The Role of N-Glycosylation in Submandibular Gland Branching Morphogenesis

Objectives: The submandibular gland (SMG) develops through branching morphogenesis from an epithelial bud into an array of ducts terminating in secretory acini. Pivotal to SMG development is E-cadherin, the primary salivary N-glycoprotein cell-cell adhesion receptor that functions through the formation of dynamic multiprotein complexes linked to the actin cytoskeleton, named adherens junctions (AJs). We have shown that during embryonic development, SMG buds comprise distinct cell populations with AJs of varying stability. In other studies, we demonstrated that AJ stability was regulated by the N-glycosylation status of E-cadherin. Here, we investigated the role of N-glycans in E-cadherin adhesive function during SMG branching morphogenesis. Our strategy was to use siRNA to partially inhibit DPAGT1, a key regulator of protein N-glycosylation. Results: Treatment of E12.5 SMGs with siRNA to DPAGT1 resulted in a 40% knockdown of its transcript levels and diminished N-glycosylation of E-cadherin. This correlated with aberrant morphology and reduced outgrowth of the glands. Confocal microscopy of F-actin staining revealed more organized acinar and ductal structures in siRNA-treated SMGs. This coincided with increased colocalization of gamma-catenin with E-cadherin at cell-cell contact sites and changes in the molecular organization of E-cadherin junctions consistent with their stabilization. Methods: SMGs were transfected with DPAGT1 siRNA and non-silencing control for 48 – 72 h and examined by phase contrast and confocal microscopy for F-actin, E-cadherin and gamma-catenin distribution. The composition of AJ was determined by immunoprecipitation of E-cadherin followed by Western blot for associated proteins. Conclusion: Partial inhibition of N-glycosylation using siRNA to DPAGT1 interferes with branching morphogenesis of the SMG by prematurely stabilizing E-cadherin junctional complexes resulting in precocious differentiation of the gland. This work was supported by Grants DE14437 and DE10183 from the NIH/NIDCR to MAK