Mass spectrometric approach for characterization of kinetics and site-specificity of a glycosyltransferase initiating O-glycosylation of IgA1 hinge region

摘要

We produced recombinant GalNAc-T2 in a baculovirus expression system and developed an in vitro system to study the kinetics of site-specific glycosylation of IgAl HR using high-resolution MS. Time course analysis of GalNAc-T2 activity with sHR showed that the number of GalNAc residues added to the sHR increased with time: within 10 min, all sHR molecules were glycosylated with at least one GalNAc and within 15 min, sHR had 3 to 6 GalNAc residues attached. The GalNAc-T2 transferred GalNAc residues to T7 first and then T15, followed by S11, T4, and S9 sites. These sites were consistent with the O-glycosylation sites previously identified on IgAl. The alternative sites (S3 and T12) in the two adjacent S-T sites were the last ones to be glycosylated.