INHIBITORY EFFECT OF ACETYLATED LECTIN DOMAINS ON GLYCOSYLTRANSFERASE ACTIVITY OF ppGalNAc-T2

LORENZ V, IRAZOQUI FJ.

Potreros de los Funes - San Luis

Congreso; 47th Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2011

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Polypeptide GalNAc transferases (ppGalNAc-Ts) are a family ofenzymes that catalyze initiation of mucin-type O-glycosylation. AllppGalNAc-Ts in mammals are type II transmembrane proteinshaving a Golgi lumenal region that contains a catalytic domain withglycosyltransferase activity, and a C-terminal "ricin-like" lectindomain. We investigated the effect of acetylated lectin domains ofppGalNAc-T3 and ppGalNAc-T4 on GalNAc-transferase activityof ppGalNAc-T2. Lectin domains and ppGalNAc-T2 wereexpressed as soluble recombinant proteins in insect cells.Constructs contain 6xHis and T7 tags. Recombinant proteins werepurified to homogeneity using Ni2+ affinity chromatography. Lectindomains were acetylated in vitro, and this chemical modificationwas detected by western-blot using anti-acetylated lysine antibody.Enzymatic activity of ppGalNAc-T2 was analyzed by massspectrometry and colorimetric assay. The presence of acetylatedlectin domains from ppGalNAc-T3 and ppGalNAc-T4 produced aclear inhibitory effect on catalytic activity of ppGalNAc-T2. Directbinding of lectin domains to (glycosylated) acceptor peptides wasnot affected by acetylation. In addition, competitive lectin assaysshowed that acetylation do not change the carbohydrate recognitionof these lectin domains. Taken together, these findings suggest acontrol of ppGalNAc-T2 activity by acetylated lectin domains