Influence of acetylation on biological activity of polypeptide GalNAc-transferase

ZLOCOWSKI N; LORENZ, VIRGINIA; IRAZOQUI, FJ

Conferencia; GlycoAr 2014; 2014

Post-translational acetylation is an important molecular regulatory mechanism affecting the biological activity of proteins. Polypeptide GalNAc transferases (ppGalNAc-Ts) are a family of enzymes that catalyze initiation of mucin-type O-glycosylation. All ppGalNAc-Ts in mammals are type II transmembrane proteins having a Golgi lumenal region that contains a catalytic domain with glycosyltransferase activity, and a C-terminal R-type ("ricin-like") lectin domain. We investigated the effect of acetylation on catalytic activity of glycosyltransferase, and on fine carbohydrate-binding specificity of the R-type lectin domain of ppGalNAc-T2. Mass spectrometric analysis of acetylated ppGalNAc-T2 revealed seven acetylated amino acids (K103, S109, K111, K363, S373, K521, S529); the first five are located in the catalytic domain. Specific glycosyltransferase activity of ppGalNAc-T2 was reduced 95% by acetylation. The last two amino acids, K521 and S529, are located in the lectin domain, and their acetylation results in alteration of the carbohydrate-binding ability of ppGalNAc-T2. Direct binding assays, competitive assays and site-directed mutagenesis approaches showed that acetylation of ppGalNAc-T2 modifies the fine GalNAc-binding form of the lectin domain. Taken together, these findings indicate that catalytic capacity and glycan-binding ability of ppGalNAc-T2 is regulated by acetylation