HCT116 cells as experimental model to study nuclear fucosylation

ANGELONI, GENARO; IRAZOQUI, FERNANDO J.

Mendoza

Congreso; LVIII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research; 2022

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular

Glycosylation has been found to be involved in tumor malignancy, developmental processes, andimmune system, which has led to new insights into many human diseases. In cases of colon cancer, manyreports have suggested that changes in glycosylation are involved in carcinogenesis and metastasis.Particularly, aberrant glycosylation is associated with the acquisition of all characteristic features oftumor cells. Fucosylation represents the transfer of a fucose residue (from GDP-fucose) tooligosaccharide chains. Most GDP-fucose is synthesized by the de novo pathway, in which GDP-mannoseis transformed into GDP-fucose through three steps catalyzed by GDP-mannose-4,6-dehydratase(GMDS), and FX, an epimerase-reductase enzyme. The salvage pathway synthesizes GDP-fucose fromfree fucose derived from extracellular or lysosomal sources. The deficiency of GMDS has been reportedfor HCT116 cell line, caused by a deletion of 400 bp, which corresponds to three exons encodingfunctionally critical domains for enzyme activity. In the present work, we evaluate HCT116 cells as an invivo model for the study of nuclear fucosylation. Since GDP-fucose is deficient in HCT116 cells, this cell lineshows a marked decrease in the fucosylation level, which we characterize by western-blot andmicroscopy analysis. In addition, we were able to recover the fucosylation phenotype by takingadvantage of the salvage pathway, supplementing the culture medium of the cell line with free fucose.This caused an increase in the fucosylation levels not only in the whole cell but also in purified nuclei.These results place the HCT116 cell line as a good model for fucosylation studies at the cellular and nuclearlevel, where we are able to “turn on and off” the fucosylation pathway as needed just by supplementingwith free fucose