This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goals of this study are to determine the role of the first N-glycosylation gene, ALG7, in the mouse submandibular gland (SMG) development and the role of E-cadherin glycosylation in cancer cell proliferation. By focusing on a downstream target, the N-linked glycoprotein E-cadherin, we are investigating the role of N-glycosylation in E-cadherin mediated cell-cell adhesion during SMG morphogenesis and cytodifferentiation. Cadherins constitute a class of transmembrane adhesion receptors that mediate cell-cell adhesion in solid tissues of the body. E-cadherin is a classical epithelial cadherin that functions in the formation of homotypic cell-cell contact, or adherens junctions. Adherens junctions have a prominent role in remodeling epithelial cell-cell interactions and are especially critical in development during the morphological transitions of tissue formation; and function in the maintenance of adult tissue architecture. Changes in the extent of E-cadherin N-glycosylation suggest that this modification plays a role in the formation of cell-cell contacts and this research study is investigating N-glycan site occupancy and oligosaccharide glycoform composition of E-cadherin during SMG development and during carcinogenesis, and the makeup of protein complexes that involve E-cadherin. E-cadherin and its complexes are being isolated from tissue homogenate and/or cell lysates and partially purified using ion-exchange and affinity chromatography. During early morphogenesis, highly N-glycosylated E-cadherin is found in transient unstable cell-cell contacts, while in the cytodifferentiated gland, hypo-N-glycosylated E-cadherin forms stable cell-cell contacts. Our results suggest that, during differentiation when cell-cell contacts are more stable, E-cadherin in a hypo-N-glycosylated form. MS analysis of an excised SDS-PAGE E-cadherin band from immunoprecipitated E-cadherin containing cell lysate identified E-cadherin and confirmed that MS identification and analysis of E-cadherins is feasible. Preliminary experimental results have verified that the isolation and detection of E-cadherin and its glycans can be accomplished for samples obtained in limited amounts from functionally relevant biological sources. Since E-cadherin is a membrane glycoprotein that provides strong, Ca2+-dependent epithelial intercellular adhesion. E-cadherin functions in various morphogenic processes through mediation of cell-cell contacts, as well as cell transformation.(1-4) E-cadherin cell-cell contacts are dynamic and undergo continuous rearrangement depending on cell context. Therefore, the recruitment of various proteins to the adhesion complex define the overall stability of E-cadherin mediated adhesion. Separation and identification of individual adhesive complex components provide insight on E-cadherin cellular activities. Ion Exchange chromatography with Q Sepharose has been used to separate several classes of E-cadherin protein complexes from cancerous (A253) and normal (MDCK) epithelial cells. Immunoprecipitation to capture the E-cadherin complex followed by protein analysis with Immunoblot techniques and MALDI-TOF MS, was performed. Our results indicate that E-cadherin exists in different types of protein complexes that vary among cell types (i.e., normal and cancerous epithelial tissue), and even within the same cell type; these changes are accompanied by differences in the aggregation properties of the cells. Additionally, fractionation of these E-cadherin complexes, using ion exchange chromatography, appears to be a useful tool to profile cell type. These findings suggest that the dynamic balance among such complexes governs the overall stability of E-cadherin mediated cell-cell contacts and that charge differences can be used to isolate distinct E-cadherin protein complexes. Further work in this area is being pursued
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