posted on 2022-01-05, 17:34authored byChung-Yao Wang, Edmund Bergström, Jennifer Southgate, Jane Thomas-Oates
This study aimed to investigate the
highly differentiated urothelial
apical surface glycome. The functions of the mammalian urothelium,
lining the majority of the urinary tract and providing a barrier against
toxins in urine, are dependent on the correct differentiation of urothelial
cells, relying on protein expression, modification, and complex assembly
to regulate the formation of multiple differentiated cell layers.
Protein glycosylation, a poorly studied aspect of urothelial differentiation,
contributes to the apical glycome and is implicated in the development
of urothelial diseases. To enable surface glycome characterization,
we developed a method to collect tissue apical surface N- and O-glycans. A simple, novel device using basic laboratory supplies
was developed for enzymatic shaving of the luminal bladder urothelial
surface, with subsequent release and mass spectrometric analysis of
apical surface O- and N-glycans, the first normal
mammalian urothelial N-glycome to be defined. Trypsinization
of superficial glycoproteins was tracked using immunolabeling of the
apically expressed uroplakin 3a protein to optimize enzymatic release,
without compromising the integrity of the superficial urothelial layer.
The approach developed for releasing apical tissue surface glycans
allowed for comparison with the N-glycome of the
total porcine bladder urothelial cells and thus identification of
apical surface glycans as candidates implicated in the urothelial
barrier function. Data are available in MassIve: MSV000087851.