Enzymatic acetylation of ppGalNAc-T2 controls its glycosyltransferase activity

LORENZ, VIRGINIA; ZLOCOWSKI, NATACHA; IRAZOQUI, FERNANDO J

Puerto Madryn, Argentina

Congreso; SAIB; 2010

Sociedad Argentina de Investigaciones Bioquímicas (SAIB)

Mucin-type O-glycosylation is initiated by UDP-Nacetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts). ppGalNAc-Ts are type II transmembrane proteins with a Golgi luminal region containing a catalytic domain and a C-terminal R-type lectin domain. Here we study the enzymatic acetylation effect on the GalNAc-transferase activity of ppGalNAc-T2. Recombinant human ppGalNAc-T2 was expressed in insect cells and purified to homogeneity by affinity chromatography. Purified ppGalNAc-T2 was acetylated in presence of p300 acetyltransferase or without acetyltransferase. ppGalNAc-T2 acetylation degree was demonstrated by western blots using anti-acetylated lysine antibody. The mutant ppGalNAc-T2 K521Q, mimicking an acetylated amino acid in lectin domain, was also analyzed. Glycosyltransferase activity was studied using several mucin peptide acceptors. ppGalNAc-T2 and ppGalNAc-T2 K521Q showed similar glycosyltransferase activity with MUC1, MUC2 and MUC5B peptide acceptors. ppGalNAc-T2 acetylated with p300 or auto-acetylated showed lower enzymatic activity using MUC1 and MUC5B peptide acceptor, and no difference on MUC2 acceptor. Thus, the presence of acetyl residues on defined positions of ppGalNAc-T2 controls its glycosyltransferase activity depending on the mucin peptide acceptor.