A dual-color FRET assay for detection and quantitative analysis of O-glycopeptidases

Mammalian mucosal surfaces are coated with excreted mucus composed primarily of large, heavily O-glycosylated proteins known as mucins. Such extensive O-glycosylation often makes mucin recalcitrant to conventional proteases, making mucins challenging to study by techniques requiring proteolysis. Many mucosa-associated microbes, however, encode mucolytic proteases (O-glycopeptidases or mucinases) that are O-glycan dependent. O-glycopeptidases display substantial diversity in both glycan and peptide sequence recognition, constituting a versatile toolkit for selective cleavage of glycoproteins bearing distinct glycan structures. Exploiting this diversity requires efficient methods for O-glycopeptidase discovery and characterization of substrate selectivity. Here, we describe a high-throughput FÖrster resonance energy transfer (FRET)-based assay for screening O-glycopeptidase activity and glycan selectivity.