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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.
Bibliographical noteFunding Information:
The authors are grateful to Dr. Daniel Ungar for helpful discussions and his insights on glycobiology throughout the development of this study. Dr. Ashley Ward is thanked for his expert contribution to the design of the bladder stretch device described herein. We gratefully acknowledge the assistance of Timothy Ayers (Electronics Workshop, Department of Chemistry, University of York) for his technical expertise with fabricating the reaction device for stretching and sampling bladder surfaces. Dr. Jenny Hinley and Rosalind Duke of the Jack Birch Unit are thanked for their expert technical support with cell biological and immunohistochemical aspects of the study. The York Centre of Excellence in Mass Spectrometry was created thanks to a major capital investment through Science City York, supported by Yorkshire Forward with funds from the Northern Way Initiative, and subsequent support from EPSRC (EP/K039660/1; EP/M028127/1). JS and other members of the Jack Birch Unit receive support from York Against Cancer.
- mass spectrometry
- permethylated glycan
- tryptic shaving