O-glycosylation is a complex post-translational protein modification, involved in a series of cellular processes including protein folding, stability and secretion. It involves an α-linkage of a sugar moiety on serine or threonine residues via an oxygen atom. Mucin-type O-glycan synthesis starts with the formation of Tn antigen (Ser/Thr-O-GalNAc), which is catalysed by polypeptide N-acetylgalactosaminyltransferases (ppGalNAcTs). The Tn antigen gets modified in the T-synthase catalysed reaction to form an O-linked disaccharide made of GalNAc and Gal.
In a study from 2018, a group of scientists showed that impaired O-linked glycosylation leads to pancreatic exocrine dysfunction and diabetes in mice. The study showed that the activity of a key β3galactosyltransferase (T-synthase) in O-glycan synthesis depends on its essential chaperon Cosmc (C1galt1c1). The T-synthase activity in wild type and Cosmc-knock out mice tissues was detected with an O-glycosidase-coupled fluorescent assay.
UDP-Gal and GalNAc-α-4MU from Carbosynth were mixed with protein extracts from pancreatic tissues containing T-synthase. The reaction yielded Galβ1-3GalNAcα1-4MU disaccharide, which was then incubated with O-glycosidase. This enzyme specifically cleaves core 1 O-glycans and releases 4MU, yielding a fluorescent signal that can be detected at Ex 355/Em 460 nm.
For more details, read the original paper published in Experimental and Molecular Medicine: Wolters-Eisfeld et al., 2018.