Methods and compositions for detections and modulating O-glycosylation

A method of identifying a compound that modulates polypeptide O-glycosylation, the method comprising:

• (a) contacting a glucosyltransferase enzyme, a sugar donor, and a candidate modulating compound with a molecule comprising a detectably labeled polypeptide having:
• (i) an O-glycosylation site and
• (ii) a specific protease cleavage site positioned in the polypeptide such that the rate of specific protease cleavage of the polypeptide glycosylated at the O-glycosylation site is different than the rate of specific protease cleavage of the polypeptide not glycosylated at the O-glycosylation site,
• wherein the detectable label is positioned in the polypeptide such that a change in the amount of detectable label correlates with cleavage of the polypeptide by the specific protease at the specific protease cleavage site;
• (b) adding to the contacted polypeptide the specific protease;
• (c) monitoring the rate of specific protease cleavage of the polypeptide;
• and,
• (d) comparing the rate of specific protease cleavage of the polypeptide to a control rate of specific protease cleavage
• wherein a difference in the control rate of specific protease cleavage compared to the rate of cleavage of the polypeptide contacted with the candidate modulating compound identifies the candidate modulating compound as modulating O-glycosylation of the polypeptide.