A MUTATION MIMICKING ACETYL RESIDUE ON LECTIN DOMAIN OF ppGalNAc-T2 AFFECTS ITS BIOLOGICAL ACTIVITY

ZLOCOWSKI N; 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

ppGalNAc-T2 is a type II membrane protein with an intraluminal glycosyltransferase domain followed by R-type lectin domain. We previously described the effect of acetylation on catalytic and glycan-binding abilities of ppGalNAc-T2. Acetylation on K103, S109, K111, K363, S373, K521 and S529 of ppGalNAc-T2 reduced the enzymatic activity and modified the carbohydrate recognition of lectin domain. (QXW)3 motive is a main characteristic of R-type lectin family and K521 is part of QKW motif of ppGalNAc-T2 lectin domains. This work studies the relevance of K521 on catalytic activity of glycosyltransferase ppGalNAc-T2 as well as its contribution on the carbohydrate-binding ability of lectin domain when K521 is mutated mimicking an acetyl residue. Mutant ppGalNAc-T2K521Q was cloned in Baculovirus expression vector pAcGP67, expressed in Sf9 insect cells, purified by affinity chromatography to homogeneity and acetylated in vitro. Competitive assays revealed a carbohydrate-binding specificity of lectin domain from mutant ppGalNAc-T2K521Q similar to acetylated ppGalNAc-T2. In addition, catalytic activity of enzyme was reduced by mutation, as observed by acetylation on ppGalNAc-T2. In conclusion, the mutation K521Q of ppGalNAc-T2 modifies the protein in similar form to acetylation. Acetylation on K521 could have a regulatory role on biological function of ppGalNAc-T2.