Relevance of a post-translational modification site in β-trefoil fold of ppGalNAc-T3

LORENZ, VIRGINIA; CEJAS, RB; IRAZOQUI FJ

Simposio; GlycoAr 2016; 2016

Lysine acetylation is the more evolutionary conserved post-translational modification (PTM) of proteins controlling molecular events. Polypeptide GalNAc-transferase (ppGalNAc-T) enzymes promote the covalent linkage of GalNAc to Ser/Thr of acceptor protein in the beginning of O-GalNAc glycan biosynthesis. In the present work we study the importance of a PTM site in the β-trefoil fold of the isoform ppGalNAc-T3 on catalytic and binding properties by using site-directed mutagenesis. Mutants of ppGalNAc-T3 in K626 showed lower GalNAc-transferase activity. Also, K626Q and K626A ppGalNAc-T3 mutants showed reduced binding recognition to O-GalNAc glycopeptide in comparison with wild-type enzyme. Competitive assays revealed that these mutations affect the carbohydrate recognition capacity of lectin domain as well as the protein-protein interaction of β-trefoil fold modulated by the presence of glycosides. Also GlcNAc glycosides also reduced the enzyme activity in ppGalNAc-T3K626Q without influence in wild-type ppGalNAc-T3. In conclusion, we described that a mutation in lectin domain of ppGalNAc-T3 reduced its GalNAc-transferase activity and presence of GlcNAc glycosides increased the inhibition on enzyme activity. Taken together, this data show the important role of PTMs in lectin domain on biological activity of human ppGalNAc-T3.