Deconvoluting the functions of polypeptide N-α-acetylgalactosaminyltransferase family members by glycopeptide substrate profiling

The polypeptide N-alpha-acetylgalactosaminyltransferases (ppGaINAcTs) play a key role in mucin-type O-linked glycan biosynthesis by installing the intial GaINAc residue on the protein scaffold. The preferred substrates and functions of the >20 isoforms in mammals are not well understood. However, current data suggest that glycosylated mucin domains are created by the successive, often hierarchical, action of several specific ppGaINAcTs. Herein we analyzed the glycopeptide substrate preferences of several ppGaINAcT family members using a library screening approach. A 56-member glycopeptide library designed to reflect a diversity of glycan clustering was assayed for substrate activity with ppGaINAcT isoforms using an azido-ELISA. The data suggest that the ppGaINAcTs can be classified into at least four types, which working together, are able to produce densely glycosylated mucin glycoproteins.