Role of ppGalNAc-Ts lectin domains on mucin-type O-glycosylation initiation.

LORENZ VIRGINIA ; IRAZOQUI FERNANDO JOSÉ

Buenos Aires

Simposio; First Argentinian Symposium on Glycobiology "GlycoAR 2014"; 2014

ppGalNAc-Ts (polypeptide GalNAc transferases) are involvedin the first step of mucin-type O-glycosylation. ppGalNAc-T2and T3 are members of this extended enzyme family. They aremammalians type II transmembrane proteins with a Golgilumenal region that contains a catalytic domain withglycosyltransferase activity. Particularly, they are the onlyglycosyltransferases having a C-terminal "ricin-like" lectindomain. ppGalNAc-Ts and the lectin domains were expressedas soluble recombinant proteins in Sf9 insect cells. Constructscontain 6xHis and T7 tags. Recombinant proteins were purifiedto homogeneity using Co++ affinity chromatography. Weevaluated enzyme activity of ppGalNAc-T2 and T3 in presenceppGalNAc-T3 and T4 lectin domains with two methodologies:colorimetric assay and MALDI-TOF MS against differentpeptide acceptors. Kinetics parameters were measured byusing a colorimetric assay. We found the lectin domains havean inhibitory effect on these ppGalNAc-Ts activity andinhibitory constants (Ki) were measured. Binding assaysshowed the recognition of lectin domains to ppGalNAc-Ts andwe were able to characterize the type of inhibition. Theseresults indicate that lectin domains could have an importantrole in regulation of mucin-type O-glycosylation.