Mass spectrometry analysis of epithelial cadherin

E-cadherin functions as the primary epithelial intercellular adhesion receptor and forms cell-cell contacts known as adherens junctions. With key roles in development and cancer progression, alterations in the assembly or disassembly of adherens junctions occur in association with major changes in cell states. These studies have investigated the N-glycosylation of E-cadherin and the role E-cadherin N-glycosylation has on its association with protein binding partners to mediate cellular adhesion.;Using anion exchange chromatography, charge distinct E-cadherin complexes were isolated. Our results have determined that more positively charged E-cadherin complexes are involved in more stable cell-cell adhesions. Additionally, E-cadherin from these pools contains more high mannose and hybrid-type N-glycans as compared to negatively charged E-cadherin complexes, which contain mostly complex type N-glycans.;By affinity purification methods and reversed phase chromatography coupled to tandem mass spectrometry, we have investigated the molecular organization of E-cadherin complexes from dense benign and malignant cell cultures, which display characteristically different adherens junctions. We identified many cytoskeletal proteins known to associate with E-cadherin, as well as proteins of other cellular functions. In malignant cultures, E-cadherin was associated with an increased number of proteins involved in cellular processes, cell development and cell metabolism and a reduced number of proteins involved in cell regulation and cell responses to stimuli, as compared to E-cadherin from benign cell cultures. Analyses of complexes isolated from E-cadherin with diminished N-glycosylation detected differences in the association of E-cadherin with proteins involved in cell responses to stimuli, cell regulation and cell localization as compared to wild-type E-cadherin.;Finally, we used mass spectrometry and endoglycosidases to determine E-cadherin N-glycan structures from a recombinant E-cadherin protein. The N-glycans are of both high mannose and complex type, with some being sialylated and fucosylated.;These studies have confirmed the relationship between E-cadherin N-glycosylation and its protein binding partners to provide further insight into Ecadherin mediated function.;This work was supported by NIH grants P41 RR10888 and 510 RR15942 (C. E. Costello) and R01 DE10183 and ROI DE 14437 (M. A. Kukuruzinska). Graduate training assistance was provided by T32 AG000115 (PI. P. Polgar)...