proximity-biotinylation.

Results:

Overexpression of hyperactive glycogen synthase 2 (ha-GYS2) resulted in massive glycogen loading, evident in PAS-stained HepG2-cells and electron microscopy. After fusion of TurboID to ha-GYS2 glycogen overload failed, but the co-transfection with ha-GYS2 at a molar ratio of 1:5 – 1:6 could restore glycogen overload. By western blot analysis we confirmed that enough ha-GYS2-TurboID was expressed to obtain sufficient biotinylation. We also could prove co-localization of ha-GYS2/ha-GYS2-TurboID, indicating that ha-GYS2-TurboID is not mislocalized. Fluorescence microscopy with fluorescent streptavidin in combination with PAS-staining showed that in approximately 75% of cells containing biotinylated proteins also glycogen had accumulated. In further investigations could indicate that the glycogen was soluble and metabolically accessible: It could be depleted by culture in glucose-free medium or digestion with alpha-amylase before PAS staining and biotin-labeled proteins were detected within glycogen masses.

In summary, our results show that labeled proteins co-localize with ha-GYS2 but also biotinylation is diffusely associated with glycogen. Therefore, we are able to label proteins in the proximity of glycogen, giving us the opportunity to purify biotinylated proteins and determine the glycogen-associated proteome in further investigations.