The biological activity of ppGalNAc-T2 is regulated by chemical acetylation

ZLOCOWSKI, NATACHA; IRAZOQUI, FERNANDO J

Puerto Madryn, Argentina

Congreso; SAIB; 2010

Sociedad Argentina de Investigaciones Bioquímicas (SAIB)

Acetylation is an important molecular regulatory mechanism on the biological activity of proteins. Polypeptide GalNAc transferases (ppGalNAc-Ts) are a family of enzymes which catalyze the initiation of mucin-type O-glycosylation. ppGalNAc-Ts are type II transmembrane proteins with a Golgi luminal region containing a catalytic domain and a C-terminal R-type lectin domain. We investigate the acetylation effect on the catalytic activity and the carbohydrate-binding specificity of ppGalNAc-T2. Mass spectrometry of purified human recombinant ppGalNAc-T2 acetilated in vitro revealed acetylation of K103, S109, K111, K363, S373, K521 and S529. The first five amino acids are located in the catalytic domain. As a consequence of acetylation, ppGalNAc-T2 reduced its specific catalytic activity by 95% to different peptide acceptors (MUC1, MUC2 and MU5Ac). K521 and S529, belong to the lectin domain, their acetylation modified the carbohydrate-binding ability of ppGalNAc-T2. Direct binding assays showed that acetylated ppGalNAc-T2 enhanced the interaction to GalNAc-MUC1. The GalNAc-binding form of lectin domain was also changed by acetylation as demonstrated by competitive assays. In conclusion, the biological activity of ppGalNAc-T2 was clearly regulated by acetylation. In addition, immunoprecipitation on HeLa cell lysate demonstrated the in vivo presence of acetylated ppGalNAc-T2.