O-GalNAc
glycans are important structures in cellular homeostasis. Their biosynthesis is initiated by members of the
polypeptide GalNAc-transferase (
ppGalNAc-T)
enzyme family. Mutations in ppGalNAc-T3
isoform cause diseases (
congenital disorders of glycosylation) in humans. The K626 residue located in the C-terminal β-trefoil fold of ppGalNAc-T3 was predicted to be a site with high likelihood of acetylation by CBP/
p300 acetyltransferase. We used a site-directed mutagenesis approach to evaluate the role of this acetylation site in
biological properties of the
enzyme. Two K626 mutants of ppGalNAc-T3 (T3K626Q and T3K626A) had GalNAc-T activities lower than that of wild-type
enzyme. Direct and competitive interaction assays revealed that GalNAc recognition by the
lectin domain was altered in the mutants. The presence of GlcNAc
glycosides affected the interaction of the three
enzymes with
mucin-derived
peptides. In GalNAc-T activity assays, the presence of GlcNAc
glycosides significantly inhibited activity of the mutant (T3K626Q) that mimicked acetylation. Our findings, taken together, reveal the crucial role of the K626 residue in the C-terminal β-trefoil fold in
biological properties of human ppGalNAc-T3. We propose that acetylated residues on ppGalNAc-T3 function as control points for
enzyme activity, and high level of GlcNAc
glycosides promote a synergistic regulatory mechanism, leading to a metabolically disordered state.