The initiation of mucin-type O-glycosylation is modulated by ppGalNAc-T lectin domains

LORENZ, VIRGINIA; IRAZOQUI FJ

Wageningen, The Netherlands

Congreso; 23rd Joint Glycobiology Meeting 2012; 2012

ppGalNAc-Ts (polypeptide GalNAc transferases) are involved in the first step of mucin-type O-glycosylation. ppGalNAc-T2 and T3 are members of this extended enzyme family. They are mammalians type II transmembrane proteins with a Golgi lumenal region that contains a catalytic domain with glycosyltransferase activity. Particularly, they are the only glycosyltransferases having a C-terminal "ricin-like" lectin domain. Here, we study the influence of lectin domains on mucin-type GalNAc-transferase initiation. ppGalNAc-Ts and the lectin domains were expressed as soluble recombinant proteins in Sf9 insect cells. Constructs contain 6xHis and T7 tags. Recombinant proteins were purified to homogeneity using Co++ affinity chromatography. We evaluated enzyme activity of ppGalNAc-T2 and T3 in presence ppGalNAc-T3 and T4 lectin domains with MUC1 and MUC2 as peptide acceptors. Kinetics parameters were measured by using a colorimetric assay. We found the lectin domains have an inhibitory effect on these catalytic ppGalNAc-T activities and inhibitory constants (Ki) were measured. Binding assays showed the recognition of lectin domains to ppGalNAc-Ts and we were able to characterize the type of inhibition. The influence of ppGalNAc-T lectin domains on mucin-type O-glycosylation is also appreciated in in vivo assay. These results indicate that lectin domains could have an important role in regulation on initiation of mucin-type O-glycosylation