Inhibitory effect of acetylated lectin domains on glycosyltransferase activity of ppGalNAc-T2

LORENZ, VIRGINIA; IRAZOQUI FJ

Potrero de los Funes, San Luis

Congreso; XLVII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2011

SAIB

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 "ricin-like" lectin domain. We investigated the effect of acetylated lectin domains of ppGalNAc-T3 and ppGalNAc-T4 on GalNAc-transferase activity of ppGalNAc-T2. Lectin domains and ppGalNAc-T2 were expressed as soluble recombinant proteins in insect cells. Constructs contain 6xHis and T7 tags. Recombinant proteins were purified to homogeneity using Ni++ affinity chromatography. Lectin domains were acetylated in vitro, and this chemical modification was detected by western-blot using anti-acetylated lysine antibody. Enzymatic activity of ppGalNAc-T2 was analyzed by mass spectrometry and colorimetric assay. The presence of acetylated lectin domains from ppGalNAc-T3 and ppGalNAc-T4 produced a clear inhibitory effect on catalytic activity of ppGalNAc-T2. Direct binding of lectin domains to (glycosylated) acceptor peptides was not affected by acetylation. In addition, competitive lectin assays showed that acetylation do not change the carbohydrate recognition of these lectin domains. Taken together, these findings suggest a control of ppGalNAc-T2 activity by acetylated lectin domains