The Urothelial Glycome: Towards Differential Analysis of N- and O-glycans at the Apical Surface

Normal mammalian urothelium lines the majority of the urinary tract performing a barrier function against penetration of toxic substances and adventitious microorganisms from the urine. These protective functions of the urothelium are dependent on the correct differentiation of urothelial cells, relying on protein expression, modification and complex assembly to regulate the basal to apical organisation of multiple cell layers, including the highly-differentiated lumen-facing superficial cell. As a poorly studied part of the urothelial differentiation programme, protein glycosylation is reported to contribute to the apical glycome, with possible roles in urothelial cancer and infection by uropathogenic E. coli. It is thus important to investigate the apical surface glycome of the normal superficial urothelial cell that is presumed to act in healthy urothelial function and disease.

The aim of this study was to investigate the urothelial cell apical surface glycome using MALDI-MS to analyze permethylated glycans. The first step was to define the N-glycome of the porcine urothelial total cell lysate using the established filter-aided N-glycan separation approach; this represents the first normal mammalian urothelial N-glycome to be defined. Moreover, this thesis presents development of a method to differentially collect the urothelial cell apical surface N- and O-glycans. An in-house designed and built device for trypsinization of the luminal urothelial surface, with subsequent β-elimination and PNGase F digestion was used to release apical surface O- and N-glycans. The effect of trypsinization on superficial glycoproteins was post-monitored using immunolabelling of apically-expressed uroplakin 3a protein to optimize the period of trypsinization, without compromising the integrity of the superficial urothelial layer. 47 N- and 12 O-glycan species were observed on the apical surface of porcine urothelium. The approach developed for releasing apical cell surface N- and O-glycans allowed identification of apical surface glycans as candidates implicated in urothelial barrier function.